AU726734B2 - Mycobacterium cell wall compositions - Google Patents

Description

WO 98/20900 PCT/AU97/00770 -1- MYCOBACTERIUM CELL WALL COMPOSITIONS FIELD OF THE INVENTION The present invention relates generally to a method of immunomodulating therapy and pharmaceutical compositions useful for same. More particularly, the present invention provides a method of ameliorating the effects of autoimmune conditions. Even more particularly, the present invention contemplates a method for preventing, delaying onset of or otherwise ameliorating the effects of insulin-dependent diabetes mellitus (IDDM) by administering a cell wall subunit or a chemical or functional equivalent thereof from Mycobacterium or a related organism or other suitable biological source. The present invention is further directed to a pharmaceutical composition useful in preventing, delaying onset of, curing, curing in association with islet replacement or otherwise ameliorating the effects of autoimmune conditions such as IDDM or for enhancing an immune response against melanoma or other cancer comprising a cell wall subunit or a chemical or functional equivalent thereof from Mycobacterium or a related organism or other suitable biological source.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

BACKGROUND OF THE INVENTION Insulin-dependent diabetes mellitis (IDDM) is a debilitating, chronic, cell-mediated autoimmune disease characterised by lymphocytic infiltration of the pancreatic islets and T lymphocyte- WO 98/20900 PCT/AU97/00770 -2mediated destruction of insulin-producing P cells 2).

Non-obese diabetic (NOD) mice are a valuable model in studying IDDM as they spontaneously develop the disease which has many immunological and pathological similarities to human IDDM It has been previously shown that administration of Freund's complete adjuvant (CFA) or Mycobacterium bovis (Bacillus Calmette-Guerin (BCG) prevents development of diabetes in NOD mice However, Baxter et al showed the administration of BCG, although preventing diabetes in NOD mice, precipitated a syndrome similar to systemic lupus erythematosus (SLE), precluding its use in humans.

In accordance with the present invention, it has been shown that a subunit complex from the cell wall of Mycobacterium prevents diabetes in NOD mice without risk of precipitating SLE. The subunit complex, or one or more of its components, are useful, therefore in immunomodulatory therapy for autoimmune diseases and for enhancing an immune response to various cancers.

SUMMARY OF THE INVENTION One aspect of the present invention contemplates a method of immunomodulatory therapy in a mammal said method comprising administering to said mammal an immunomodulating effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source.

Another aspect of the present invention provides a method of preventing, delaying onset of, curing or otherwise ameliorating the effects of an autoimmune disease in a mammal said method comprising administering to said mammal an autoimmune-preventing effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source.

Still another aspect of the present invention is directed to a method of preventing, delaying onset WO 98/20900 PCT/AU97/00770 -3of, curing or otherwise ameliorating the effects of insulin-dependent diabetes mellitus (IDDM) in a mammal said method comprising administering to said mammal an autoimmune-preventing effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source.

Yet another aspect of the present invention contemplates a method of enhancing an immune response against melanoma or other cancer in a mammal said method comprising administering to said mammal an immunomodulatory effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source.

Still yet another aspect of the present invention contemplates immunomodulatory therapy in a mammal said method comprising administering to said mammal an immunomodulatory effective amount of mycolyl-arabinogalactan-peptidoglycan (MAPG) or a component thereof with or without other associated cell wall components and submolecular components from a Mycobacterium species such as but not limited to Mycobacterium bovis or a chemical equivalent of said MAPG or of a component thereof.

Even yet another aspect of the present invention contemplates a method for isolating components of MAPG for use in a therapeutic composition for preventing, delaying the onset of or otherwise ameliorating the effects of diabetes in a mammal or for use in immunomodulatory therapy said method comprising preparing cell envelopes from a species of Mycobacterium or related organism or other suitable biological source, subjecting said cell envelopes to glycolipid removing means to remove soluble glycolipids, treating the product so obtained to break the mycolic acids linkage and isolating said mycolic acids, treating the remaining complex to cleave linkage at rhamnose residue connecting arabinogalactan to the peptidoglycan backbone and separating and isolating arabinogalactan and peptidoglycan.

Another aspect of the present invention provides a composition of matter comprising MAPG or a derivative or a component thereof or its derivative.

WO 98/20900 PCT/AU97/00770 -4- Yet another aspect of the present invention relates to the use of a cell wall component of Mycobacterium in immunomodulatory therapy.

BRIEF DESCRIPTION OF THE FIGURES In the Figures: Figure 1 is a diagrammatic representation of a mycobacterial cell wall.

Figure 2 is a graphical representation showing the percentage incidence of diabetes over time (days) following administration of phosphate buffered saline

(PBS).

Figure 3 is a graphical representation showing the percentage incidence of diabetes over time (days) following administration of BCG.

El 0.1 mg/mouse 0.2 mg/mouse O 0.4 mg/mouse 0 0.8, 1.6, 4 mg/mouse Figure 4 is a graphical representation showing the percentage incidence of diabetes over time (days) following administration of N-CFAglucosaminyl-N-acetylmuramyl-L-alanyl-Disoglutamine I.V. (1.0-10gg).

0 1.0 /g/mouse 2.5 /g/mouse O 5.0 /g/mouse 0 10 /g/mouse Figure 5 is a graphical representation showing the percentage incidence of diabetes over time (days) following administration of lipoarabinomannan 6 2 5 -50tg WO 98/20900 PCT/AU97/007 70 D 6.25 /g/mouse 12.5 /g/mouse O 25 /ag/mouse 0 50 /g/mouse Figure 6 is a graphical representation showing the percentage incidence of diabetes over time (days) following administration of mycolyl-arabinogalactan-peptidoglycan (0.125-1.0 mg I.V).

0 0.125 mg/mouse 0.25 mg/mouse O 0.5 mg/mouse 0 1.0 mg/mouse Figure 7 is a graphical representation showing the percentage incidence of diabetes over time (days) following administration of mycolic acids (13-200 g C 12.5 /g/mouse 50 /g/mouse 0 200 /g/mouse WO 98/20900 PCT/AU97/00770 -6- DETAILED DESCRIPTION OF PREFERRED

EMBODIMENTS

The present invention contemplates a method of immunomodulatory therapy in a mammal said method comprising administering to said mammal an immunomodulating effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source or chemical equivalents of said components.

In one aspect, the present invention is directed to a method of preventing, delaying onset of, curing or otherwise ameliorating the effects of an autoimmune disease in a mammal said method comprising administering to said mammal an autoimmune-preventing effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source or chemical equivalents of said components.

In another aspect, the present invention provides a method of enhancing an immune response against melanoma or other cancer in a mammal said method comprising administering to said mammal an immunomodulatory effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source or chemical equivalents of said components.

Autoimmune conditions contemplated by the present invention include but are not limited to IDDM, thyroiditis, atrophic gastritis (type pernicious anaemia, Addison's disease, pemphigus vulgaris, pemphigoid, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, haemolytic anaemia, sympathetic ophthalmia, uveitis, idiopathic thrombocytopenia, idiopathic leucopenia, primary biliary cirrhosis, autoimmune chronic active hepatitis, ulcerative colitis, Sjogren's syndrome, dermatomyositis, scleroderma and mixed connective tissue disease.

Cancers contemplated for immunomodulatory therapy include but are not limited to bladder cancer, carcinoma, melanoma amongst many others.

The present invention is hereinafter described in relation to IDDM but this is done with the WO 98/20900 PCT/AU97/00770 -7understanding that the invention extends to autoimmune diseases such as contemplated above as well as the immunomodulatory therapy of cancers.

Accordingly, the present invention particularly contemplates a method of preventing, delaying onset of, curing, curing in association with islet replacement and/or pancreas transplant or otherwise ameliorating the effects of IDDM in a mammal said method comprising administering to said mammal an IDDM-preventing effective amount of one or more components of the cell wall of Mycobacterium or a related organism or analogous components from another biological source or chemical equivalents of said components.

The major components of the mycobacterial cell wall are: 1) mycolyl-arabinogalactanpeptidoglycan (MAPG), a polymer which provides the structural framework of the cell wall, 2) lipids, 3) phosphatidylinositol mannosides, and 4) lipoarabinomannan (LAM). MAPG has three major subdomains which are: 1) peptidoglycan, 2) arabinogalactan and 3) mycolic acids. Nacetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) and N-acetylmuramyl-Lalanyl-D-isoglutamine (MDP) are peptidoglycan subunits considered to be responsible for most of the adjuvant activity of CFA.

The components of the Mycobacterium cell wall or of another suitable source or their chemical equivalents contemplated for use in preventing IDDM in mammals include MAPG or components or chemical equivalents thereof with or without other associated cell wall components and submolecular components or such as but not limited to mycolate, arabinogalactan and/or peptidoglycan or derivatives or chemical equivalents thereof (see Figure MAPG or its components may be in native or chemically synthetic form. MAPG is a complex of covalently attached macromolecules. Mycolic acids are covalently attached to arabinogalactan which is in turn covalently attached to peptidoglycan. Reference herein to "MAPG" includes the MAPG complex isolated from Mycobacterium or related organism or other suitable biological source or to a chemically or functionally equivalent complex as well as submolecular components including mycolic acids, peptidoglycan or arabinogalactan or chemical or functional equivalents thereof. The submolecular components may be in isolated form or in partial complex forms such as comprising mycolic acids and arabinogalactan, arabinogalactan and WO 98/20900 PCT/AU97/00770 -8peptidoglycan or mycolic acids and peptidoglycan or chemical or functional equivalents thereof.

A particular complex may also comprise, for example, mycolic acids covalently linking to arabinogalactan and this may in turn be covalently linked to a portion or derivative of peptidoglycan.

A convenient source of MAPG or its components is Mycobacterium bovis or BCG. The present invention, however, extends to MAPG or its components from any species of Mycobacterium or from physiologically, genetically, biochemically or structurally related microorganisms.

Examples of similar organisms include Actinomycetes, Nocardia and Corynebacterium.

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similar molecule or natural complex or components thereof may also be isolatable for other biological sources such as plants and coral. The MAPG complex or its components may be isolated from mycobacterial cell envelopes prepared, for example, by the method of Azuma et al (8) According to this aspect of the present invention, there is provided a method for isolating components of MAPG for use in a therapeutic composition for preventing, delaying the onset of or otherwise ameliorating the effects of diabetes in a mammal, said method comprising preparing cell envelopes from a species of Mycobacterium or related organism or other suitable biological source, subjecting said cell envelopes to glycolipid removing means to remove soluble glycolipids, treating the product so obtained to break the mycolic acids linkage and isolating said mycolic acids, treating the remaining complex to cleave linkage at rhamnose residue connecting arabinogalactan to the peptidoglycan backbone and separating and isolating arabinogalactan and peptidoglycan.

The soluble glycolipids are conveniently removed by repeated centrifugation in the presence of sodium dodecyl sulphite (SDS). The resulting insolvent envelope MAPG complex is then subjected to fractionation.

The mycolic acids linkage is preferably cleaved by saponification, base-catalysed methanolysis or ammonolysis. The remaining complex of arabinogalactan and peptidoglycan is preferably then subjected to a Smith degradation comprising periodate followed by borohydride reduction and WO 98/20900 PCT/AU97/0077 0 -9mild acid treatment.

The MAPG complex or its component parts or derivatives thereof may be in any convenient form such as vacuum dried, powder, liquid or slurry.

The present invention further contemplates a composition of matter comprising MAPG or a derivative thereof or a component thereof or its derivative or chemical equivalents of MAPG or its components. These components are referred to herein as "active ingredients".

Preferably, the composition is a pharmaceutical composition for use in preventing, delaying onset of, curing, curing in association with islet replacement or otherwise ameliorating the effects of IDDM in mammals or for enhancing an anti-tumour immune response in mammals. The pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers and/or diluents.

According to this and other aspects of the present invention, preferred mammals include humans, primates, livestock animals (eg. cows, horses, sheep, pigs, donkeys), laboratory test animals (eg.

mice, rabbits, guinea pigs, hamsters), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, foxes, deer).

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. The present invention also contemplates administration via topically applied compositions where molecules are used to permit entry via the skin. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier may be a solvent medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) or suitable mixtures thereof and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, WO 98/20900 PCT/AU97/007 7 0 chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

S. 15 When the active ingredients are suitably protected they may be orally administered, for example, o with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft S: shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with food material (including solid or liquid products). For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions Sand preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 )ug and about 4000 mg of active compound. Other ranges contemplated herein include from about Ijig to about 1000 mg, from about 10 Ag to about 100 mg and from about 100 jg to about 50 mg. Effective amounts may also be provided as an amount per kilogram of body weight of the recipient. For example, from about 0.01 ng to about 10,000 mg/kg body weight or may be administered from about 0.1 ng to about 500 mg/kg y I dy weight.

WO 98/20900 PCT/AU97/007 7 0 -I1 The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels.

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Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Parental compositions may be administered by, for example, intravenous intradermal, subcutaneous deep SC or intramuscular (IM) routes amongst other routes. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with

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WO 98/20900 PCT/AU97/00770 -12required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active material and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail. Other routes of administration are also contemplated by the present invention including intracerebral intraperitoneal, intranasal, buccal, rectal, implant, infusion, inhalation administration in addition to intravenous drip.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active Scompound in amounts ranging from about 0.1 pg to about 4000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 pg to about 2000 mg/ml of carrier.

15 In the case of compositions containing supplementary active ingredients, the dosages are S. determined by reference to the usual dose and manner of administration of the said ingredients.

The active ingredients of the present invention may be administered alone or in combination with other therapeutic molecules such as molecules which reduce effects of the autoimmune pathology :20 associated with IDDM. Alternatively, the active ingredients may be administered with anticancer agents or functionally unrelated but nevertheless useful molecules such as antibodies, analogues or the like. A single dose may be administered or multiple doses may be required with intervals of from minutes to hours, daily to weekly or monthly to yearly.

Reference herein to "preventing" IDDM includes total prevention of IDDM or substantial prevention for a limited time (eg. from about 0.5 to about 10 years) or delaying onset of IDDM or reducing the severity or otherwise ameliorating the effects of IDDM.

The present invention further contemplates use of a cell wall component of Mycobacterium in immunomodulatory therapy. Preferably, the therapy is the treatment of an autoimmune disease as diabetes. Alternatively, the therapy is the treatment of melanoma, bladder cancer or WO 98/20900 PCT/AU97/00770 -13other cancer, preferably by enhancing an immune response.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 Components of mycobacterial cell wall Components of the mycobacterial cell wall are shown in Figure 1.

EXAMPLE 2 Mice Female NOD/Lt/Arc mice were obtained from the Animal Resources Centre (Canning Vale, WA, Australia) and maintained in clean conditions in the Centenary Institute Animal House.

S 15 Sentinel mice were tested by serology at four-monthly intervals for the following pathogens: mouse hepatitis virus, rotavirus, ectomclia, mouse cytomegalovirus, polyoma virus, murine adenovirus, lymphocytic choriomeningitis virus, mouse pneumonia virus, retrovirus, Sendai virus, S Theiler's murine encephalitis virus, Bacillus piliformis, Mycoplasma pulmonis, Bordetella bronchiseptica, Corynebacterium kutscheri, Klebsiella species, Pasturella multocide, Pasturella 20 pneumotropica, Pseudomonas aeruginosa, Straphylococcus aureus, Streptococcus pneumoniae SCitrobacterfreundii and salmonella species. No mice tested positive for any of these pathogens.

Mice were housed at 21C and 40% humidity and were fed Barastock mouse chow (Melbourne, VIC, Australia) and acidified water ad libitum.

Under these conditions, about 75% of female NOD mice spontaneously developed IDDM by weeks of age. The disease process involved a progressive preclinical phase of islet destruction which commenced at 4-6 weeks of age, and concluded with the onset of clinical diabetes between 14 and 35 weeks of age. Within a population, disease onset occurred in a sigmoidal fashion with the peak incidence of IDDM at 22 weeks of age, and a plateau at 35 weeks after which few previously unaffected mice ever progressed to diabetes WO 98/20900 PCTIAU97/00 7 7 0 14 NOD mice intravenously injected with a single dose of 0.4-4.0 mg of heat-killed BCG did not become diabetic, but developed a lupus-like disease characterised by haermolytic anaernia (indicated by a lowered haematocrit and positive Coombs' test), increased titres of anti-nuclear antibodies (demonstrated by immunofluorescence of HEp-2 cells) and glomerulonepAfi i (demonstrated by immunofluorescence of C3c bound to the renal glomeruli) EXAMPLE 3 BCG therapy Evans (Langhurst, UK) or CSL (Parkville, Australia) live freeze dried and attenuated Mycobacterium bovis (bacillus Calmette-Gucrin; BCG) vaccine was dissolved in isotomic saline and heat inactivated at 65 C for 45 minutes.

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a 15 EXAMPLE 4 Mycobacterial subfractjons Mannose-capped lipoarabinomannan (ManLAM) and mYcoly-abarbinogaactanppidglca complex (MAPO) may be Prepared as Previously described N-acetylglucosaminyl-NacetyilmuramyLalaylyD-isglutnj (GMDP) was purchased from a commercial source 20 (GERBU Biotechnik, Gaibery, Germany). The Applicant acknowledges with appreciation receiving samples of ManLAMv and MAPG from the Tube~rcul osis Reposifory by Drs P. J.

Brennan and J.T. Belisle through NJAD, 13 November, 1996 [NUT Contracts NOI-AUI-25 147 and NOI1-AI-753 EXAMPLE Random blood glucose estimations Each mouse was bled by retro-orbital venepuncture of 100-150 jzl and the serum glucose concentration measured by the glucose oxidase technique on a Glucostix reagent strip (Ames, Basingstoke, UK). A mouse was considered to be diabetic if it was found to have a random __blood glucose level 11. 1lmMolI.

WO 98/20900 PCT/AU97/00770 EXAMPLE 6 Haematocrit measurement Seventy-five microlitres of blood were drawn up into a heparinized capillary tube (Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged at 1000g for 15 minutes. The height of the column of packed red cells was divided by the total height of the column of blood and expressed as a percentage.

EXAMPLE 7 Direct Coombs test Mice were bled and the plasma removed. Ten microlitres of packed cells were resuspended in PBS with 0.3% w/v bovine serum albumin, washed and resuspended in Iml of the same solution. Triplicates of 100 aliquots were placed in 96 well round bottom plates (Nunc, Denmark) with 3 serial V2 dilutions. The plates were vortexed and incubated at 37 C for Ihr.

Wells were then assessed for false positive results. Ten microlitres of 10 gg/ml polyclonal goat anti-mouse IgG (Sigma Chemical Company, MO, USA) added. Plates were then vortexed gently and incubated at 37C for a further two hours. Wells in which the cells collected in a button were recorded as negative, while those in which the cells remained spread of the surface over the surface of the well were recorded as positive.

EXAMPLE 8 Assessment of antinuclear antibodies Sera were assessed for binding to HEp-2 slides (Quantafluor, Chaska, MN, USA). Slides were incubated in phosphate buffered saline (PBS) for 10 minutes. Sera diluted in PBS (starting concentration 1:100) were incubated on the slides at room temperature (RT) for 30 minutes in a moist chamber. Slides were then washed 3 times for 5 minutes in PBS and incubated for minutes at RT with 1:50 FITC conjugated rat anti-mouse Ig (Serotec, Oxford, UK). Slides were again washed 3 times for 5 minutes with PBS, mounted and examined on an Axiophot fluorescence microscope (Zeiss, DDR). Sera from MRL/lpr-lpr and BALB/c mice were used WO 98/20900 PCT/AU97/00770 16as controls.

EXAMPLE 9 Detection of glomerular immune complex deposits Kidneys were embedded in Tissue-Tek OCT Compound (Miles, Elkhart, IN) and frozen for histological analysis. Sections of 6 Mm were cut on a Microm cryostat (Waldorf, Germany) and mounted on microscope slides, air dried, acetone fixed for 10 minutes and stored at -80C in an air-tight bag containing silica desiccant. When slides were stained, they were thawed to room temperature, fixed in acetone for a further five minutes and blocked with 4% v/v foetal calf serum (FCS; CSL, Melbourne, VIC, Australia). Sections were stained with Goat anti-mouse C3c polyclonal IgG (Nordic Immunological Laboratories, Tilburg, Holland) at a 1:10 dilution in PBS for 45 minutes. Slides were then washed three times in PBS, and coverslipped with polyvinyl alcohol mounting media (Sanofi Diagnostics Pasteur Inc., Chaska, MN) and examined on a Leica fluorescence microscope (Leica Mikoskopie, Postfach, Germany).

EXAMPLE Effects of heat killed BCG on incidence of diabetes Varying concentrations of heat killed Mycobacterium bovis (BCG) were administered intravenously to NOD mice at 0.1 mg, 0.2 mg, 0.4 mg, 0.8 mg, 1.6 mg, 4 mg relative to a control of phosphate buffered saline (PBS; Figure No diabetes was detected in mice given 0.8, 1.6 or 4 mg of BCG (Figure Doses of 0.1 mg, 0.2 mg and 0.4 mg were not completely effective although they did provide some protection.

EXAMPLE 11 Effects of mycobacterial cell wall components on incidence of diabetes In order to attempt to separate the activity of BCG which prevented IDDM from that which precipitated lupus, the mycobacterial subfractions GMDP, MAPG and ManLAM were tested for these activities. GMDP (10, 5.0, 2.5 and 1.0 ug), ManLAM (50, 25, 12.5 and 6.25 gg), and WO 98/20900 PCT/AU97/00770 -17- MAPG 0.5, 0.25 and 0.125 mg) were each suspended in saline and injected intravenously into eight week old female NOD mice. These doses of MAPG and ManLAM were based on estimates of the equivalent quantities in mng of BCG, while the dose of GMDP was based on that used for immunoadjuvant activity; 10g being the maximum dose tolerated by mice. Groups of 5 mice were tested and the incidence of diabetes compared to 17 saline treated and 14 BCG treated control mice. The results are shown in Figures 2 to 7.

While 10/17 PBS treated mice (Figure 12/20 of GMDP treated mice (Figure 4) and 13/18 of ManLAM treated mice developed diabetes (Figure 5) no mice receiving MAPG did so (Figure Haematocrits and Coombs' tests indicated that no mice receiving MAPG developed haemolytic anaemia whereas 11/14 of BCG treated (0.8-1.6 mg) control mice had a haematocrit below 46% and 7/14 mice were Coombs' test positive. Similarly there was no increase in the expression of antinuclear antibodiesintheMAPG treatedmice The S effects of mycolic acids administration on the incidence of diabetes is shown in Figure 7. 15 EXAMPLE 12 Effects of mycobacterial cell wall components on incidence of diabetes

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MAPG was injected intravenously into 8 week old NOD mice in doses ranging from 1 mg to 20 0.125 mg/mouse. All doses tested prevented IDDM. MAPG treated mice were tested for haematocrit, Coombs' test, antinuclear antibodies (ANA) and C3c complement component deposition in the renal glomeruli. No mice developed haemolytic anaemia as detected by lowered haematocrit and positive Coombs' test. ANA levels were not raised above those detected in PBS treated mice. Mild C3c deposition was found in a minority of mice. It was concluded that MAPG administration prevented IDDM without inducing lupus in NOD mice.

It is significant that while MAPG was still effective at preventing IDDM when used at a dose of 0.125 mg/mouse, BCG was ineffective at this dose. This indicates that it is likely that the effect of the MAPG preparation used in these experiments is due to MAPG itself and not to a contaminant from BCG.

WO 98/20900 PCT/AU97/00770 -18- EXAMPLE 13 Purification of components of MAPG Mycobacterium cell envelopes maybe prepared by the method of Azuma et al and the soluble glycolipids removed by repeated centrifugation in the presence of SDS. The insoluble envelope component, the peptidoglycan conjugated with mycolic acids-substituted arabinogalactan, is fractionated into its constituent domains by the following procedure.

1. the mycolic acids linkage is cleaved by saponification, base-catalysed methanolysis or ammonolysis and separated from the insoluble residue.

2. the residue is submitted to Smith degradation (periodate, followed by borohydride reduction and mild acid treatment) to cleave the linkage at the rhamnose residue connecting the galactan to the peptidoglycan backbone.

Due to the nature of the furanosyl linkages in the galactan and arabinan domains, these residues are not affected by this treatment. The soluble arabinogalactan is separated from the peptidoglycan by centrifugation.

EXAMPLE 14 Testing components of MAPG The previous example shows the effectiveness of MAPG in preventing development of diabetes relative to other components of the mycobacterial cell wall. The individual constituents of MAPG are purified according to Example 13 and tested at varying concentrations in NOD mice.

The incidence of diabetes is then determined over time.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, WO 98/20900 PCTIAU97/00770 -19individually or collectively, and any and all combinations of any two or more of said steps or features.

WO 98/20900 PCT/AU97/00770

BIBLIOGRAPHY

Bach, J.F. Enclocrine Rev 15: 516, 1994.

2. Honeyman, M.C. and Harrison L.C. Springer Semn Immunopathol 14: 253, 1993.

3. Makino S et al Exp. Anim 29: 1-13, 1980.

4. Kanazawa Y, et al Diabetologia 27: 113-115, 1984.

Harada M et al Diabetes Res Cmn Pract 8: 85, 1990.

6. Sadelain M, W. J. et al Diabetes 39: 583, 1990.

7. Baxter, A.G. et al Immunology 83: 227-23 1, 1997.

8. Azumna, 1. et al J. Bacterol. 96: 1885-1887, 1968.

9. Baxter A.G. et al Diabetes 38: 1296-1300, 1989.

Claims (14)

1. A method of immunomodulatory therapy in a mammal, said method comprising administering to said mammal an immunomodulating effective amount of a cell wall of Mycobacterium or a related organism, said cell wall component comprising mycolyl- arabinogalactan-peptidoglycan (MAPG) or a component thereof or chemical equivalent thereof with or without other associated cell wall components and submolecular components or their chemical equivalents wherein said immunomodulatory therapy is not for the treatment of infection by Mycobacterium tuberculosis or Mycobacterium leprae.

2. A method according to Claim 1 wherein the immunomodulatory therapy is for the treatment of an autoimmune disease. S 3. A method according to Claim 2 wherein the autoimmune disease is one or more of 00: IDDM, thyroiditis, atrophic gastritis (type pernicious anaemia, Addison's disease, pemphigus vulgaris, pemphigoid, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, haemolytic anaemia, sympathetic ophthalmia, uveitis, idiopathic thrombocytopenia, idiopathic leucopenia, primary biliary cirrhosis, autoimmune chronic active hepatitis, ulcerative colitis, Sjogren's syndrome, dermatomyositis, scleroderma and mixed connective tissue disease. A method according to Claim 3 wherein the autoimmune disease is IDDM. A method according to Claim 1 wherein the immunomodulatory therapy is for enhancing an anti-tumour immune response.

6. A method according to Claim 5 wherein the immunomodulatory therapy is for enhancing an immune response against melanoma or bladder cancer. I- -tk 7. A method according to any one of Claims 1 to 6 wherein the MAPG is administered in combination with one or more of mycolic acids, peptidoglycan or P\Op rEjl\AMENDED\2177l97.cwims doc-19/09/00 -22- arabinogalactan or chemical or functional equivalents thereof.

8. A method according to Claim 1 wherein MAPG or its components are derived from Mycobacterium bovis.

9. A method according to Claim 1 wherein the mammal is a human. A method for preventing, delaying onset of, curing, curing in association with islet and/or pancreas transplant replacement or otherwise ameliorating the effects of IDDM in a mammal, said method comprising administering to said mammal an IDDM treating effective amount of MAPG from Mycobacterium or a related organism or a derivative or .**component of MAPG or a chemical equivalent thereof.

11. A method according to Claim 10 wherein the mammal is a human.

12. A method according to Claim 10 or 11 wherein the MAPG or its derivatives or components is from M bovis.

13. A composition of matter comprising MAPG or a derivative or a component thereof or its derivative or chemical equivalents thereof when used in accordance with the method any one of Claims 1 to 12. t* 14. A composition according to Claim 13 further comprising one or more pharmaceutically acceptable carriers and/or diluents. A method for isolating components of MAPG for use in a therapeutic composition for preventing, delaying the onset of or otherwise ameliorating the effects of diabetes in a mammal or for use in immunomodulatory therapy, said method comprising preparing cell envelopes from a species of Mycobacterium or related organism or other suitable biological source, subjecting said cell envelopes to glycolipid removing means to remove A L, soluble glycolipids, treating the product so obtained to break the mycolic acids linkage and P:\Oper\Ejlh\AMENDED\2177197.ncwlaims doc- 19/09/00 -23- isolating said mycolic acids, treating the remaining complex to cleave linkage at rhamnose residue connecting arabinogalactan to the peptidoglycan backbone and separating and isolating arabinogalactan and peptidoglycan.

16. A method according to Claim 15 wherein the glycolipids are removed by repeated centrifugation in the presence of sodium dodecyl sulphate (SDS).

17. A method according to Claim 15 wherein the mycolic acids linkage is cleaved by saponification, base-catalysed methano lysis or ammonolysis.

18. Use of MAPG or a derivative or a component thereof from Mycobacterium in immunomodulatory therapy provided said immunomodulatory therapy is not for the treatment of Mycobacterium tuberculosis or Mycobacterium leprae.

19. Use according to Claim 18 wherein the immunomodulatory therapy is for the treatment of diabetes.

20. Use according to Claim 18 wherein the immunomodulatory therapy is for the treatment of carcinoma.

21. A method according to any one of Claims 1 to 14 or 17 to 19 or a composition according to Claim 15 or 16 or a use according to Claim 20, 21 or 22 substantially as hereinbefore defined with reference to the Figures and/or Examples.