Note: Descriptions are shown in the official language in which they were submitted.
CA 02378285 2002-02-05
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VACCINES FOR THE TREATMENT OF AUTOIMMUNE DISEASE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to USSN 60/147,626, filed August 6,
1999, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
The present invention relates to compositions and methods for treating
autoimmune diseases. The compositions comprise as an active ingredient
Mycobacteria
vaccae.
A number of pathological responses involving unwanted immune
responses are known. For instance, autoimmune disease is a particularly
important class
of deleterious immune response. In autoimmune diseases, self tolerance is lost
and the
immune system attacks "self' tissue as if it were a foreign target. More than
30
autoimmune diseases are presently known, including rheumatoid arthritis (RA),
insulin-
dependent diabetes mellitus (IDDM), multiple sclerosis (MS), myasthenia gravis
(MG),
systemic lupus erythematosis (SLE), and scleroderma.
Although many advances have been made in the treatment of autoimmune
disease, new therapeutic approaches to treatment and prevention of these
diseases are
needed. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides pharmaceutical compositions comprising
killed M. vaccae cells or P-VAC for treatment of autoimmune diseases. P-VAC is
a
delipidated and deglycolipidated fraction derived from M. vaccae cells. The
compositions of the invention may further comprise an adjuvant. The
compositions may
be administered according to standard methods well known in the art.
Typically, the
compositions are administered parenterally, for example subcutaneously or
intraperitonealy. The methods of the invention can be used to treat autoimmune
diseases
such as diabetes, multiple sclerosis, and rheumatoid arthritis
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates a statistically significant reduction in the incidence
of diabetes following multiple doses of M-VAC.
Figure 2 demonstrates a statistically significant reduction in the incidence
of diabetes following multiple doses of PVAC/IFA.
Figure 3 demonstrates a statistically significant reduction in the incidence
of diabetes following either pre-cyclo OR post-cyclo doses of PVAC/IFA.
Figure 4 demonstrates a statistically significant reduction in the incidence
of diabetes following a single dose of PVAC/IFA on day 4 post-cyclophosphamide
treatment.
Figure 5 shows the protocol and results for experiments demonstrating the
ability of MVAC to lower the incidence of EAE.
Figure 6 shows the protocol for experiments that test the ability of
multiple pre- and post-disease induction injections of PVAC/IFA to block
symptoms of
EAE.
Figure 7 shows the results for experiments demonstrating that multiple
pre- and post-disease induction injections of PVAC/IFA lower the incidence of
EAE.
Figure 8 shows the protocol for experiments that test the ability of two
post-disease induction injections of PVAC/IFA to block symptoms of EAE.
Figure 9 shows the results of experiments demonstrating that two
injections of PVAC/IFA given post-disease induction lowers the symptoms of
EAE.
Figure 10 shows the results of experiments demonstrating the ability of
PVAC/IFA to act as adjuvants in the induction of EAE in the SJL mouse.
Figure 11 shows the scoring guide used in the evaluation of CIA model.
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Figure 12 shows the protocol for experiments testing the ability of PVAC
to lower the incidence of arthritis in the collagen induced arthritis (CIA)
model.
Figure 13 shows the results suggesting that PVAC/IFA lowers the severity
of arthritis in CIA model.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides pharmaceutical compositions comprising
antigenic material derived from Mycobacterium vaccae. The compositions are
generally
useful in the treatment of pathological conditions such as autoimmune
diseases.
The therapeutic compositions of the invention may comprise dead cells of
M. vaccae, referred to here as M-VAC. The means by which the cells have been
killed is
not critical and may be done by e.g. autoclaving or irradiation. Preparation
of
pharmaceutical compositions comprising such cells are described for example in
EP 630
259, and U.S. Patent No. 5,833,996. A strain of M. vaccae denoted R877R has
been
deposited under the Budapest Convention at the National Collection of Type
Cultures
(NCTC) under the number NCTC 11659. (see, Stanford and Paul, Ann. Soc. Belge
Med,
Trop. 53:141-389 (1973)).
Alternatively, the compositions of the invention may comprise delipidated
and deglycolipidated fraction derived from M. vaccae cells (DD- M.V.) referred
to here
as P-VAC. Preparation of such cells is described for instance in WO 99/32634
and WO
98/8542 (see Example 1 for details).
The M. vaccae cells or modified cells can be formulated in pharmaceutical
compositions useful for administration to mammals, particularly humans, to
treat and/or
prevent deleterious autoimmune responses. Suitable formulations are found in
Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia,
PA,
17th ed. (1985).
PHARMACEUTICAL COMPOSITIONS
The present invention concerns formulation of the M. vaccae compositions
disclosed herein in pharmaceutically-acceptable solutions for administration
to an animal,
either alone, or in combination with one or more other modalities of therapy.
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WO 01/10221 PCT/US00/21644
Formulation of pharmaceutically-acceptable excipients and carrier
solutions is well-known to those of skill in the art, as is the development of
suitable
dosing and treatment regimens for using the particular compositions described
herein in a
variety of treatment regimens, including e.g., oral, parenteral, intravenous,
intranasal, and
intramuscular administration and formulation.
I. 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 with an assimilable edible carrier, or
they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be compressed
into tablets, or
they may be incorporated directly with the food of the diet.
The active compounds may even be incorporated with excipients and used
in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions,
syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.
S. Patent
5,641,515; U. S. Patent 5,580,579 and U. S. Patent 5,792,451, each
specifically
incorporated herein by reference in its entirety). The tablets, troches,
pills, capsules and
the like may also contain the following: a binder, as gum tragacanth, acacia,
cornstarch,
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 as sucrose, lactose or saccharin may be added or
a flavoring
agent, such as peppermint, oiI of wintergreen, or cherry flavoring. 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 of elixir may contain the active
compound 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 should be pharmaceutically pure and substantially non-toxic in the
amounts
employed. In addition, the active compounds may be incorporated into sustained-
release
preparation and formulations.
Typically, these formulations may contain at least about 0.1 % of the active
compound or more, although the percentage of the active ingredients) may, of
course, be
varied and may conveniently be between about 1 or 2% and about 60% or 70% or
more
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of the weight or volume of the total formulation. Naturally, the amount of
active
compounds) in each therapeutically useful composition may be prepared is such
a way
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 will be
contemplated by one
skilled in the art of preparing such pharmaceutical formulations, and as such,
a variety of
dosages and treatment regimens may be desirable.
For oral administration the compositions of the present invention may
alternatively be incorporated with one or more excipients in the form of a
mouthwash,
dentifrice, buccal tablet, oral spray, or 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 a 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
dispersed in
a dentifrice, or added in a therapeutically-effective amount to a composition
that may
include water, binders, abrasives, flavoring agents, foaming agents, and
humectants.
Alternatively the compositions may be fashioned into a tablet or solution form
that may
be placed under the tongue or otherwise dissolved in the mouth.
2O 2. INJECTABLE DELIVERY
In certain circumstances it will be desirable to deliver the pharmaceutical
compositions disclosed herein parenterally, intravenously, intramuscularly, or
even
intraperitoneally as described in U. S. Patent 5,543,158; U. S. Patent
5,641,515 and U. S.
Patent 5,399,363 (each specifically incorporated herein by reference in its
entirety).
Solutions of the active compounds as free base or pharmacologically acceptable
salts may
be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene glycols, 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 (U. S. Patent 5,466,468,
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
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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 (e.g.,
glycerol,
propylene glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for example, by the
use of a
coating, such as lecithin, by the maintenance of the required particle size in
the case of
dispersion and by the use of surfactants. The prevention of the action of
microorganisms
can be facilitated by various antibacterial and antifungal agents, for
example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, 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.
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. These particular aqueous solutions
are
especially suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal
administration. In this connection, a sterile aqueous medium that can be
employed will
be known to those of skill in the art in light of the present disclosure. For
example, one
dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to
1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g.,
Remington's
Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 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. Moreover, for human administration,
preparations should
meet sterility, pyrogenicity, and the general safety and purity standards as
required by
FDA Office of Biologics standards.
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
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vacuum-drying and freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously sterile-
filtered solution
thereof.
The compositions disclosed herein may be formulated in a neutral or salt
form. Pharmaceutically-acceptable salts, include the acid addition salts
(formed with the
free amino groups of the protein) and which are formed with inorganic acids
such as, for
example, hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic,
tartaric, mandelic, and the like. Salts formed with the free carboxyl groups
can also be
derived from inorganic bases such as, for example, sodium, potassium,
ammonium,
calcium, or ferric hydroxides, and such organic bases 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 easily administered in a variety of dosage
forms such as
injectable solutions, drug-release capsules, and the like.
As used herein, "carrier" includes any and all solvents, dispersion media,
vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic
and absorption
delaying agents, buffers, carrier solutions, suspensions, colloids, 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, its use in the 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 untoward reaction when
administered to a human. The preparation of an aqueous composition that
contains a
protein as an active ingredient is well understood in the art. Typically, such
compositions
are prepared as injectables, either as liquid solutions or suspensions; solid
forms suitable
for solution in, or suspension in, liquid prior to injection can also be
prepared. The
preparation can also be emulsified.
3. NASAL DELIVERY
In certain embodiments, the pharmaceutical compositions may be
delivered by intranasal sprays, inhalation, and/or other aerosol delivery
vehicles.
Methods for delivering genes, nucleic acids, and peptide compositions directly
to the
lungs via nasal aerosol sprays has been described e.g., in U. S. Patent
5,756,353 and U. S.
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Patent 5,804,212 (each specifically incorporated herein by reference in its
entirety).
Likewise, the delivery of drugs using intranasal microparticle resins
(Takenaga et al.,
1998) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871,
specifically
incorporated herein by reference in its entirety) are also well-known in the
pharmaceutical
arts. Likewise, transmucosal drug delivery in the form of a
polytetrafluoroetheylene
support matrix is described in U. S. Patent 5,780,045 (specifically
incorporated herein by
reference in its entirety).
4. LIPOSOME-, NANOCAPSULE-, AND MICROPARTICLE-MEDIATED DELIVERY
In certain embodiments, the inventors contemplate the use of liposomes,
nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the
like, for the
introduction of the compositions of the present invention into suitable host
cells. In
particular, the compositions of the present invention may be formulated for
delivery
either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere,
or a~
nanoparticle 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 is generally known to those of skill in the art
(see for
example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes
the use of
liposomes and nanocapsules in the targeted antibiotic therapy for
intracellular bacterial
infections and diseases). Recently, liposomes were developed with improved
serum
stability and circulation half times (Gabizon & Papahadjopoulos, 1988; Allen
and Choun,
1987; U. S. Patent 5,741,516, specifically incorporated herein by reference in
its entirety).
Further, various methods of liposome and liposome like preparations as
potential drug
carriers have been reviewed (Takakura, 1998; Chandran et al., 1997; Margalit,
1995; U.
S. Patent 5,567,434; U. S. Patent 5,552,157; U. S. Patent 5,565,213; U. S.
Patent
5,738,868 and U. S. Patent 5,795,587, each specifically incorporated herein by
reference
in its entirety).
Liposomes have been used effectively to introduce genes, drugs (Heath &
Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta & Puglisi,
1996),
radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a;
Imaizumi et
al., 1990b), viruses (Falter & Baltimore, 1984), transcription factors and
allosteric
effectors (Nicolau & Gersonde, 1979) into a variety of cultured cell lines and
animals. In
addition, several successful clinical trials examining the effectiveness of
liposome-
WO 01/10221 CA 02378285 2002-02-05 PCT/US00/21644
mediated drug delivery have been completed (Lopez-Berestein et al., 1985a;
1985b;
Coupe, 1988; Sculier et al., 1988). Furthermore, several studies suggest that
the use of
liposomes is not associated with autoimmune responses, toxicity or gonadal
localization
after systemic delivery (Mori & Fukatsu, 1992).
Liposomes are formed from pliospholipids that are dispersed in an aqueous
medium and spontaneously form multilamellar concentric bilayer vesicles (also
termed
multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to
4 Vim.
Sonication of MLVs results in the formation of small unilamellar vesicles
(SLJVs) with
diameters in the range of 200 to 500 ~, containing an aqueous solution in the
core.
Liposomes bear resemblance to cellular membranes and are contemplated
for use in connection with the present invention as carriers for the
compositions. They
are widely suitable as both water- and lipid-soluble substances can be
entrapped, i.e. in
the aqueous spaces and within the bilayer itself, respectively. It is possible
that the drug-
bearing liposomes may even be employed for site-specific delivery of active
agents by
selectively modifying the liposomal formulation.
In addition to the teachings of Couvreur et al. (1977, 1988), the following
information may be utilized in generating liposomal formulations.
Phospholipids can
form a variety of structures other than liposomes when dispersed in water,
depending on
the molar ratio of lipid to water. At low ratios the liposome is the preferred
structure.
The physical characteristics of liposomes depend on pH, ionic strength and the
presence
of divalent canons. Liposomes can show low permeability to ionic and polar
substances,
but at elevated temperatures undergo a phase transition which markedly alters
their
permeability. The phase transition involves a change from a closely packed,
ordered
structure, known as the gel state, to a loosely packed, less-ordered
structure, known as the
fluid state. This occurs at a characteristic phase-transition temperature and
results in an
increase in 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, deform and
penetrate
the bilayer, thereby causing changes in permeability. Cholesterol inhibits
this penetration
of proteins, apparently by packing the phospholipids more tightly. It is
contemplated that
the most useful liposome formations for antibiotic and inhibitor delivery will
contain
cholesterol.
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WO 01/10221 PCT/US00/21644
The ability to trap solutes varies between different types of liposomes. For
example, MLVs are moderately efficient at trapping solutes, but SUVs are
extremely
inefficient. SUVs offer the advantage of homogeneity and reproducibility in
size
distribution. However, a compromise between size and trapping efficiency is
offered by
large unilamellar vesicles (LUVs). These are prepared by ether evaporation and
are three
to four times more efficient at solute entrapment than MLVs.
In addition to liposome characteristics, an important determinant in
entrapping compounds is the physicochemical properties of the compound itself.
Polar
compounds are trapped in the aqueous spaces and nonpolar compounds bind to the
lipid
bilayer of the vesicle. Polar compounds are released through permeation or
when the
bilayer is broken, but nonpolar compounds remain affiliated with the bilayer
unless it is
disrupted by temperature or exposure to lipoproteins. Both types show maximum
efflux
rates at the phase transition temperature.
Liposomes interact with cells via four different mechanisms: endocytosis
by phagocytic cells of the reticuloendothelial system such as macrophages and
neutrophils; adsorption to the cell surface, either by nonspecific weak
hydrophobic or
electrostatic forces, or by specific interactions with cell-surface
components; fusion with
the plasma cell membrane by insertion of the lipid bilayer of the liposome
into the plasma
membrane, with simultaneous release of liposomal contents into the cytoplasm;
and by
transfer of liposomal lipids to cellular or subcellular membranes, or vice
versa, without
any association of the liposome contents. It often is difficult to determine
which
mechanism is operative and more than one may operate at the same time.
The fate and disposition of intravenously injected liposomes depend on
their physical properties, such as size, fluidity, and surface charge. They
may persist in
tissues for hours or days, depending on their composition, and half lives in
the blood
range from minutes to several hours. Larger liposomes, such as MLVs and LUVs,
are
taken up rapidly by phagocytic cells of the reticuloendothelial system, but
physiology of
the circulatory system restrains the exit of such large species at most sites.
They can exit
only in places where large openings or pores exist in the capillary
endothelium, such as
the sinusoids of the liver or spleen. Thus, these organs are the predominant
site of uptake.
On the other hand, SUVs show a broader tissue distribution but still are
sequestered
highly in the liver and spleen. In general, this in vivo behavior limits the
potential
targeting of liposomes to only those organs and tissues accessible to their
large size.
These include the blood, liver, spleen, bone marrow, and lymphoid organs.
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WO 01/10221 PCT/US00/21644
Targeting is generally not a limitation in terms of the present invention.
However, should specific targeting be desired, methods are available for this
to be
accomplished. Antibodies may be used to bind to the liposome surface and to
direct the
antibody and its drug contents to specific antigenic receptors located on a
particular cell-
type surface. Carbohydrate determinants (glycoprotein or glycolipid cell-
surface
components that play a role in cell-cell recognition, interaction and
adhesion) may also be
used as recognition sites as they have potential in directing liposomes to
particular cell
types. Mostly, it is contemplated that intravenous injection of liposomal
preparations
would be used, but other routes of administration are also conceivable.
Alternatively, the invention provides for pharmaceutically-acceptable
nanocapsule formulations of the compositions of the present invention.
Nanocapsules can
generally entrap compounds in a stable and reproducible way (Henry-Michelland
et al.,
1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid side
effects due to
intracellular polymeric overloading, such ultrafine particles (sized around
0.1 Vim) should
be designed using polymers able to be degraded in vivo. Biodegradable
polyalkyl-
cyanoacrylate nanoparticles that meet these requirements are contemplated for
use in the
present invention. Such particles may be are easily made, as described
(Couvreur et al.,
1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et
al., 1995
and U. S. Patent 5,145,684, specifically incorporated herein by reference in
its entirety).
VACCINES
In certain preferred embodiments of the present invention, vaccines are
provided. The vaccines will generally comprise one or more pharmaceutical
compositions, such as those discussed above, in combination with an
immunostimulant.
An immunostimulant may be any substance that enhances or potentiates an immune
response (antibody and/or cell-mediated) to an exogenous antigen. Examples of
immunostimulants include adjuvants, biodegradable microspheres (e.g.,
polylactic
galactide) and liposomes (into which the compound is incorporated; see, e.g.,
Fullerton,
U.S. Patent No. 4,235,877). Vaccine preparation is generally described in, for
example,
Powell & Newman, eds., Vaccine Design (the subunit and adjuvant approach)
(1995).
Pharmaceutical compositions and vaccines within the scope of the present
invention may
also contain other compounds, which may be biologically active or inactive.
For
example, one or more immunogenic portions of other tumor antigens may be
present,
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either incorporated into a fusion polypeptide or as a separate compound,
within the
composition or vaccine.
While any suitable carrier known to those of ordinary skill in the art may
be employed in the vaccine compositions of this invention, the type of carrier
will vary
depending on the mode of administration. Compositions of the present invention
may be
formulated for any appropriate manner of administration, including for
example, topical,
oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or
intramuscular
administration. For parenteral administration, such as subcutaneous injection,
the carrier
preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For
oral
administration, any of the above carriers or a solid carrier, such as
mannitol, lactose,
starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose,
sucrose, and
magnesium carbonate, may be employed. Biodegradable microspheres (e.g.,
polylactate
polyglycolate) may also be employed as carriers for the pharmaceutical
compositions of
this invention. Suitable biodegradable microspheres are disclosed, for
example, in U.S.
Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;
5,814,344
and 5,942,252. One may also employ a carrier comprising the particulate-
protein
complexes described in U.S. Patent No. 5,928,647, which are capable of
inducing a class
I-restricted cytotoxic T lymphocyte response in a host.
Such compositions may also comprise buffers (e.g., neutral buffered saline
or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose
or
dextrans), mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants,
bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g.,
aluminum
hydroxide), solutes that render the formulation isotonic, hypotonic or weakly
hypertonic
with the blood of a recipient, suspending agents, thickening agents and/or
preservatives.
Alternatively, compositions of the present invention may be formulated as a
lyophilizate.
Compounds may also be encapsulated within liposomes using well known
technology.
Any of a variety of immunostimulants may be employed in the vaccines of
this invention. For example, an adjuvant may be included. Most adjuvants
contain a
substance designed to protect the antigen from rapid catabolism, such as
aluminum
hydroxide or mineral oil, and a stimulator of immune responses, such as lipid
A,
Bortadella pertussis or Mycobacterium species or Mycobacterium derived
proteins.
Suitable adjuvants are commercially available as, for example, Freund's
Incomplete
Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck
Adjuvant 65
(Merck and Company, Inc., Rahway, NJ); AS-2 and derivatives thereof
(SmithKline
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Beecham, Philadelphia, PA); CWS, TDM, Leif, aluminum salts such as aluminum
hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an
insoluble
suspension of acylated tyrosine; acylated sugars; cationically or anionically
derivatized
polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl
lipid
A and quit A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also
be used
as adjuvants.
Within the vaccines provided herein, the adjuvant composition is
preferably designed to induce an immune response predominantly of the Thl
type. High
levels of Thl-type cytokines (e.g., IFN-y, TNFa, IL-2 and IL-12) tend to favor
the
induction of cell mediated immune responses to an administered antigen. In
contrast,
high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to
favor the
induction of humoral immune responses. Following application of a vaccine as
provided
herein, a patient will support an immune response that includes Thl- and Th2-
type
responses. Within a preferred embodiment, in which a response is predominantly
Thl-
type, the level of Thl-type cytokines will increase to a greater extent than
the level of
Th2-type cytokines. The levels of these cytokines may be readily assessed
using standard
assays. For a review of the families of cytokines, see Mosmann & Coffman, Ann.
Rev.
Immunol. 7:145-173 (1989).
Preferred adjuvants for use in eliciting a predominantly Thl-type response
include, for example, a combination of monophosphoryl lipid A, preferably 3-de-
O-
acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL
adjuvants are available from Corixa Corporation (Seattle, WA; see US Patent
Nos.
4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing
oligonucleotides (in
which the CpG dinucleotide is unmethylated) also induce a predominantly Thl
response.
Such oligonucleotides are well known and are described, for example, in WO
96/02555,
WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory
DNA
sequences are also described, for example, by Sato et al., Science 273:352
(1996).
Another preferred adjuvant is a saponin or saponin mimetics or
derivatives, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA),
which
may be used alone or in combination with other adjuvants. For example, an
enhanced
system involves the combination of a monophosphoryl lipid A and saponin
derivative,
such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a
less
reactogenic composition where the QS21 is quenched with cholesterol, as
described in
13
CA 02378285 2002-02-05
wo omo22i PcT~soom6aa
WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion
and
tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL
and
tocopherol in an oil-in-water emulsion is described in WO 95/17210.
Other preferred adjuvants include Montanide ISA 720 (Seppic, France),
SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the
SBAS
series of adjuvants (e.g., SBAS-2, AS2', AS2," SBAS-4, or SBAS6, available
from
SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, MT), RC-529
(Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs),
such
as those described in pending U.S. Patent Application Serial Nos. 08/853,826
and
09/074,720, the disclosures of which are incorporated herein by reference in
their
entireties.
Any vaccine provided herein may be prepared using well known methods
that result in a combination of antigen, immune response enhancer and a
suitable carrier
or excipient. The compositions described herein may be administered as part of
a
sustained release formulation (i.e., a formulation such as a capsule, sponge
or gel
(composed of polysaccharides, for example) that effects a slow release of
compound
following administration). Such formulations may generally be prepared using
well
known technology (see, e.g., Coombes et al., Vaccine 14:1429-1438 (1996)) and
administered by, for example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release formulations may
contain 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 within such formulations are biocompatible, and may also
be biodegradable; preferably the formulation provides a relatively constant
level of active
component release. Such carriers include microparticles of poly(lactide-co-
glycolide),
polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed-
release carriers
include supramolecular biovectors, which comprise a non-liquid hydrophilic
core (e.g., a
cross-linked polysaccharide or oligosaccharide) and, optionally, an external
layer
comprising an amphiphilic compound, such as a phospholipid (see, e.g., U.S.
Patent No.
5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The
amount of active compound contained within a sustained release formulation
depends
upon 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
14
WO 01/10221 CA 02378285 2002-02-05 pCT/US00/21644
or mufti-dose containers, such as sealed ampoules or vials. Such containers
are
preferably hermetically sealed to preserve sterility of the formulation until
use. In
general, formulations may be stored as suspensions, solutions or emulsions in
oily or
aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may
be stored
in a freeze-dried condition requiring only the addition of a sterile liquid
carrier
immediately prior to use.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or patent
application
were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be readily
apparent to one of ordinary skill in the art in light of the teachings of this
invention that
certain changes and modifications may be made thereto without departing from
the spirit
or scope of the appended claims.
EXAMPLE 1
PREPARATION OF DELIPIDATED AND DEGLYCOLIPIDATED M. VACCAE (P-
VAC)
P-VAC was prepared as generally described in WO 99/32634. A summary
of the protocol follows.
Heat-killed M. vaccae was prepared using standard methods. To prepare
delipidated M. vaccae, the autoclaved M. vaccae was pelleted by
centrifugation, the pellet
washed with water, collected again by centrifugation and then freeze-dried. An
aliquot of
this freeze-dried M. vaccae was set aside and referred to as lyophilised M.
vaccae. When
used in experiments it was resuspended in PBS to the desired concentration.
Freeze-dried
M. vaccae was treated with chloroform/methanol (2:1 ) for 60 mins at room
temperature to
extract lipids, and the extraction was repeated once. The delipidated residue
from
chloroform/methanol extraction was further treated with 50% ethanol to remove
glycolipids by refluxing for two hours. The 50% ethanol extraction was
repeated two
times. The residue from the 50% ethanol extraction was freeze-dried and
weighed. The
delipidated and deglycolipidated M. vaccae (DD-M. vaccae) was resuspended in
phosphate-buffered saline by sonication, thus producing P-VAC. This
preparation in
WO 01/10221 CA 02378285 2002-02-05 pCT~S00/21644
preferred embodiments is used for treatment of autoimmune diseases without
further
processing. In some embodiments, the preparation is autoclaved before
administration.
EXAMPLE 2
M-VAC CAN AMELIORATE DISEASE IN A NOD MODEL FOR AUTOIMMUNE
IDDM
This example demonstrates the use of killed M. vaccae cells (M-VAC) to
ameliorate disease in NOD mice, a marine model for autoimmune IDDM. The
disease in
these animals is characterized by anti-islet cell antibodies, severe
insulitis, and evidence
for autoimmune destruction of the ~i-cells. Seventy to ninety percent of
female and 20-
30% of male animals spontaneously develop diabetes within the first six months
of life.
The disease can also be induced in these mice by administration of
cyclophosphamide.
Protocol and Results for M-VAC experiment
1. Normorglycaemic NOD male mice (age 6-8 weeks) were divided into 3
experimental
groups
2. Group 1 received SOO~g of MVAC IP
3. Group 2 received SOO~g of MVAC IP on day (-2), l, 4, 7 and 14.
Cyclophosphamide
was given on Day 0.
4. Group 3 received cyclophosphamide injections only on Day 0
5. Mice were monitored for development of diabetes by DIASTIX on day 7 and
every
Monday, Wednesday and Friday thereafter until day 21 when the experiment was
terminated.
As can be seen in Figure l, multiple injections of MVAC given by IP injection
provides statistically significant protection from the development of diabetes
in this
model (p=0.0001). The administration of MVAC alone (in the absence of
cyclophosphamide) had no effect on the mice.
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EXAMPLE 3
P-VAC ALONE CANNOT AMELIORATE DISEASE IN A NOD MODEL FOR
AUTOIMMUNE IDDM
This example evaluates the use of delipidated, deglycolipidated M. vaccae
(P-VAC) to ameliorate disease in NOD mice, a marine model for autoimmune IDDM.
The disease in these animals is characterized by anti-islet cell antibodies,
severe insulitis,
and evidence for autoimmune destruction of the ~-cells. Seventy to ninety
percent of
female and 20-30% of male animals spontaneously develop diabetes within the
first six
months of life. The disease can also be induced in these mice by
administration of
cyclophosphamide with between 60-80% of treated mice developing diabetes.
General Protocol
Normoglycaemic NOD (non-obese diabetic) male mice between the ages
of 8 and 10 weeks were used in this model system. Cyclophosphamide was
administered
at a single dose of 250 mg/kg body weight by intraperitoneal injection; Day 0
is the date
of cyclophosphamide injection. P-VAC suspended in 100 ~.l PBS was administered
at
varying doses, via intraperitoneal (IP) or subcutaneous (SC) injection, and
both pre- and
post-induction. Disease progression was monitored at Days 7, 9, 11, 14, 16,
and 21 by
tests for glycosuria (DIASTIX).
A common analytic strategy was used 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 (i.e., non-glycosuric) mice observed on the final
experimental day
(i.e., Day 21, following cyclophosphamide injection). For each comparison,
this results
in a 2x2 table of counts (non-glycosuric vs. glycosuric status, by
experimental vs. control
group); the proportions of non-glycosuric mice in the two groups were compared
using
Pearson's chi-square test for equality of proportions.
Second, survival analysis methods were used to look for differences
between experimental groups and the control group in the time course of the
development
of hyperglycemia. Specifically, the Kaplan-Meier estimate of the survivor
function was
computed for each experimental group, where "survivor" means (in this case) a
mouse
not yet glycosuric. That is, the survivor curve is plot of the estimated
chance of a mouse
remaining non-glycosuric, evaluated at each of the seven experimental days
following
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cyclophosphamide injection. The log-rank test was then used to test for
equality of
survivor functions between each of the experimental groups and the control
group.
Experimental Design and Results
S In the first experiment, P-VAC alone was administered IP at Day 1, 4, 7,
and 14 after disease induction using different dose regimens (1.0 ~,g, 0.1 pg,
or 0.01 fig).
The results are seen in Table 1. Statistical analyses indicate that IP
injection of 1.0 fig,
0.1 fig, or 0.01 ~,g of P-VAC after cyclophosphamide induction has no effect
on the
incidence of diabetes occurrence in this model system.
In a second experiment, the P-VAC dosing regimen was changed as
follows:
P-VAC was administered IP both pre- and post-disease induction at Day (-
5), (-2), +1, +4, +7 and +14 using different dose regimens (100 ~.g or 10
~,g). The results
are seen in Table 1. Statistical analyses indicate that IP treatment with 10
~g or 100 ~g P-
1 S VAC both pre- and post-disease induction has no effect on the incidence of
diabetes
occurrence in this model system.
In a third experiment, the route of administration was altered. P-VAC was
administered by SC injection both pre- and post-disease induction at Day (-5),
(-2), +1,
+4, +7 and +14 using different dose regimens (100 ~g or 10 fig). The results
are seen in
Table 1. Statistical analyses indicate that SC treatment with 10 ~g or 100 ~g
P-VAC both
pre- and post-disease induction has no effect on the incidence of diabetes
occurrence in
this model system.
In a fourth experiment, the previous experiment was repeated with larger
groups of animals and the first P-VAC treatment date changed to Day (-4)
before disease
induction with cyclophosphamide. The results are seen in Table 1. Statistical
analyses
indicate that SC treatment with 10 ~g or 100 ~g P-VAC both pre- and post-
disease
induction has no effect on the incidence of diabetes occurrence in this model
system
For each of Experiments 1, 2, 3, and 4, no significant difference was found
between any experimental group and the corresponding control group with
respect to the
proportion of non-glycosuric mice on Day 21 (Pearson chi-squarep > 0.05, in
each case).
Similarly, no significant difference was found between the survivor functions
of any
experimental group and the corresponding control group (log-rankp > 0.05, in
each case).
While no significant differences were found in each of Experiments 2, 3, and
4, the
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proportion of non-glycosuric mice on Day 21 was greater in the 100 p.g PVAC
group than
in the corresponding control group.
Table 1 Analysis of PVAC effects in IDDM model in NOD mice
Table 1 summarizes the results of the statistical analyses. The first column
displays labels for the various groups, the second column shows the number of
mice (n)
in the group, and the third column shows the number of non-glycosuric mice in
that group
on the final experimental day (Day 21 ). The fourth column displays the p-
value for the
Pearson chi-square test comparing the proportion of non-glycosuric mice on Day
21 vs.
the corresponding proportion for the control group. Column five of each table
shows the
median "survival time" in each group, i.e., the median number of days until
the
appearance of hyperglycemia. (Note that, in some cases, fewer than half of the
animals in
a group became glycosuric during the experimental period. In those cases, the
median
time is shown as ">21".) Finally, column six in each table shows thep-value
for the log-
rank test for equality of survivor functions between an experimental group and
the control
group.
25
Table 1 Analysis of PVAC effects in IDDM model in NOD mice
Experiment 1
ExperimentalSample Non-glycosuric Chi SquareMedian Days Log Rank
Group Size Mice on Day p-value To Glycosuriap-value
(n) 21
1.0 p,g 16 5 0.988 14 0.786
PVAC
0.1 ~,g 21 4 0.34 11 0.431
PVAC
0.01 ~g 17 4 0.585 16 0.565
PVAC
Control 29 9 14
~
Experiment 2
ExperimentalSample Non-glycosuric Chi SquareMedian Days Log Rank
Group Size Mice on Day p-value To Glycosuriap-value
(n) 21
100 ~,g 20 7 0.216 14 0.417
PVAC
10 pg PVAC 15 2 0.877 11 0.933
Control 13 ~ 2 11
~
Experiment 3
Experimental Sample Non-glycosuric Chi Square Median Days Log Rank
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WO 01/10221 PCT/US00/21644
Group Size Mice on Day p-value To Glycosuriap-value
(n) 21
100 p,g PVAC20 11 0.20 >21 0.325
~,g PVAC 20 13 0.058 >21 0.139
Control 20 7 16
Experiment 4
ExperimentalSample Non-glycosuricChi SquareMedian Days Log Rank
Group Size Mice on Day p-value To Glycosuriap-value
(n) 21
100 ~g PVAC 24 14 0.474 >21 0.552
10 pg PVAC 25 12 0.967 >21 0.974
Control 19 9 >21
5
EXAMPLE 4
PVAC/IFA ADMINISTERED PRE- AND POST-DISEASE INDUCTION
AMELIORATES DISEASE IN AN NOD MODEL FOR AUTOIMMUNE IDDM
This example demonstrates the use of multiple pre-and post- PVAC/IFA
injections to lower the incidence of disease in NOD mice, a marine model for
autoimmune IDDM. The disease in these animals is characterized by anti-islet
cell
antibodies, severe insulitis, and evidence for autoimmune destruction of the
(3-cells.
Seventy to ninety percent of female and 20-30% of male animals spontaneously
develop
diabetes within the first six months of life. The disease can also be induced
in these mice
by administration of cyclophosphamide.
Protocol and Results for experiments desiuned to evaluate the ability of
PVAC/IFA to
lower incidence of diabetes in an NOD model for autoimmune IDDM.
1. Normoglycaemic male NOD mice (age 6-8 weeks) were divided into 3 groups
2. Group 1 was given 100~g PVAC emulsified in IFA on days (-5), (-2), l, 4 and
7
days post cyclophosphamide administration
3. Group 2 was given PBS emulsified in IFA on days (-S), (-2), 1, 4 and 7 days
post
cyclophosphamide adminstration
4. Group 3 was given cyclophosphamide only on day 0.
5. Mice were monitored for the development of diabetes by DIASTIX on day 7 and
every other day thereafter until day 21 when the experiment was terminated.
CA 02378285 2002-02-05
WO 01/10221 PCT/US00/21644
As seen in Figure 2, a statistically significant decrease in the incidence of
IDDM was evident in the mice receiving multiple injections of PVAC/IFA
(p=0.006).
EXAMPLE 5
PVAC/IFA ADMINISTERED POST-DISEASE INDUCTION AMELIORATES
DISEASE IN AN NOD MODEL FOR AUTOIMMUNE IDDM
This example demonstrates the use of multiple post- PVAC/IFA injections to
lower the
incidence of disease in NOD mice, a murine model for autoimmune IDDM. The
disease
in these animals is characterized by anti-islet cell antibodies, severe
insulitis, and
evidence for autoimmune destruction of the (3-cells. Seventy to ninety percent
of female
and 20-30% of male animals spontaneously develop diabetes within the first six
months
of life. The disease can also be induced in these mice by administration of
cyclophosphamide.
Protocol and Results for comparison ofpre- and post- cyclophosphamide
injections of
PVAC emulsified in IFA.
1. Normoglycaemic male NOD mice (age 6-8 weeks) were divided into 4 groups
2. Group 1 received injections of 100~.g PVAC/IFA on days (-5), (-2), 1, 4, 7
and 14
post cyclophosphamide injection.
3. Group 2 received injections of 100~g PVAC/IFA on days (-5) and (-2) post
cyclophosphamide injection.
4. Group 3 received injections of 100~g PVAC/IFA on days 1, 4, 7 and 14 post
cyclophosphamide injection
5. Group 4 received injections of PBS/IFA on days (-5), (-2), 1, 4, 7 and 14
post
cyclophamide.
6. Mice were monitored for the development of diabetes by DIASTIX on day 7 and
every other day thereafter until day 21 when the experiment was terminated.
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As seen in Figure 3, injections of PVAC/IFA given pre-cyclophosphamide
(p=0.04) OR post-cyclophosphamide (p=0.02) are equally as efficacious as the
pre-
AND post-cyclophosphamide treatment (p=0.04).
S
EXAMPLE 6
ADMINISTRATION OF PVAC/IFA ON DAY 4 POST DISEASE INDUCTION
AMELIORATES DISEASE IN AN NOD MODEL FOR AUTOIMMUNE IDDM
This example demonstrates the use of a single administration of
PVAC/IFA to lower the incidence of disease in NOD mice, a marine model for
autoimmune IDDM. The disease in these animals is characterized by anti-islet
cell
antibodies, severe insulitis, and evidence for autoimmune destruction of the
(3-cells.
Seventy to ninety percent of female and 20-30% of male animals spontaneously
develop
diabetes within the first six months of life. The disease can also be induced
in these mice
by administration of cyclophosphamide
Protocol and Results for determination of the adminstration date for PVAC/IFA
which provides maximum protection.
1. Normoglycaemic male NOD mice (age 6-8 weeks) were divided into 5 groups.
2. Group 1 received 100pg PVAC/IFA on day 4 post cyclophosphamide injection
3. Group 2 received 100p,g PVAC/IFA on day 7 post cyclophosphamide injection
4. Group 3 received 100pg PVAC/IFA on day 10 post cyclophosphamide injection
5. Group 4 received 100pg PVAC/IFA on day 13 post cyclophosphamide injection.
6. Group 5 received cyclophosphamide alone on day 0.
7. Mice were monitored for the development of diabetes by DIASTIX on day 7 and
every other day thereafter until day 21 when the experiment was terminated.
As seen in Figure 4, administration of PVAC/IFA on day 4 provides
maximum protection from the development of diabetes in this model (p=0.006).
22
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EXAMPLE 7.
M-VAC CAN AMELIORATE DISEASE 1N AN EAE MODEL FOR MULTIPLE
SCLEROSIS
This example demonstrates the use of killed M. vaccae cells (M-VAC) to
ameliorate disease in experimental allergic encephalomyelitis (EAE) a model
for multiple
sclerosis. EAE is an autoimmune inflammatory disorder of genetically
susceptible mice
that is mediated by autoantigen-specific CD4+ MHC class II restricted T cells.
In
susceptible SJL/J mice, the disease can display a relapsing-remitting clinical
course of
paralysis, which makes it an ideal system to study the efficacy of various
immunoregulatory strategies both in the prevention and treatment of disease.
EAE can be induced by using either crude preparations of spinal cord
homogenate or myelin or immunodominant encephalitogenic peptides derived from
myelin, such as PLP 139-151 peptide. In this case, remitting-relapsing (RR)
EAE is
induced in female SJL mice (8-12 wks old) by immunization with 150 pg/mouse of
the
immunodominant peptide PLP 139-151. The peptide is dissolved in PBS and
emulsified
with an equal volume of CFA containing 600 pg of Mycobacterium tuberculosis
H37Ra.
Each mouse receives 0.2 ml of the emulsion, which will be administrated
subcutaneously
(sc~ and distributed over three sites on the flank and base of tail. Mice are
also given an
injection of the coadjuvant pertussis toxin (200 ng/mouse intraperitoneally
(ip)) on the
day of immunization and 48 hrs later. Beginning approximately 9 days after
immunization, animals are weighed daily and observed for the onset of
neurological
dysfunction. Disease is graded as follows: 0, normal; 0.5, stiff tail; 1, limp
tail; 1.5, limp
tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one
limb and
weakness of one other limb; 3, complete paralysis of both hind limbs; 4,
moribund; 5,
death. Mice are usually followed for up to 75-80 days after immunization for
relapses.
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 marine EAE model.
In the first experiment, EAE was induced in SJL mice according to the
protocol used previously and M-VAC was given at the dose of 500 p,g/mouse ip
in PBS at
day -2, +1, +4, +7 and +14 after disease induction. By day +14, 12 out of 16
mice
treated with M-VAC were dead whereas only 1 mouse out of 15 dead in the
untreated
23
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WO 01/10221 PCT/US00/21644
control. This result raised the question of whether M-VAC had any toxic effect
in
conjunction with the co-injection of pertussis toxin (delivered IP as well).
Therefore we
repeated the same experiment in SJL mice where EAE was induced without the co-
administration of pertussis toxin. In addition, 2 different doses of M-VAC,
500 and 100
~g/mouse in PBS were used in this experiment. The results (see, Figure 5)
indicated that
the toxic effect observed previously was indeed caused by the coinjection of
pertussis and
not by M-VAC itself (mice with no disease induction and treated with M-VAC do
not
show toxic effect as well, data not shown). Furthermore, mice receiving 500
~g/mouse of
M-VAC showed a significant reduction of disease incidence.
EXAMPLE 8
PVAC ALONE CANNOT AMELIORATE DISEASE IN AN EAE MODEL FOR
MULTIPLE SCLEROSIS
This example demonstrates the use of delipidated, deglycolipidated M.
vaccae to ameliorate disease in experimental allergic encephalomyelitis (EAE),
a model
for multiple sclerosis. EAE is an autoimmune inflammatory disorder of
genetically
susceptible mice that is mediated by autoantigen-specific CD4+ MHC class II
restricted T
cells. In susceptible SJL/J mice, the disease can display a relapsing-
remitting clinical
course of paralysis, which makes it an ideal system to study the efficacy of
various
immunoregulatory strategies both in the prevention and treatment of disease.
General Protocol
EAE can be induced by using either crude preparations of spinal cord
homogenate or myelin or immunodominant encephalitogenic peptides derived from
myelin, such as PLP 139-151 peptide. In this case, remitting-relapsing (RR)
EAE is
induced in female SJL mice (8-12 wks old) by immunization with 200 ~ g/mouse
of the
immunodominant peptide PLP 139-151. The peptide is dissolved in PBS and
emulsified
with an equal volume of CFA containing 600 ~g of Mycobacterium tuberculosis
H37Ra.
Each mouse receives 0.2 ml of the emulsion, which will be administrated
subcutaneously
(sc) and distributed over three sites on the flank and base of tail. Mice are
also given an
injection of the coadjuvant pertussis toxin (200 ng/mouse intraperitoneally
(ip)) on the
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day of immunization and 48 hrs later. Beginning approximately 9 days after
immunization, animals are weighed daily and observed for the onset of
neurological
dysfunction. Disease is graded as follows: 0, normal; 0.5, stiff tail; 1, limp
tail; 1.5, limp
tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one
limb and
weakness of one other limb; 3, complete paralysis of both hind limbs; 4,
moribund; 5,
death. Mice are usually followed for 60-70 days after immunization for
relapses.
A common analytic strategy was used for these experiments. The worst
disease status (highest severity grade) was calculated for each mouse. Each of
the
experimental groups was then compared to the control groups with respect to
the worst
disease status observed, in two ways. First, the groups were compared in terms
of
whether any disease-related symptoms were observed, i.e. worst disease status
= 0 vs.
worst disease status > 0. Second, the groups were compared in terms of whether
any
paralysis (or a worse symptom) was observed, i.e., worst disease status < 2
vs. worst
disease status >_ 2. Each possible comparison resulted in a 2x2 table of
counts (control vs.
experimental group, by two types of worst disease status). Fisher's exact test
was used to
test the hypothesis of no difference between groups in the proportions of mice
in the two
worst disease status categories.
Experimental Design and Results
In the first experiment, EAE was induced in female SJL mice (8-12 wks
old) by sc injection of PLP 139-151/CFA + Pt (day 0) and P-VAC was
administrated IP at
day +1, +4, +7 and +14 after disease induction using different dose regimens
(0.01
~,g/mouse; 0.1 ~g/mouse, 1 ~g/mouse).
The results, expressed as the number of symptomatic and paralytic mice,
showed no significant effect of any of the doses tested in the development of
disease
(Table 3). No statistically significant differences were found between the
control group
and any of the experimental groups comparing the worst disease status of each
mouse
during the entire experimental period.
In a second experiment, the P-VAC regimen and route of administration
were changed as follows:
SJL mice received either 10 or 100 ~g/mouse of P-VAC delivered ip or sc
at day -5, -2, +l, +4, +7 and +14 after disease induction. The responses of
the mice in
this experiment were bi-phasic; consequently, the disease comparisons were
performed
CA 02378285 2002-02-05
WO 01/10221 PCT/US00/21644
for the time periods Days 1-31 and Days 32-62 post-disease induction. There
was a
statistically significant difference between the 10 p.g P-VAC (ip) treatment
groups and the
control group during the first 31 days of the experiment; fewer mice in the P-
VAC
treatment groups showed signs of paralysis (p=0.048) (Table 3). These results
indicate
that P-VAC can protect against the development of EAE when animals are treated
both
before and after disease induction.
Table 3 Analysis of PVAC Effects in EAE Model
Experiment 1
ExperimentalSample Symptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact Mice Days Exactp-
(n) 1- p-value 1- value
65 65
1.0 ~g PVAC12 12 * 9 0.70
0.1 pg PVAC12 12 * 9 0.70
0.01 p,g 11 11 * 10 0.20
PVAC
PBS 12 12 * 11 0.18
Control 15 15 ~ * ~ 10
*No mouse in this group was symptom-free
Experiment 2 (Phase 1 )
Experimental SampleSymptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact p- Mice Days Exact p-
(n) 1- value 1- value
31 31
100 p,g PVAC,15 15 * 15
ip
10 pg PVAC, 16 16 * 11 0.048
ip
100 p,g PVAC,16 16 * 14 0.488
sc
10 pg PVAC, 15 15 * 14 1.000
sc
100 p.1 PBS, 16 16 * 15 1.000
ip
Control 13 13 * 13
*No mouse in this group was symptom-free
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Experiment 2 (Phase 2)
ExperimentalSample Symptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact p- Mice Days Exact p-
(n) 32- value 32-62 value
62
100 ~.g PVAC,1 S 15 * 8 0.276
ip
~,g PVAC,16 16 * 8 0.451
ip
100 ~,g PVAC,16 16 * 6 1.000
sc
10 p,g PVAC,15 15 * 7 0.460
sc
100 ~,l PBS,16 16 * 9 0.264
ip
Control ~ 13 ~ 13 * 4
*No mouse in this group was symptom-free
5
EXAMPLE 9
10 ADMINISTRATION OF PVAC/IFA PRE- AND POST-DISEASE INDUCTION CAN
AMELIORATE DISEASE IN AN EAE MODEL OF MULTIPLE SCLEROSIS
This example demonstrates the use of delipidated, deglycolipidated M.
vaccae to ameliorate disease in experimental allergic encephalomyelitis (EAE),
a model
for multiple sclerosis. EAE is an autoimmune inflammatory disorder of
genetically
susceptible mice that is mediated by autoantigen-specific CD4+ MHC class II
restricted T
cells. In susceptible SJL/J mice, the disease can display a relapsing-
remitting clinical
course of paralysis, which makes it an ideal system to study the efficacy of
various
immunoregulatory strategies both in the prevention and treatment of disease.
General Protocol
EAE can be induced by using either crude preparations of spinal cord
homogenate or myelin or immunodominant encephalitogenic peptides derived from
myelin, such as PLP 139-151 peptide. In this case, remitting-relapsing (RR)
EAE is
induced in female SJL mice (8-12 wks old) by immunization with 200 ~,g/mouse
of the
immunodominant peptide PLP 139-151. The peptide is dissolved in PBS and
emulsified
with an equal volume of CFA containing 600 ~g of Mycobacterium tuberculosis
H37Ra.
Each mouse receives 0.2 ml of the emulsion, which will be administrated
subcutaneously
(sc) and distributed over three sites on the flank and base of tail. Mice are
also given an
injection of the coadjuvant pertussis toxin (200 ng/mouse intraperitoneally
(ip)) on the
27
WO 01/10221 CA 02378285 2002-02-05 PCT~$00/21644
day of immunization and 48 hrs later. Beginning approximately 9 days after
immunization, animals are weighed daily and observed for the onset of
neurological
dysfunction. Disease is graded as follows: 0, normal; 0.5, stiff tail; 1, limp
tail; 1.5, limp
tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one
limb and
weakness of one other limb; 3, complete paralysis of both hind limbs; 4,
moribund; 5,
death. Mice are usually followed for 60-70 days after immunization for
relapses.
A common analytic strategy was used for these experiments. The worst
disease status (highest severity grade) was calculated for each mouse. Each of
the
experimental groups was then compared to the control groups with respect to
the worst
disease status observed, in two ways. First, the groups were compared in terms
of
whether any disease-related symptoms were observed, i.e. worst disease status
= 0 vs.
worst disease status > 0. Second, the groups were compared in terms of whether
any
paralysis (or a worse symptom) was observed, i.e., worst disease status < 2
vs. worst
disease status >_ 2. Each possible comparison resulted in a 2x2 table of
counts (control vs.
experimental group, by two types of worst disease status). Fisher's exact test
was used to
test the hypothesis of no difference between groups in the proportions of mice
in the two
worst disease status categories.
In this experiment, the dose was altered to 50 pg/mouse P-VAC emulsified
in IFA. Mice were treated with P-VAC/IFA and injected sc in the flank of the
animal at
Days -5, -2, l, 4, 7, and 14 (see Figure 6). An additional group of mice were
treated on
the same schedule with IFA alone. Control mice did not receive any treatment
(Table 3).
Only one significant difference was found in this experiment. Significantly
fewer mice in
the P-VAC/IFA treatment group displayed disease-related symptoms than did mice
in the
control group (p = 0.033) (Table 4). The results indicate that P-VAC
emulsified in IFA
has a protective effect when animals are treated both before and after~disease
induction.
These results are also shown in bar graph form in Figure 7.
Table 4.
ExperimentalSample Symptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact p- Mice Days Exact p-
(n) 1-SO value 1- value
50
PVAC/IFA 16 6 0.033 4 0.149
IFA 16 13 0.669 9 1.000
Control 16 12 9
I
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EXAMPLE 10
POST DISEASE INDUCTION ADMINISTRATION OF PVAC/IFA CAN
AMELIORATE DISEASE IN AN EAE MODEL OF MULTIPLE SCLEROSIS
This example demonstrates the use of delipidated, deglycolipidated M.
vaccae to ameliorate disease in experimental allergic encephalomyelitis (EAE),
a model
for multiple sclerosis. EAE is an autoimmune inflammatory disorder of
genetically
susceptible mice that is mediated by autoantigen-specific CD4+ MHC class II
restricted T
cells. In susceptible SJL/J mice, the disease can display a relapsing-
remitting clinical
course of paralysis, which makes it an ideal system to study the efficacy of
various
immunoregulatory strategies both in the prevention and treatment of disease.
General Protocol
EAE can be induced by using either crude preparations of spinal cord
homogenate or myelin or immunodominant encephalitogenic peptides derived from
myelin, such as PLP 139-151 peptide. In this case, remitting-relapsing (RR)
EAE is
induced in female SJL mice (8-12 wks old) by immunization with 200 ~g/mouse of
the
immunodominant peptide PLP 139-151. The peptide is dissolved in PBS and
emulsified
with an equal volume of CFA containing 600 ~g of Mycobacterium tuberculosis
H37Ra.
Each mouse receives 0.2 ml of the emulsion, which will be administrated
subcutaneously
(sc) and distributed over three sites on the flank and base of tail. Mice are
also given an
injection of the coadjuvant pertussis toxin (200 ng/mouse intraperitoneally
(ip)) on the
day of immunization and 48 hrs later. Beginning approximately 9 days after
immunization, animals are weighed daily and observed for the onset of
neurological
dysfunction. Disease is graded as follows: 0, normal; 0.5, stiff tail; l, limp
tail; 1.5, limp
tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one
limb and
weakness of one other limb; 3, complete paralysis of both hind limbs; 4,
moribund; 5,
death. Mice are usually followed for 60-70 days after immunization for
relapses.
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w0 01/10221 CA 02378285 2002-02-05 PCT/US00/21644
Protocol and Results for determining a minimum dose of PVAC/IFA that provides
protection in the EAE model.
Protocol is outlined in Figure 8
1. Sixty SJL/J mice were divided into three groups of 20.
2. Disease was induced as described..
3. Group 1 received SOp,g PVAC/IFA administration on day 1 and 7 after disease
induction on day 0.
4. Group 2 received IFA administration on day 1 and day 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 demonstrate a protective effect of PVAC/IFA with only 2
injections of
SODg PVAC emulsified in IFA on two days after disease induction (Figure 9)
EXAMPLE 11
P-VAC/IFA CAN ACT AS AN ADJUVANT FOR ENCEPHALITOGENIC PLP
PEPTIDE
This example demonstrates the use of delipidated, deglycolipidated M.
vaccae cells (P-VAC) as an adjuvant for encephalitogenic PLP peptide.
To better understand the properties of P-VAC as an adjuvant, we have
asked the question whether P-VAC alone or in emulsion with IFA could
substitute for
CFA in delivering the 200 ~g encephalitogenic PLP 139-151 peptide and inducing
EAE
in SJL mice. Peptide + IFA alone is not sufficient to immunize mice and induce
EAE.
PLP 139-151 peptide was delivered subcutaneously (sc) to SJL mice (8-12
wks old) in either CFA (control), P-VAC alone, or P-VAC + IFA. Monitoring of
disease
appearance and statistical analyses were performed as described in Example S
(page 23,
lines 11-21). In this experiment, significantly fewer mice in the group
treated with 100
~g P-VAC showed any disease-related symptoms (p<0.001) or signs of paralysis
(p =
0.005) than did mice in the control group (Table 4). These results demonstrate
that P-
VAC alone does not substitute for the adjuvant properties of CFA in induction
of EAE.
W~ ~l/1~221 CA 02378285 2002-02-05 pCT/US00/21644
In a second experiment, the protocol was slightly modified. The groups
were expanded and PLP 139-151 peptide was delivered sc in either CFA, IFA, P-
VAC
alone, or P-VAC with IFA. In addition, 35 days after disease induction,
animals were
rechallenged with PLP 139-151 delivered in CFA.
S For statistical analysis of this experiment, the entire set of worst status
comparisons was performed twice; once for each of the two experimental phases
(i.e. pre-
and post-rechallenge at Day 35) (Table 4). In Phase I (Days 1-35),
significantly fewer
mice treated with IFA displayed disease-related symptoms (p=0.001 ) or signs
of paralysis
(p=0.035) than did mice on the control group. Mice treated with 100 ~,g P-VAC
also
responded in a similar manner; fewer mice in this group displayed disease-
related
symptoms (p=0.001 ) or signs of paralysis than mice in the control group. No
significant
differences were found between the 100 p,g P-VAC/IFA and control groups.
During Phase II of this experiment (after rechallenge at day 35) only one
significant difference was noted. More mice treated with 100~y P-VAC showed
signs of
paralysis than did mice in the control group (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 induction of EAE. It appears
from these
experiments that P-VAC in combination with IFA can substitute for CFA in
inducing
disease.
Table 4 Analysis of use PVAC as adiuvant in EAE Model
The following table (Table 4) summarizes the results of the analyses in a
common format. The first column displays labels for the various groups, the
second
column shows the number of mice (n) in the group, and the third column shows
the
number of symptomatic mice in that group, i.e., mice with severity grade > 0
during the
comparison period. The fourth column displays the p-value for the Fisher's
exact test
comparing the proportion of symptomatic mice in the experimental group vs. the
corresponding proportion for the control group. Column 5 of each table shows
the
number of mice that experienced paralysis or worse (i.e., severity grade >2)
during the
comparison period. The sixth column displays the p-value for the Fisher's
exact test
comparing the proportion of paralyzed mice in the experimental group vs. the
corresponding proportion for the control group.
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WO 01/10221 PCT/fJS00/21644
Table 5 Analysis of PVAC Effects in EAE Model
Experiment 1
Experimental Sample Symptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact p- Mice Days Exact p-
(n) 1-62 1-
value 62 value
100 pg 12 12 * 9 0.667
PVAC/IFA
100 p,g PVAC 12 0 0.000 0 0.005
Control ~ 12 12 * 7
* No mouse in this group was symptom-free
Experiment 2 (Phase 1 )
Experimental Sample Symptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact p- Mice Days Exact p-
(n) 1-35 value 1- value
35
100 p,g 11 6 0.659 4 1.000
PVAC/IFA
100 ~,g PVAC 12 0 0.001 0 0.029
IFA 11 0 0.001 0 0.035
Control (CFA) 10 I 7 -I - _ _
~
Experiment 2 (Phase 2)
Experimental Sample Symptomatic Fisher's Paralytic Fisher's
Group Size Mice Days Exact p- Mice Days Exact p-
(n) 36- value 36-70 value
70
100 ~g 11 10 0.586 1 0.311
PVAC/IFA
100 ~g PVAC 12 12 0.195 11 0.006
IFA 11 7 0.635 S 0.659
Control (CFA) 10 8 3
EXAMPLE 12
P-VAC/IFA, BUT NEITHER M-VAC NOR M-VAC/IFA, CAN ACT AS AN
ADJUVANT
This example demonstrates the use of killed M. vaccae cells (M-VAC) and
delipidated, deglycolipidated M. vaccae (P-VAC) as adjuvants for
encephalitogenic PLP
peptide.
To better understand the properties of P-VAC and M-VAC as adjuvants,
we have asked the question whether P-VAC or M-VAC alone or in emulsion with
IFA
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WO 01/10221 CA 02378285 2002-02-05 PCT/US00/21644
could substitute for CFA in delivering the encephalitogenic PLP 139-151
peptide and
inducing EAE in SJL mice. Peptide + IFA alone is not sufficient to immunize
mice and
induce EAE.
PLP 139-151 peptide was delivered subcutaneously (sc) to SJL mice (8-12
wks old) in either CFA, IFA, P-VAC alone, M-VAC alone, P-VAC + IFA or M-VAC +
IFA and animals were monitored for disease appearance and progression. The
results are
shown in Figure 10. 3 S days after immunization, none of the mice immunized
with PLP
139-151 in P-VAC alone showed sign of disease, whereas animal immunized with
PLP
139-151 in IFA/P-VAC developed disease (60%), suggesting that IFA+P-VAC can
replace the CFA effect in inducing EAE. Interestingly, none of the mice
immunized with
either M-VAC alone or IFA/M-VAC developed disease, indicating again
differences in
M-VAC and P-VAC effects.
EXAMPLE 13
P-VAC/IFA CAN AMELIORATE DISEASE IN A CIA MODEL FOR HUMAN
RHEUMATOID ARTHRITIS
This example demonstrates the use of killed M. vaccae cells (M-VAC) and
delipidated, deglycolipidated M. vaccae (P-VAC) to ameliorate disease in
collagen-
induced arthritis (CIA) a model for human RA because of 1 ) observed
similarities with
respect to synovial inflammation and cartilage/bone destruction; 2)
involvement of class
II restricted T cell activation in the pathogenesis.
CIA is induced in male DBA/1 mice (8-12 weeks) (10 mice/group) by
intradermal (id) injections in the base of the tail (at day= 0 and boost at
day 21) of chick
collagen type II (C11) in CFA. The mean onset is 27-28 days. Animals are
assessed for
redness and swelling of limbs and clinical score allocated for each mouse >3
times per
week. The scoring system is based on the progression of the swelling and /or
erythema of
the joints up to the stage of joint distortion and/or rigidity (see Figure 11
for RA scoring
guide).
Experimental Design and Results
CIA is one of the most widely used polyarthritis model for assessing the
role of effects and for determining the efficacy of therapeutic drugs. We have
used this
protocol to evaluate the effect of P-VAC and M-VAC on this autoimmune disease.
Mice
received P-VAC (100 Og/mouse intraperitoneally (ip) in PBS) or M-VAC (500
33
CA 02378285 2002-02-05
wo omoz2l PcT~soom6aa
Og/mouse ip in PBS) at the time of the 2"d collagen type II injection
according to the
protocol described before (e.g. day -2, +1, +4, +7 and +14 after 2"d collagen
injection)
Experimental protocol is seen in Figure 12.. The results from a first
experiment (Figure
13) indicate a reduction in the number of mice with severe arthritis (defined
as MAT) in
the group treated with P-VAC compared with the mice treated with M-VAC or
untreated
control.
The above examples are provided to illustrate the invention but not to limit
its
scope. Other variants of the invention will be readily apparent to one of
ordinary skill in
the art and are encompassed by the appended claims. All publications, patents,
and patent
applications cited herein are hereby incorporated by reference.
34
