JP2003510335A - Vaccines for the treatment of autoimmune diseases - Google Patents

Abstract

  (57) [Summary] The present invention is directed to M. et al. Pharmaceutical compositions comprising vaccae cells are provided. These compositions can include either dead cells or delipidated and deglycolipidated cells. The present invention provides a method of treating an autoimmune disease, the method comprising administering an immunologically effective dose of a pharmaceutical composition comprising P-VAC. The present invention is a method of treating an autoimmune disease, the method comprising the steps of: A method comprising administering an immunologically effective dose of a pharmaceutical composition comprising vaccae cells.

Description

Detailed Description of the Invention

(Cross Reference of Related Applications) This application is filed on Aug. 6, 1999 in USSN 60 / 147,626 (
Priority, which is incorporated herein by reference).

BACKGROUND OF THE INVENTION The present invention relates to compositions and methods for treating autoimmune diseases. These compositions contain Mycobacterium vaccae as the active ingredient.

Many pathological responses are known, including unwanted immune responses. For example, autoimmune diseases are a particularly important class of adverse immune responses. In autoimmune diseases, self-tolerance is lost and the immune system attacks "self" tissues as if they were foreign targets. More than thirty autoimmune diseases are currently known and these include:
Rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), myasthenia gravis (MG), systemic lupus erythematosus (SLE) and scleroderma.

Although many advances have been made in the treatment of autoimmune diseases, new therapeutic approaches to the treatment and prevention of these autoimmune diseases are needed. The present invention addresses these and other needs.

SUMMARY OF THE INVENTION The present invention relates to killed M. et al. Provided is a pharmaceutical composition comprising vaccae cells or P-VAC. P-VAC is an M.P. vac
Defatted and deglycolipidated fraction from cae cells. The composition of the present invention may further include an adjuvant. These compositions may be administered according to standard methods well known in the art. Typically, these compositions are administered parenterally (eg subcutaneously or intraperitoneally). The methods of the invention can be used to treat autoimmune diseases such as diabetes, multiple sclerosis and rheumatoid arthritis.

DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention provides a pharmaceutical composition comprising an antigenic material derived from Mycobacterium vaccae. These compositions are generally useful for treating pathological conditions such as autoimmune diseases.

The therapeutic compositions of the present invention are described in M. dead cells of vaccae (herein M-VA
Called C). The means by which these cells are killed is not critical and can be done, for example, by autoclaving or irradiation. The preparation of pharmaceutical compositions containing such cells is described, for example, in EP 630 259 and US Pat. No. 5,833.
, 996. The M.R. The vaccae strain is N
national Collection of Type Cultures (
NCTC) under the Budapest Treaty under the number NCTC11659 (Stanford and Paul, Ann. Soc. Belge Med.
Trop. 53: 141-389 (1973)).

Alternatively, the composition of the present invention is referred to herein as P-VAC, M. va
The delipidated and deglycolipidated fraction (DD-MV) from ccae cells may be included. The preparation of such cells is described, for example, in WO99 / 32634 and WO9.
8/8542 (see Example 1 for details).

M. Vaccae cells or modified cells may be formulated in a pharmaceutical composition useful for administration to mammals, particularly humans, for treating and / or preventing adverse autoimmune responses. A suitable formula is Remington's Pharmac
electrical Sciences, Mack Publishing Com
Pany, Philadelphia, PA, 17th edition (1985).

PHARMACEUTICAL COMPOSITIONS The present invention provides the present invention in a pharmaceutically acceptable solution for administration to an animal, either alone or in combination with one or more other therapeutic modalities. The M.S. v
It relates to formulations of accae compositions.

The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those of ordinary skill in the art and employs various treatment regimens (eg, oral, parenteral, intravenous, intranasal and intramuscular administration and formulation). The development of suitable doses and treatment regimens for using the particular compositions described herein (including) is also well known to those of skill in the art.

1. Oral Delivery In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be tablets. Or they can be incorporated directly with the food of the diet.

The active compounds can even be incorporated within excipients and used in the form of tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, cachets and the like for ingestion ( Mathiowitz et al., 1997; Hwang et al., 19
98; U.S. Pat. No. 5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451, each of which is specifically incorporated herein by reference in its entirety. Be done)). Tablets, troches, pills, capsules, etc. may also include: Binders (eg, gum tragacanth, gum arabic, corn starch, or gelatin); Excipients (eg, dicalcium phosphate); Disintegrants (eg, corn starch). , Potato starch, alginic acid, etc.); lubricants (eg magnesium stearate); and sweeteners (eg sucrose, lactose or saccharin may be added); or flavoring agents (eg peppermint, oil of pearl millet, or cherries) Flavoring agent). When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier.
Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. For example, tablets, pills or capsules
It can be coated with shellac, sugar, or both. An elixir syrup may contain the active compounds sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form must be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

Typically, these formulations may contain at least about 0.1% or more of the active compound, although the proportion of active ingredient may, of course, vary and, conveniently, the total formulation. Can be between about 1 or 2% and about 60% or 70% or more by weight or volume. Of course, the amount of active compound with which each therapeutically useful composition is prepared is such that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations, are contemplated by those of skill in the art of preparing such pharmaceutical formulations. , And that various dosages and treatment regimens may be desirable by those of skill in the art.

For oral administration, the compositions of the present invention may alternatively comprise one or more excipients,
It may be incorporated into a mouthwash, a dentifrice, a buccal tablet, an oral spray, or a sublingual orally administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be incorporated into an oral solution, such as one containing sodium borate, glycerin, and potassium bicarbonate, or
Or it may be dispersed in a dentifrice or added in a composition which may include water, a binder, an abrasive, a flavoring agent, a foaming agent, and a wetting agent in a therapeutically effective amount. Alternatively, the composition can be placed under the tongue or formed into a tablet or solution which is otherwise dissolved in the mouth.

2. Injectable Delivery In certain circumstances, the pharmaceutical compositions disclosed herein may be administered as described in US Pat.
No. 5,543,158; US Pat. No. 5,641,515 and US Pat. No. 5,3.
99,363, each of which is specifically incorporated herein by reference in its entirety, for parenteral, intravenous, intramuscular, or even intraperitoneal delivery. desirable. Solutions of the active compound as a free base or as a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersion is also
It can be prepared in glycerol, polyethylene glycol liquids, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (US Pat. No. 5,466). , 468, which is specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
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 can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and polyethylene glycol liquids), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as a lectin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microbial action is achieved by various antibacterial and antifungal agents (eg, paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc.)
Can be promoted by. In many cases, it will be preferable to include isotonic agents, for example sugars and sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use of agents in the composition that delay absorption such as aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Must be done. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection
Sterile aqueous media that can be used are known to those of skill in the art in light of the present disclosure. For example,
One dose can be dissolved in 1 ml of isotonic NaCl solution, and 1000 ml
Either added to a hypodermoclysis fluid or injected at the site of application of the infusion (eg, Remin
gton's Pharmaceutical Sciences, 15th Edition, 10
See pages 35-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Furthermore, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity as required by FDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. To be done. 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. To be done. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are the suction-drying and freeze-drying techniques, which are based on the active ingredient and any further desired from the previously sterile-filtered solution. Produces a powder of the ingredients of.

The compositions disclosed herein may be formulated in a neutral or salt form. Possible pharmaceutically important salts include acid addition salts (formed with the free amino groups of the protein), which are inorganic acids (eg hydrochloric acid or phosphoric acid) or acetic acid, oxalic acid, tartaric acid. , Formed with organic acids such as mandelic acid.
Salts formed with free carboxyl groups may also be derived from, for example, inorganic salts such as sodium, potassium, ammonium, calcium or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. . Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are conveniently administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

As used herein, “carrier” refers to any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents. , Buffer solutions, carrier solutions, suspensions, colloids and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
Except to the extent that any conventional vehicle or drug is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an allergic or similar annoying reaction when administered to a human. The preparation of an aqueous composition containing a protein as an active ingredient is well understood in the art.
Typically, such compositions are prepared as either injectable liquid solutions or suspensions; solid forms suitable for liquid solutions or suspensions are also prepared prior to injection. obtain. The preparation can also be emulsified.

3. Nasal Delivery In certain embodiments, the pharmaceutical composition comprises intranasal spray, inhalation, and / or
Or it may be delivered by other aerosol delivery vehicles. Methods for delivering gene, nucleic acid, and peptide compositions directly to the lung by nasal aerosol spray are described in US Pat. No. 5,756,353 and US Pat. No. 5,804,212 (each of which is herein incorporated by reference). The entire contents of which are incorporated by reference in detail). Similarly, drug delivery using intranasal particulate resin (Takena
Ga et al., 1998), and drug delivery using lysophosphatidylglycerol compounds (US Pat. No. 5,725,871, which is specifically incorporated herein by reference in its entirety). Well known in the art. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene loaded matrix is described in US Pat. No. 5,780,045, which is specifically incorporated herein by reference in its entirety.

4. Liposomes, Nanocapsules, and Microparticle-Mediated Delivery In certain embodiments, we will introduce liposomes, nanocapsules, microparticles for introducing the compositions of the invention into suitable host cells. The use of microspheres, lipid particles, vehicles and the like is contemplated. In particular, the composition of the present invention is for delivery
It can be formulated encapsulated in any of lipid particles, liposomes, vehicles, nanospheres, nanoparticles, or the like.

Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the compositions disclosed herein. The formation and use of liposomes generally involves
Known to those of skill in the art (eg, Couvreur et al., 1997; Couvreu
r, 1988; Lasic, 1998, which describes the use of liposomes and nanocapsules in targeted antibiotic therapy against intracellular bacterial infections and diseases). Recently, liposomes with improved serum stability and circulating half-life have been developed (Gabizon and Papahadjopoul).
Os, 1988; Allen and Choun, 1987; US Patent No. 5,74.
No. 1,516, which is specifically incorporated herein by reference in its entirety. In addition, various methods have been reviewed as potent drug carriers for liposomes and liposome-like preparations (Takakura, 1998; Chandran et al., 19).
97; Margalit, 1995; US Pat. No. 5,567,434; US Pat. No. 5,552,157; US Pat. No. 5,565,213; US Pat.
738,868 and US Pat. No. 5,795,587 (each of which is
Are hereby incorporated by reference in their entirety in detail).

Liposomes consist of genes, drugs (Heath and Martin, 1986; H
eath et al., 1986; Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),
An enzyme (Imaizumi et al., 1990a; Imaizumi et al., 1990b),
Viruses (Faller and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau and Gersonde, 19).
79) has been effectively used to introduce various cultured cell lines and animals. In addition, several successful clinical trials have been completed to test the efficacy of liposome-mediated drug delivery (Lopez-Brestein et al., 1985a; 19).
85b; Cune, 1988; Sculier, 1988). Moreover, some studies have suggested that the use of liposomes is not associated with autoimmune response, toxicity, or gonadal localization after systemic delivery (Mori and Fukatsu, 1
992).

Liposomes are dispersed in an aqueous medium and contain multilamellar concentric bilayer vesicles (
Phospholipids that naturally form multilamellar vesicles (MLVs)).
MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs forms small unilamellar vesicles (SUVs) with diameters in the range 200-500Å and containing aqueous solution in the core.

Liposomes have similarities to cell membranes and are contemplated for use with the present invention as carriers for compositions. These are broadly suitable as both water-soluble and fat-soluble substances, and soluble substances can be entrapped (ie in the aqueous space and in the bilayer respectively). Drug-loaded liposomes can further be used for site-specific delivery of active agents by selectively modifying liposome formulations.

In addition to the teaching of Couvreur et al. (1977, 1988), the following information can be utilized in making liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, based on the lipid to water molar ratio. In small proportions, liposomes are the preferred structure. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes
Although it may exhibit low permeability to ionic and polar substances, at elevated temperatures it undergoes a phase transition that significantly alters their permeability. This phase transition is associated with a change from a tightly packed regular structure (known as the gel state) to a loosely packed, less ordered structure (known as the fluid state). It occurs at a characteristic phase transition temperature and results in increased permeability to ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter the permeability of liposomes. Certain soluble proteins, such as cytochrome c, bind to the bilayer and deform,
It then penetrates, which causes a change in permeability. Cholesterol apparently inhibits the penetration of this protein by packing it more tightly with phospholipids.
It is contemplated that the most useful liposomal formulations for antibiotic and inhibitor delivery include cholesterol.

The ability to trap solutes varies between different types of liposomes. For example, MLV
Is moderately effective in trapping solutes, but SUVs are not. SU
V offers the advantage of uniformity and reproducibility in size distribution. However, the trade-off between size and capture capacity is provided by large unilamellar vesicles (LUVs). They are prepared by evaporation of ether and are 3-4 times more effective than MLVs in trapping solutes.

In addition to the properties of liposomes, an important determinant in entrapment of compounds is the physicochemical properties of the compound itself. Polar compounds are trapped in the aqueous space and non-polar compounds bind to the lipid bilayer of vesicles. Polar compounds are released by osmosis or when the bilayer collapses, while non-polar molecules remain in the bilayer unless destroyed by temperature or exposure to lipoproteins. Both types show a maximum efflux rate at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system (eg, macrophages and neutrophils); nonspecific weak hydrophobicity or Adsorption to the cell surface, either by electrostatic forces or specific interactions with cell surface components; traits due to the insertion of liposome lipid bilayers into the plasma membrane with simultaneous release of liposome contents into the cytoplasm. By fusion with cell membranes; and by the transfer of liposomal lipids to cell membranes or cell component membranes (or vice versa) without any association of liposome contents. It is often difficult to determine which mechanism works, and whether more than one mechanism can work at the same time.

The developmental fate and placement of intravenously injected liposomes depends on their physical properties (eg,
Details, fluidity and surface charge). They persist in tissues for hours or days, depending on their composition, and half-lives in blood range from minutes to hours. Larger liposomes (eg MLV and LUV)
Although rapidly taken up by phagocytes of the reticuloendothelial system, the physiology of the circulatory system limits the exit of such large species at most sites. They can only emerge where large openings or pores are present in the capillary endothelium (eg sinusoids of the liver or spleen). Therefore, these organs are the predominant sites of uptake. In other words, SUVs show a broader tissue distribution but remain more highly isolated in the liver and spleen. In general, this in vivo behavior limits the possible targeting of liposomes to only those organs and tissues that are acceptable for their large size. These include blood, liver, spleen, bone marrow, and lymphocyte organs.

Targeting is generally not a limitation in terms of the present invention. However, specific targeting should be desired and methods are available to achieve this. Antibodies can be used to bind to the surface of liposomes and to direct the antibody and its drug content to specific antigen receptors located on specific cell type surfaces. Carbohydrate determinants, cell surface components of glycoproteins or glycolipids that play a role in cell-cell recognition, interaction and adhesion, also recognize sites where they have the potential to direct liposomes to specific cell types. Can be used as. Intravenous injection of liposomal preparations is primarily used, but it is contemplated that other routes of administration are also contemplated.

Alternatively, the present invention provides a pharmaceutically acceptable nanocapsule formulation of the composition of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible manner (Henry-Michelland et al., 1987; Quintanar).
-Guerrero et al., 1998; Douglas et al., 1987). In order to avoid side effects due to the overloading of intracellular polymerization, such ultrafine particles (size about 0.1 μm) have to be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl cyanoacrylic acid nanoparticles that meet these requirements are contemplated for use in the present invention. Such particles can be readily made as described below (Couvreur et al., 1980; 198).
8; zur Muhlen et al., 1998; Zambaux et al., 1998; Pin.
to-Alphandry et al., 1995, and US Pat. No. 5,145,684.
No., which are specifically incorporated herein by reference in their entirety).

Vaccines In certain preferred embodiments of the invention, vaccines are provided. Vaccines generally include one or more pharmaceutical compositions such as those described above in combination with an immunostimulatory factor. The immune stimulator can be any substance that enhances or enhances the immune response (antibody-mediated and / or cell-mediated) to exogenous antigens. Examples of immunostimulators are adjuvants, biodegradable microspheres (eg polymeric galactides), and liposomes (wherein the compound is incorporated; eg F.
Ullerton, U.S. Pat. No. 4,235,877). Vaccine preparations are generally described, for example, by Powell and Newman, eds., V.
accine Design (subunit and adjuvant approach) (
1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also include other compounds that may be biologically active or inactive. For example, there may be one or more immunogenic portions of other tumor antigens, either incorporated into the fusion polypeptide or incorporated into the composition or vaccine as separate compounds.

Any suitable carrier known to those of skill in the art may be used in the vaccine compositions of the present invention, the type of carrier varying based on the mode of administration. The compositions of the invention are administered in any suitable manner, including topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
Can be prescribed for. For parenteral administration (eg subcutaneous injection), preferably the carrier comprises water, saline, alcohols, fats, waxes or buffers. For oral administration, any of the above carriers or solid carriers can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate. Biodegradable microspheres such as polylactic acid polyglycolate can also be used as carriers for the pharmaceutical compositions of the invention.
Suitable biodegradable microspheres are described, for example, in US Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,1.
No. 28; No. 5,820,883; No. 5,853,763; No. 5,81.
No. 4,344, and No. 5,942,252. US Patent No. 5,
Carriers including the particle-protein complex described in 928,647, which can induce a class I-restricted cytotoxic T lymphocyte response in the host, can also be used.

Such compositions may also include buffers (eg neutral buffered saline or phosphate buffered saline), carbohydrates (eg glucose, mannose, sucrose or dextran), mannitol, proteins. , Polypeptides or amino acids (eg glycine), antioxidants, bacteriostats, chelating agents (eg EDT
A or glutathione), an adjuvant (eg, aluminum hydroxide), a solute, suspending agent, thickening agent and / or preservative which renders the formulation isotonic, hypotonic or weakly hypertonic with the blood of the recipient. May be included. Alternatively, the composition of the invention may be formulated as a lyophilizate. The compounds can also be encapsulated within liposomes using well known techniques.

Any of a variety of immune stimulators may be used in the vaccine of the present invention. For example, an adjuvant can be included. Most adjuvants are substances designed to protect the antigen from rapid catabolism (eg aluminum hydroxide or mineral oil) and stimulators of the immune response (eg Lipid A), Bordetel.
la pertussis or Mycobacterium species or Myc
(Obacterium-derived protein). Suitable adjuvants include, for example, Freund's Incomplete Adjuvant.
Adjuvant) and Freund's complete adjuvant (Freund's)
Complete Adjuvant) (Difco Laboratorie)
s, Detroit, MI), Merck Adjuvant 65 (Merc)
k and Company, Inc. , Rahway, NJ); AS-2 and their derivatives (SmithKline Beecham, Philadel).
PIA, PA); CWS, TDM, Leif, aluminum hydroxide gel (alum) or aluminum salts such as aluminum phosphate; calcium, iron,
Or zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatives of polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A (monophosphoryl lipid A).
) And Quil A. Cytokines such as GM-CSF or interleukin-2, interleukin-7 or interleukin-12 can also be used as adjuvants.

In the vaccines provided herein, the adjuvant composition is preferably designed to induce a predominantly Th1 type immune response. High level T
h1 type cytokines (eg, IFN-γ, TNFα, IL-2 and IL-1
2) tends to favor the induction of a cell-mediated immune response to the administered antigen. In contrast, high levels of Th2-type cytokines (eg IL-4, IL-5,
IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of the vaccines provided herein, patients support an immune response that includes a Th1-type response and a Th2-type response. In a preferred embodiment, where the response is predominantly Th1 type, the levels of Th-1 type cytokines are increased to a greater extent than the levels of Th2 type cytokines. The levels of these cytokines can be easily assessed using standard assays. For a review of the family of cytokines, see Mosmann & Coffman, Ann. Rev. Immun
ol. 7: 145-173 (1989).

Preferred adjuvants, primarily for use in inducing a Th1-type response, include, for example, monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), and aluminum. A combination of salts may be mentioned. MPL adjuvant is available from Corixa Corporation (Seattle, WA; US Pat. Nos. 4,436,727; 4,8,8).
77,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides, where the CpG dinucleotides are not methylated, also predominantly induce a Th1-type response. Such oligonucleotides are well known, for example WO96 / 02555, WO99 / 3.
3488 and US Pat. Nos. 6,008,200 and 5,856,462.
No. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Scie.
nce 273: 352 (1996).

Another preferred adjuvant is saponin or a mimetic or derivative of saponin (preferably QS21 (Aquila Biopharmaceutica).
ls Inc. , Framingham, MA)), which can be used alone or in combination with other adjuvants. For example, an enhanced system is a combination of monophosphoryl lipid A and a saponin derivative (eg WO94 /
Combination of QS21 and 3D-MPL as described in 00153), or a less reactive composition in which QS21 is cholesterol quenched, as described in WO 96/33739. Other preferred formulations include oil-in-water emulsions and tocopherols. A particular potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is W
O95 / 17210.

Other preferred adjuvants include Montanide ISA720 (Sep
pic, France), SAF (Chion, California, Uni)
Ted States), ISCOMS (CSL), MF-59 (Chiron)
), SBAS series of adjuvants (eg SBAS-2, AS2 ′, AS
2 '', SBAS-4 or SBAS6, SmithKline Beecha
m, available from Rixensart, Belgium), Detox (Corix
a, Hamilton, MT), RC-529 (Corixa, Hamilto
n, MT) and other aminoalkyl glucosaminide 4-phosphate (AGP
) (Eg, those described in pending US patent application serial numbers 08 / 853,826 and 09 / 074,720, the disclosures of which are incorporated herein by reference in their entireties). )).

Any of the vaccines provided herein can be prepared using well known methods that result in the combination of antigen, immune response enhancer and a suitable carrier or excipient. The compositions described herein include sustained release formulations (ie, formulations such as capsules, sponges or gels (eg, composed of polysaccharides) that provide a slow release of the compound after administration. Can be administered as part of a product. Such formulations may generally be prepared using well known techniques (see, eg, Combes et al.
Vaccine 14: 1429-1438 (1996)), and, for example, by oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may include a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate controlling membrane.

Carriers for use in such formulations may be biocompatible and also biodegradable; preferably, the formulation provides a relatively constant level of active ingredient release. provide. Such carriers include particulates such as poly (lactide-co-glycolide), polyacrylates, latex, starch, cellulose, dextran. Other delayed release carriers include supramolecular biovectors, which include a non-liquid hydrophilic core (eg, cross-linked polysaccharides or oligonucleotides) and optionally an amphipathic compound (eg, phospholipids) Layers (eg, US Pat. No. 5,151,254 and PCT application WO94 / 2).
0078, WO / 94/23701 and WO 96/06638).
. The amount of active compound contained within a sustained release formulation depends on the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

Vaccines and pharmaceutical compositions may be presented in unit-dose containers or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably sealed to maintain the sterility of the formulation until use. In general, the formulations can be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the vaccine or pharmaceutical composition may be stored in lyophilized form requiring only the addition of a sterile liquid carrier just before use.

All publications and patent applications cited herein are hereby specifically and individually indicated to be incorporated by reference in their respective publications or patent applications. Incorporated as a reference.

While the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be made without departing from the spirit or scope of the appended claims. What can be done will be readily apparent to one of ordinary skill in the art in view of the teachings of the present invention.

Example 1 Delipidated and deglycolipidated M. Preparation of vaccae (P-VAC)) P-VAC was prepared as generally described in WO99 / 32634. The summary of the protocol is as follows.

Heat-killed M. vaccae was prepared using standard methods. Delipidated M. v
In order to prepare accae, autoclaved M. The vaccae were pelleted by centrifugation, the pellet washed with water, collected again by centrifugation and then lyophilized. This lyophilized M. An aliquot of V. vaccae was set aside and freeze-dried. I called it vaccae. When used in experiments, it was resuspended in PBS to the desired concentration. Lyophilized M. vaccae 6
Lipids were extracted by treatment with chloroform / methanol (2: 1) for 0 minutes at room temperature and the extraction was repeated once. The delipidated residue from the chloroform / methanol extract was further treated with 50% ethanol and refluxed for 2 hours to remove glycolipids. The 50% ethanol extraction was repeated twice. The residue from the 50% ethanol extraction was lyophilized and weighed. This delipidated and deglycolipidized M. vaccae (DD-M. vaccae) was resuspended in phosphate buffered saline by sonication, thus producing P-VAC. In a preferred embodiment, this preparation was used for the treatment of autoimmune diseases without further processing. In some embodiments, this preparation was autoclaved prior to administration.

Example 2 M-VAC Can Ameliorate Disease in the NOD Model for Autoimmune IDDM This example demonstrates that killed M. NOD using vaccae cells (M-VAC)
It is shown to ameliorate the disease in mice (mouse model for autoimmune IDDM). The disease in these animals is characterized by anti-islet cell antibodies, severe insulinitis, and evidence for autoimmune destruction of β-cells. 70-90% of females and 20-30% of males spontaneously develop diabetes within the first 6 months of life. This disease can also be introduced into these mice by administration of cyclophosphamide.

(Protocol and Results of M.-VAC Experiment) 1. 1. Normoglycemic NOD mice (6-8 weeks old) were divided into 3 experimental groups. Group 1 received 500 μg MVAC IP. Group 2 received 500 μg MVAC IP on day (-2), day 1,
Acceptance was on days 4, 7, and 14. Cyclophosphamide was given on day 0.

4. Group 3 received cyclophosphamide injection only on day 0 5. Mice on day 7 and then DI every Monday, Wednesday and Friday until Day 21
The development of diabetes was monitored by ASTIX, and the experiment was completed on the 21st day.

As can be seen in FIG. 1, multiple injections of MVAC given by IP injection provide statistically significant protection from the development of diabetes in this model (P
= 0.0001). Administration of MVAC alone (in the absence of cyclophosphamide) had no effect on mice.

Example 3 (P-VAC alone cannot ameliorate disease in NOD model of autoimmune IDDM) This example is intended to ameliorate disease in NOD mice, a murine model of autoimmune IDDM. Delipidated and deglycolipidized M. vaccae (P-VA
Evaluate the use of C). The disease in these animals is characterized by anti-islet cell antibodies, severe islet inflammation, and evidence of autoimmune destruction of β-cells. 70-
90% of females and 20-30% of males spontaneously develop diabetes within the first 6 months of survival. The disease can also be induced in these mice by administering cyclophosphamide between 60-80% of treated mice that develop diabetes.

General Protocol Male mice with normoglycemic NOD (non-obese diabetic) between 8 and 10 weeks of age were used in this model system. Cyclophosphamide was administered by intraperitoneal injection in a single dose of 250 mg / kg body weight; Day 0 is the day of cyclophosphamide injection. Various doses of P-VAC suspended in 100 μl PBS were given intraperitoneally (
IP) or subcutaneous (SC) injection and both before and after induction. The progression of the disease can be seen on the 7th, 9th, 11th, 14th, 16th day,
And on day 21 were monitored by the test for diabetes (DIASTIX).

The general analytical strategy was used in each of the four experiments. First,
Each of the experimental groups was compared to the control group with respect to the number of non-diabetic (ie, non-diabetic) mice observed on the last day of the experiment (ie, 21 days after cyclophosphamide injection). For each comparison, this result in a 2 × 2 count table (non-diabetic vs. diabetic status (experimental vs. control group)); the ratio of non-diabetic mice in the two groups, for ratio equivalence. Pearson's Chi-square test was used for comparison.

Second, the survival analysis method was used to see the difference between the experimental and control groups in the time course of the onset of hyperglycemia. Specifically, the survivor function Kapla
An n-Meier estimate was calculated for each experimental group. Where "survivor"
Means (in this case) a mouse that is not yet diabetic. That is, the survivor curve is a plot of the estimated chances of mice remaining non-diabetic,
Evaluated on each of 7 experimental days after cyclophosphamide injection. The log rank test was then used to test for quality of survivor function between each of the experimental and control groups.

Experimental Design and Results In the first experiment, P-VAC alone was administered at different dose regimens (1.0 μg).
, 0.1 μg, 0.01 μg), followed by day 1, 4 and 7 after induction of the disease.
IP was administered on Days 14 and 14. The results are shown in Table 1. Statistical analysis is 1.0 μg, 0.1 μg, or 0.01 μg after cyclophosphamide induction
2 shows that IP injection of P-VAC has no effect on the frequency of diabetes onset in this model system.

[0061]   In the second experiment, the P-VAC dosing regimen changed as follows.

P-VAC with IP, both before and after induction of disease, −5 days, −2 days, +
Different dosing regimens (100 μg on day 1, +4, +7, and +14)
Or 10 μg) was used for administration. The results are shown in Table 1. Statistical analysis indicates that 10 μg or 100 before and after induction of disease
It is shown that IP treatment with μg P-VAC has no effect on the frequency of onset of diabetes in this model system.

In the third experiment, the route of administration was changed. P-VAC was administered at −5 days, −2 days, +1 day, +4 days, +7 days, and +1 both before and after induction of disease.
SC on day 4 using different dose regimens (100 μg or 10 μg)
It was administered by injection. The results are shown in Table 1. Statistical analysis shows that SC treatment with 10 μg or 100 μg P-VAC has no effect on the frequency of diabetes onset in this model system, both before and after induction of the disease.

In a fourth experiment, the previous experiment was modified with a larger group of animals and the day of first P-VAC treatment was changed to-4 days prior to induction of disease with cyclophosphamide. And repeated. The results are shown in Table 1. Statistical analysis is 10 μg or 10
It is shown that SC treatment with 0 μg of P-VAC, both before and after disease induction, has no effect on the frequency of onset of diabetes in this model system.

For each of Experiments 1, 2, 3, and 4, no significant difference was found on day 21 between the proportion of non-diabetic mice between any experimental group and the corresponding control group (Pearson). Chi-square p> 0.05, in each case). Similarly, no significant difference was found between survivor function in any experimental group and the corresponding survivor function in the control group (log rank p> 0.05, in each case). No significant differences were found in each of Experiments 2, 3 and 4, but the proportion of non-diabetic mice on day 21 was greater in the 100 μg PVAC group than in the corresponding control group.

Table 1 Analysis of PVAC effects in the IDDM model in NOD mice Table 1 summarizes the results of the statistical analysis. The first column displays the labels for the various groups, the second column shows the number of mice in that group (n), and the third column.
Column indicates the number of non-diabetic mice in that group on the day of the final experiment (day 21). The fourth column displays the p-value for the Pearson chi-square test comparing the proportion of non-diabetic mice on day 21 with the corresponding proportion of the control group. The fifth column of each table shows the median “survival time” in each group, that is, the median number of days until the appearance of hyperglycemia. (Note that in some cases, less than half of the animals in the group became diabetic during the duration of the experiment.
In such a case, the median time is shown as ">21". )Finally,
The sixth column of each table is the l for quality of survivor function between experimental and control groups.
The p-value for the og rank test is shown.

[0067]

[Table 1] Example 4 Pre- and post-disease induction of PVAC / IFA ameliorates disease in a NOD model for autoimmune IDDM This example demonstrates this in NOD mice, a murine model for autoimmune IDDM. , Illustrates the use of multiple pre- and post-PVAC / IFA infusions to reduce the incidence of disease. The disease in these animals is characterized by anti-islet cell antibodies, severe pancreatitis, and evidence for autoimmune destruction of β-cells.
Seventy to ninety percent of female animals and twenty to thirty percent of male animals spontaneously develop diabetes within the first six months of survival. The disease can also be induced in these mice by administration of cyclophosphamide.

Protocols and results for experiments designed to assess the ability of PVAC / IFA to reduce the incidence of diabetes in the NOD model for autoimmune IDDM 1. Normoglycemic male NOD mice (6-8 weeks old) were divided into 3 groups. 2. Group 1 received 100 μg PVAC emulsified in IFA on days -5, -2, 1, 4 and 7 of cyclophosphamide administration. 3. Group 2 received PBS emulsified in IFA on days -5, -2, 1, 4 and 7 of cyclophosphamide administration. 4. Group 3 received cyclophosphamide only on day 0. 5. Mice were placed on day 7 and every other day thereafter until day 21 when the experiment was terminated.
Diabetes was monitored for development of diabetes.

As shown in FIG. 2, there is a statistically significant decrease in the incidence of IDDM,
Obvious in mice receiving multiple PVAC / IFA injections (
p = 0.006).

Example 5 (PVAC / IFA Administered Post-Illness Induction for Autoimmune IDDM
This example shows that multiple PVAC / IFA post-injections were used in NOD mice (autoimmune I).
It is demonstrated to reduce the number of disease occurrence in the DDM mouse model). The disease in these animals is characterized by anti-islet cell antibodies, severe insulitis, and evidence of β-cell autoimmune destruction. Seventy to ninety percent of females and twenty to thirty percent of males develop sudden diabetes within the first six months of life. Disease can also be induced in these mice by administration of cyclophosphamide.

Protocols and results for comparison of PVAC emulsified in IFA before and after cyclophosphamide injection. Normoglycemic male NOD mice (6-8 weeks old) were divided into 4 groups. 2. Group 1 was (-5), (-2), 1, 4, 7 after cyclophosphamide injection.
And on day 14 received an injection of 100 μg PVAC / IFA. 3. Group 2 had 1 on days (-5) and (-2) after cyclophosphamide injection.
Received an injection of 00 μg PVAC / IFA. 4. Group 3 received 10 on days 1, 4, 7 and 14 post cyclophosphamide injection.
Received an injection of 0 μg PVAC / IFA Group 4 is cyclophosphamide (-5) followed by (-5
), (−2), 1, 4, 7, and 14 days received PBS / IFA injections. 6. Mice were monitored every day for the onset of diabetes with DIASIX from day 7 onwards until day 21 when the experiment was completed.

As shown in FIG. 3, injection of PVAC / IFA given prior to cyclophosphamide (p = 0.04) or after cyclophosphamide (p = 0.02) was prior to cyclophosphamide. And as effective as post-cyclophosphamide treatment (p = 0.04).

Example 6 Administration of PVAC / IFA on Day 4 After Disease Induction Improves Disease in NOD Model for Autoimmune IDDM This example demonstrates the use of a single dose of PVAC / IFA. Reduce the incidence of disease in NOD mice, a mouse model of autoimmune IDDM. Diseases in these animals include anti-islet cell antibodies, severe insulitis, and β
Characterized by evidence of autoimmune destruction of cells. Seventy to ninety percent of females and twenty to thirty percent of males develop sudden diabetes within the first six months of life. Disease can also be induced in these mice by administration of cyclophosphamide.

Protocols and Results for Determination of PVAC / IFA Dosing Dates that Confer Maximum Protection 1. Normal glycemic male NOD mice (6-8 weeks old) were divided into 5 groups. 2. Group 1 received 100 μg PVAC / 4 days after cyclophosphamide injection.
I received an IFA. 3. Group 2 received 100 μg PVAC / 7 days after cyclophosphamide injection.
I received an IFA. 4. Group 3 received 100 μg PVAC 10 days after cyclophosphamide injection
/ I received IFA. 5. Group 4 received 100 μg PVAC 13 days after cyclophosphamide injection
/ I received IFA. 6. Group 5 received cyclophosphamide only on day 0. 7. Mice were monitored by DIASIX for the onset of diabetes every other day after the 7th day until the 21st day when the experiment was terminated.

As shown in FIG. 4, administration of PVAC / IFA on day 4 was performed in this model (p
= 0.006) which gave the greatest protection from the development of diabetes.

Example 7 M-VAC Can Improve Disease in the EAE Model of Multiple Sclerosis This example demonstrates that killed M. We demonstrate that the use of Vaccae cells (M-VAC) ameliorates disease in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. EAE is an autoantigen-specific CD4 + MHC class I
It is an autoimmune inflammatory disorder of genetically susceptible mice mediated by I-restricted T cells. In susceptible SJL / J mice, this disease may exhibit paralysis of relapsing-reducing clinical processes, making this system ideal for studying the efficacy of various immunomodulatory strategies in both prevention and treatment of the disease. It is a system.

EAE can be induced using either spinal cord homogenates or myelin or crude preparations of myelin-derived immunodominant encephalitis-inducing peptides such as the PLP139-151 peptide. In this case, relapse-reducing (RR) EAE is induced in female SJL mice (8-12 weeks of age) by immunizing with 150 μg / mouse of the immunodominant peptide PLP139-151. This peptide, PB
Dissolved in S, and 600 μg of Mycobacterium tubercu
Emulsify with an equal volume of CFA containing loss H37Ra. 0.2 for each mouse
Receiving ml of emulsion, the emulsion was administered subcutaneously (sc) and distributed over three sites, the flank and the base of the tail. Mice were also injected with coadjuvant pertussis toxin (200 ng /
Mice were given ip), on the day of immunization and 48 hours later. Approximately 9 days after immunization, animals were weighed daily and observed for signs of neurological dysfunction. Diseases were graded as follows: 0, normal; 0.5 ankylosing tail; 1, caudal lameness;
1.5, caudal claudication with inability to erect; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of another limb; 3, complete paralysis of both hind legs; 4, moribund; 5 ,death. Mice are usually followed for relapse for up to 75-80 days post immunization.

Experimental Design and Results Based on the data obtained with M-VAC treatment in the NOD mouse model, we decided to evaluate the effect of M-VAC on the mouse EAE model.

In the first experiment, EAE was induced in SJL mice according to the protocol used previously, and M-VAC at a dose of 500 μg / mouse ip in PBS, −2, +1, +4, +7 and after disease induction. It was given on the 14th day. By +14 days, 12 out of 16 mice treated with M-VAC died, whereas in untreated controls only 1 out of 15 mice died. This result indicates that M-VAC is co-injected with pertussis toxin.
) (Also delivered IP) has had some toxic effects. Therefore, we repeated the same experiment in SJL mice. Here, EAE was induced without co-administration of pertussis toxin. In addition, two different doses of M-VAC (500 and 100 μg / mouse in PBS) were used for this experiment. The results (see FIG. 5) showed that the previously observed toxic effects were actually caused by co-injection of pertussis and not by M-VAC itself (no disease induction and Mice treated with M-VAC also showed no toxic effect (data not shown)). Moreover, mice receiving 500 μg / mouse of M-VAC showed a significant reduction in disease incidence.

Example 8 PVAC alone cannot ameliorate the disease in the EAE model of multiple sclerosis This example was delipidated and deglycolipidated (d
E.g. We demonstrate that the use of vaccae ameliorates disease in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. EAE is a genetically susceptible autoimmune inflammatory disorder in mice mediated by self-antigen specific CD4 + MHC class II restricted T cells. Sensitivity S
In JL / J mice, this disease may exhibit paralysis of relapsing-reducing clinical processes, making this system ideal for studying the efficacy of various immunomodulatory strategies in both prevention and treatment of the disease. It is a system.

General Protocol EAE can be induced using crude preparations of spinal cord homogenates or myelin or myelin-derived immunodominant encephalitis-inducing peptides (eg, PLP139-151 peptide). In this case, recurrence-reducing (RR) EAE was performed in female SJL mice (8-12 weeks of age) at 200 □ μg / mouse of the immunodominant peptide PLP.
Triggered by immunizing with 139-151. The peptide was dissolved in PBS and 600 μg of Mycobacterium tuberculosi.
emulsify with an equal volume of CFA containing s H37Ra. Each mouse received 0.2 ml of emulsion, which was administered subcutaneously (sc) and distributed over three sites, the flank and the base of the tail. Mice were also given coadjuvant pertussis toxin (200 ng / mouse intraperitoneally (ip)) on the day of immunization and 48 hours later. About 9 at the beginning after immunization,
Animals were weighed daily and observed for signs of neurological dysfunction. Diseases were graded as follows: 0, normal; 0.5 ankylosing tail; 1, caudal lameness; 1.5.
Caudal lameness with inability to erect; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of another limb; 3, complete paralysis of both hind legs; 4, moribund; 5, death. Mice are usually followed for relapse for up to 60-70 days post immunization.

The general analytical strategy was used for these experiments. Worst disease state (
The highest severity) was calculated for each mouse. Each of the experimental groups was then compared to the control group for the worst observed disease state in two ways. First, the groups were compared in terms of whether disease-related symptoms were observed (ie, (worst disease state = 0) vs. (worst disease state> 0)). Second, these groups were compared in terms of whether any paralysis (or worse symptoms) was observed (ie, (worst disease state <2) vs. (worst disease state 2 or more)). Each possible comparison yielded a 2x2 count table (control vs. experimental group, with two types of worst disease states.) Two worst disease states using Fisher's exact test. We tested the hypothesis that there is no difference between groups in the proportion of mice in this category.

Experimental Design and Results In the first experiment, EAE was induced in female SJL mice (8-12 weeks of age) by subcutaneous injection of PLP 139-151 / CFA + Pt (day 0),
Then, +1 day, +4 day, +7 day, and +14 day after the induction of the disease, P-VAC was intraperitoneally administered (0.01 μg / mouse;
1 μg / mouse, 1 μg / mouse).

Results were presented as the number of symptomatic and paralytic mice, but did not show a significant effect on disease development at any of the doses tested (Table 3). Comparing the worst disease states of each mouse throughout the experimental period, no statistically significant difference was found between the control group and any experimental group.

In a second experiment, the P-VAC regimen and route of administration were modified as follows: SJL mice had 5 days, 2 days, 1 day, 4 days, 7 days, and 14 days before disease induction. 10 or 100 (μg / mouse) intraperitoneally or subcutaneously after day
P-VAC was administered. The response of mice in this experiment was biphasic, therefore disease comparisons were performed between 1-31 and 32-62 days after disease induction. There was a statistically significant difference between the 10 μg P-VAC (intraperitoneal) and control groups during the first 31 days of the experiment; only a few mice in the P-VAC treated group showed signs of paralysis. None (p = 0.048) (Table 3). These results indicate that when animals were treated both before and after induction of disease, P-VAC showed EA
It shows that the occurrence of E can be prevented. Table 3 (Analysis of the effect of PVAC in the EAE model)

[0086]

[Table 3] Example 9 Pre-disease and Post-disease Administration of PVAC / IFA Can Alleviate Disease in the EAE Model of Multiple Sclerosis This example is a model of multiple sclerosis, an experiment Allergic encephalomyelitis (E
AE), delipidated deglycolipidized M. vaccae
Demonstrate the use of. EAE is an autoimmune inflammatory disorder in genetically susceptible mice that is mediated by self-antigen specific CD4 + MHC class II restricted T cells. In susceptible SJT / J mice, this disease may show a clinical course of recurrent and remission paralysis, thus studying the efficacy of various immunomodulatory strategies in both prevention and treatment of the disease. Is an ideal system for.

General Protocol EAE refers to crude preparations of spinal cord homogenates or myelin or immunodominant encephalitis-inducing peptides derived from myelin (eg, PLP 139).
-151 peptide). In this case, remission-recurrence (
Remoting-relapsing (RR) EAE is induced in female SJL mice (8-12 weeks of age) by immunization with 200 μg of the immunodominant peptide PLP 139-151 per mouse. This peptide is P
600 μg of Mycobacterium dissolved in BS and containing an equal volume of CFA
It was emulsified with ium tuberculosis H37Ra. Each mouse receives 0.2 ml of emulsion. The emulsion is administered subcutaneously (sc) and distributed over the flank and base of the tail over three sites. Mice are also dosed with pertussis toxin (200 ng / mouse, ip) as a co-adjuvant on the day of immunization and 48 hours later. Start the animals approximately 9 days after immunization,
Weigh and observe for the development of neurological dysfunction. Diseases are graded as follows: 0, normal; 0.5, stiff tail; 1,
The tail is flexible (1.5); the tail is too flexible to stand (l)
imp tail with inability to right); 2,
Paralysis of one limb; 2.5, paralysis of one limb and weakening of the other limb; 3, complete paralysis of both hind limbs; 4, moribundity; 5, death. Mice are usually followed for relapse up to 60-70 days post immunization.

For these experiments, the usual analytical strategy was used. The worst disease state (highest severity) was calculated for each mouse. Then, each experimental group,
The worst disease state observed was compared with the control group in two ways. Initially, this group was compared in terms of whether disease-related symptoms were observed (ie, worst disease state = 0 vs. worst disease state> 0). Second, this group is paralyzed (
Or whether worse symptoms are observed (ie, worst disease state <2 vs.
The comparison was made in terms of the worst disease state ≧ 2). Each possible comparison is 2 total
X2 occurred (control group versus experimental group, depending on the two types of worst disease states). Two
The Fisher's exact test was used to test the hypothesis that there was no difference between the groups in the proportion of mice in the two worst disease state categories.

In this experiment, the dose was changed to 50 μg per mouse of P-VAC emulsified in IFA. Mice were treated with P-VAC / IFA on the flanks of animals on day 5, 2, 2, 1, 4, 7 and 14 and injected subcutaneously (FIG. 6).
checking). An additional group of mice was treated with IFA alone on the same schedule. Control mice received no treatment (Table 3). In this experiment, only one significant difference was found. In the P-VAC / IFA treated group, significantly fewer mice exhibited disease-related symptoms than in the control group (p = 0.033) (Table 4). The results show that P-VAC emulsified in IFA has a protective effect when the animals are treated both before and after induction of the disease. These results are also shown in the bar graph of FIG.

[0090]

[Table 4] Example 10 Post-disease Administration of PVAC / IFA Can Alleviate Disease in an EAE Model of Multiple Sclerosis This example is an experimental allergic cerebral spinal cord, a model of multiple sclerosis. Flame (E
AE), delipidated deglycolipidized M. vaccae
Demonstrate the use of. EAE is an autoimmune inflammatory disorder in genetically susceptible mice that is mediated by self-antigen specific CD4 + MHC class II restricted T cells. In susceptible SJT / J mice, this disease may show a clinical course of recurrent and remission paralysis, thus studying the efficacy of various immunomodulatory strategies in both prevention and treatment of the disease. Is an ideal system for.

General Protocol EAE refers to crude preparations of spinal cord homogenates or myelin or immunodominant encephalitis-inducing peptides derived from myelin (eg PLP 139).
-151 peptide). In this case, remission-recurrence (
RR) EAE is 200 μg of immunodominant peptide P per mouse.
Female SJL mice (8-12 weeks old) by immunization with LP 139-151
Is guided by. This peptide was dissolved in PBS and 6 volumes containing an equal volume of CFA.
00 μg of Mycobacterium tuberculosis H37R
emulsified with a. Each mouse receives 0.2 ml of emulsion. The emulsion is administered subcutaneously (sc) and distributed over the flank and base of the tail over three sites. Mice are also given pertussis toxin (200 ng / mouse, ip) as an adjunct on the day of immunization and 48 hours later. About 9 of immunity
The animals are weighed daily and observed for the development of neurological dysfunction, starting after the day. Diseases are graded as follows: 0, normal; 0.5, tight tail;
1. Tail is flexible; 1.5, Tail is too flexible to stand; 2, Paralysis of one limb;
5, paralysis of one limb and weakening of the other limb; 3, complete paralysis of both hind limbs; 4, moribund;
5, death. Mice are usually followed for relapse up to 60-70 days post immunization.

Protocols and Results for Determining the Minimal Dose of PVAC / IFA Conferring Protection in the EAE Model The protocol is outlined in FIG. 1.60 SJL / J mice were divided into 3 groups of 20. 2. Disease was induced as described. 3. Group 1 contains 50 μg PV on day 1 and day 7 after disease induction (day 0).
AC / IFA administration was given. 4. Group 2 received IFA administration on days 1 and 7 after disease induction. 5. Group 3 received no treatment other than disease induction. 6. Mice were evaluated for disease as described above.

These results show that 50 μg PV emulsified in IFA for 2 days after disease induction.
Demonstrating the protective effect of PVAC / IFA by only two injections of AC (
(Figure 9).

Example 11 (P-VAC / IFA can Act as Adjuvant for Encephalitis-Inducing PLP Peptide) This example shows that delipidated deglycosylated lipid as an adjuvant for encephalitis-inducing PLP peptide. (Delipidated, deglycolipidated)
M. Demonstrate the use of vaccae cells (P-VAC).

To better understand the properties of P-VAC as an adjuvant, we found that P-VAC alone or in emulsion with IFA was 2
Upon delivery of 00 μg encephalitis-inducing PLP 139-151 peptide, and S
When inducing EAE in JL mice, the question was asked as to whether it could replace CFA. Peptide + IFA alone immunized mice and EAE
Is not enough to induce.

PLP 139-151 peptide was added to SJL mice (8-1) in either CFA (control), P-VAC alone, or P-VAC + IFA.
2 weeks old) delivered subcutaneously (sc). Disease appearance monitoring and statistical analysis were performed as described in Example 5 (pages 23, 11-21). In this experiment, significantly fewer mice in the group treated with 100 μg of P-VAC than those shown in the control group had some disease-related symptoms (p <0.
001) or signs of paralysis (p = 0.005) (Table 4). These results demonstrate that P-VAC alone does not replace the adjuvant properties of CFA in the induction of EAE.

In the second experiment, this protocol was slightly modified. These groups were expanded and the PLP 139-151 peptide was delivered sc either in CFA, IFA, P-VAC alone or in P-VAC with IFA. In addition, at day 35 post disease induction, animals received PLP 139-1 delivered in CFA.
I challenged again at 51.

For the statistical analysis of this experiment, worst status comparisons were made.
complete set) was performed twice; once for each of the two experimental phases (ie, pre-rechallenge and post-rechallenge on day 35) (Table 4). In Phase I (days 1-35), significantly fewer IFA-treated mice than diseased control groups showed disease-related symptoms (p = 0.001) or signs of paralysis (p = 0.035). showed that. Mice treated with 100 μg P-VAC also responded in a similar manner, with fewer mice in this group than in the control group showing disease-related symptoms (p = 0.001) or signs of paralysis. . 1
No significant difference was found between the 00 μg P-VAC / IFA group and the control group.

During Phase II of this experiment (after rechallenge on Day 35), only one significant difference was noted. More 100 μγP than the mice shown in the control group
-VAC treated mice showed signs of paralysis (p = 0.006) (Table 5).

These results confirm the initial results of Example 8 in that P-VAC alone does not replace the adjuvant activity of CFA in inducing EAE. From these experiments, it appears that P-VAC in combination with IFA could replace CFA in inducing disease.

Table 4 Analysis on Use of PVAC as Adjuvant in EAE Model The following table (Table 4) summarizes the results of the analysis in a common fashion. The first column shows the labeling for the various groups, the second column shows the number of mice in that group (n), and the third column shows the symptomatic mice in that group (ie during the comparison period). To 0
The number of mice with higher severity stages). Column 4 shows the p-value for Fisher's exact test comparing the ratio of symptomatic mice in the experimental group to the corresponding ratio for the control group. The fifth column of each table shows the number of mice that experienced paralysis or worse (ie, a severity level of 2 or greater) during the comparison period. Column 6 shows the p-value for Fisher's exact test comparing the ratio of paralyzed mice in the experimental group to the corresponding ratio for the control group.

[0102]

[Table 5] Example 12 (P-VAC / IFA can act as an Adjuvant, but M-VAC also M-
VAC / IFA may also not act as an adjuvant) This example demonstrates that killed M. vaccae cells (M-VAC) and delipidated deglycolipidated M. vacc
Demonstrate the use of ae (P-VAC).

To better understand the properties of P-VAC and M-VAC as adjuvants, we used P-VAC alone or M-VAC alone, or IFA.
Of P-VAC or M-VAC in emulsion with
During delivery of the 139-151 peptide, and in inducing EAE in SJL mice, the question was asked whether it could replace CFA. peptide
+ IFA alone is not sufficient to immunize mice and induce EAE.

The PLP 139-151 peptide was labeled with CFA, IFA, P-VAC alone, M-
SJL mice (8-12 weeks of age) were delivered subcutaneously (sc) in either VAC alone, P-VAC + IFA, or M-VAC + IFA. Animals were then monitored for disease appearance and progression. The result is shown in FIG. 35 days after immunization, PLP 139-15 in P-VAC alone.
None of the mice immunized with 1 showed signs of disease, but IFA / P-V
Animals immunized with PLP 139-151 in AC developed disease (60
%). This indicates that IFA + P-VAC induces CF in EAE induction.
It suggests that the A effect can be replaced. Interestingly, M-VAC alone or I
None of the mice immunized with FA / M-VAC developed the disease. This again shows the difference between the M-VAC and P-VAC effects.

Example 13 P-VAC / IFA Can Improve Disease in the CIA Model for Human Rheumatoid Arthritis This example is intended to improve disease in collagen-induced arthritis (CIA). , Killed M. vaccae cells (M-VAC) and delipidated deglycolipidated M. v
Demonstrate the use of accae (P-VAC). CIA is a model for human RA for the following reasons: 1) similarities observed for synovial inflammation and cartilage / bone destruction; 2) involvement of class II-restricted T cell activation in pathogenesis.

CIA was performed in male DBA / 1 mice by intradermal (id) injection (booster immunization on days 0 and 21) of type II collagen (C11) in CFA at the base of the tail of chickens. (8-12 weeks old) (10 mice / group).
The average onset is on days 27-28. Animals are evaluated for paw redness and swelling, and a clinical score assigned to each mouse more than 3 times per week. This scoring system is based on joint swelling and / or erythema progression up to the stage of joint deformity and / or stiffness (see FIG. 11 for RA scoring guide).

Experimental Design and Results CIA is one of the most widely used models of polyarthritis to assess the role of effects and to determine efficacy of therapeutic agents. We use this protocol to determine P-VAC and M-VA in this autoimmune disease.
The effect of C was evaluated. Mice were subjected to P-VAC (at 2 days before, 1 day, 4 days, 7 days, and 14 days after the second collagen injection, according to the protocol described above, at the time of the second type II collagen injection, for example. Intraperitoneally (ip) in PBS, 100 μg / mouse or M-VAC (ip in PBS)
To give 500 μg / mouse). The experimental protocol is seen in FIG. Results from the first experiment (FIG. 13) show severe arthritis (defined as MAT) in P-VAC treated groups compared to M-VAC treated mice or untreated controls. Shows a decrease in the number of mice that have.

The above examples are not provided to limit the scope of the invention, but to illustrate the invention. Other modifications of the invention will be readily apparent to those skilled in the art and are covered by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

[Brief description of drawings]

FIG. 1 demonstrates a statistically significant reduction in the onset of diabetes after multiple doses of M-VAC.

FIG. 2 demonstrates a statistically significant reduction in the onset of diabetes after multiple doses of PVAC / IFA.

FIG. 3 shows pre-cyclo treatment of PVAC / IFA with cyclophosphamide.
) Demonstrate a statistically significant reduction in the onset of diabetes after either post-dose or post-cyclo dose.

FIG. 4 demonstrates a statistically significant reduction in the onset of diabetes with a single dose of PVAC / IFA at day 4 post cyclophosphamide treatment.

FIG. 5 shows protocols and results of experiments demonstrating the ability of MVAC to reduce the development of EAE.

FIG. 6 shows multiple injections of PVAC / IFA before and after disease induction in E.
2 shows an experimental protocol for testing the ability to block the symptoms of AE.

FIG. 7 shows that multiple pre- and post-disease injections of PVAC / IFA
The result of the experiment which demonstrates that the onset of EAE is reduced is shown.

FIG. 8 shows an experimental protocol to test the ability of two post-disease injections of PVAC / IFA to block the symptoms of EAE.

FIG. 9 shows the results of experiments demonstrating that two injections of PVAC / IFA given after disease induction reduce the symptoms of EAE.

FIG. 10 shows the results of experiments demonstrating the ability of PVAC / IFA to act as an adjuvant in inducing EAE in SJL mice.

FIG. 11   FIG. 11 shows the scoring guide used to evaluate the CIA model.

FIG. 12 shows an experimental protocol to test the ability of PVAC to reduce the development of arthritis in a collagen-induced arthritis (CIA) model.

FIG. 13 shows results suggesting that PVAC / IFA reduces the severity of arthritis in the CIA model.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 37/06 A61P 37/06 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, C Z, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F term (reference) 4C085 AA03 BA09 CC07 DD51 DD86 EE01 EE06 FF02 FF11 FF14 GG04 GG06

Claims (16)

[Claims] 1. A method of treating an autoimmune disease, which method comprises P-VA.
A method comprising administering an immunologically effective dose of a pharmaceutical composition comprising C. 2. The method of claim 1, wherein the P-VAC is administered with an adjuvant. 3. The method of claim 1, wherein the P-VAC is administered parenterally. 4. The method of claim 3, wherein the P-VAC is administered subcutaneously. 5. The method of claim 3, wherein the P-VAC is administered intraperitoneally. 6. The method of claim 1, wherein the autoimmune disease is multiple sclerosis. 7. The method of claim 1, wherein the autoimmune disease is IDDM. 8. The method of claim 1, wherein the autoimmune disease is rheumatoid arthritis. 9. A method of treating an autoimmune disease, the method comprising: va
A method comprising administering an immunologically effective dose of a pharmaceutical composition comprising ccae cells. 10. The M. 10. The method of claim 9, wherein the vaccae cells are administered with an adjuvant. 11. The M. 10. The method of claim 9, wherein the vaccae cells are administered parenterally. 12. The M. 2. The vaccae cells are administered subcutaneously.
The method according to 1. 13. The M. The method of claim 11, wherein the vaccae cells are administered intraperitoneally. 14. The method of claim 9, wherein the autoimmune disease is multiple sclerosis. 15. The method of claim 9, wherein the autoimmune disease is IDDM. 16. The autoimmune disease is rheumatoid arthritis.
The method described in.