U.S. patent application number 09/849969 was filed with the patent office on 2002-01-24 for treatment of t cell mediated autoimmune disorders.
This patent application is currently assigned to Trustees of Darmouth College. Invention is credited to Claassen, Eric, Noelle, Randolph J..
Application Number | 20020009450 09/849969 |
Document ID | / |
Family ID | 23913182 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009450 |
Kind Code |
A1 |
Noelle, Randolph J. ; et
al. |
January 24, 2002 |
Treatment of T cell mediated autoimmune disorders
Abstract
Method for the treatment of multiple sclerosis and other T cell
mediated autoimmune disorders is described. The method involves
administering to a subject a therapeutically effective amount of an
antagonist of a receptor on a surface of a T cell which mediates
contact dependent helper effector functions, for example, an
anti-gp39 antibody.
Inventors: |
Noelle, Randolph J.;
(Cornish, NH) ; Claassen, Eric; ( Pijncker,
NL) |
Correspondence
Address: |
Intellectual Property Group
of Pillsbury Winthrop LLP
Ninth Floor, East Tower
1100 New York Avenue, N.W.
Washington
DC
20005-3918
US
|
Assignee: |
Trustees of Darmouth
College
Hanover
NH
|
Family ID: |
23913182 |
Appl. No.: |
09/849969 |
Filed: |
May 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09849969 |
May 8, 2001 |
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09080349 |
May 18, 1998 |
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09080349 |
May 18, 1998 |
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08481735 |
Jun 7, 1995 |
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5833987 |
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Current U.S.
Class: |
424/154.1 ;
424/142.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 3/10 20180101; A61P 37/00 20180101; C07K 16/2875 20130101;
A61P 17/02 20180101; A61P 43/00 20180101; A61K 38/00 20130101; A61P
29/00 20180101; A61P 3/08 20180101; A61P 37/06 20180101; A61P 37/02
20180101 |
Class at
Publication: |
424/154.1 ;
424/142.1 |
International
Class: |
A61K 039/395 |
Goverment Interests
[0001] The work leading to this invention was supported by one or
more grants from the U.S. government. The goverment may have rights
in the invention.
Claims
1. A method for treating a T cell mediated autoimmune disorder
comprising administering to the subject a therapeutically or
prophylactically effective amount of an antagonist of a receptor on
a surface of a T cell which mediates contact dependent helper
effector functions.
2. A method of claim 1, wherein the T cell mediated autoimmune
disorder is selected from the group consisting of multiple
sclerosis, EAE, diabetes type I, oophoritis and thyroiditis.
3. A method of claim 1, wherein the T cell mediated autoimmune
disorder is multiple sclerosis.
4. The method of claim 1, wherein the receptor on the surface of
the T cell which mediates contact-dependent helper effector
function is gp39.
5. The method of claim 4, wherein the antagonist is an anti-gp39
antibody.
6. The method of claim 5, wherein the anti-gp39 antibody is a
monoclonal antibody.
7. The method of claim 5, wherein the anti-gp39 antibody is an
anti-human gp39 antibody.
8. The method of claim 6, wherein the monoclonal antibody is
produced by 89-76 hybridoma, ATCC Accession Number HB11713 or 24-31
hybridoma, ATCC Accession Number HB11712 or an antibody having the
gp39 binding characteristics thereof.
9. The method of claim 6, wherein the monoclonal antibody is a
chimeric monoclonal antibody.
10. The method of claim 6, wherein the monoclonal antibody is a
humanized monoclonal antibody.
11. A method of treating multiple sclerosis comprising
administering to a subject a therapeutically effective amount of an
anti-human gp39 antibody.
Description
BACKGROUND OF THE INVENTION
[0002] Autoimmune diseases are characterized by attack of the
immune system of an individual against its own tissues. Autoimmune
diseases usually result from breakdown of tolerance of the immune
system to its own antigens. The specific antigens recognized by the
immune system in the various autoimmune diseases can be present
systematically or they can be organ specific. For example, systemic
lupus erthematosus (SLE) is characterized by the presence of
autoantibodies to DNA, ribonucleoproteins, histones, and other
molecules that are not organ specific. Other autoimmune diseases
are characterized by the destruction of mostly one organ. Such
autoimmune diseases include type I diabetes, in which the insulin
producing .beta. cells of the islets of Langerhans in the pancreas
are destroyed.
[0003] In some autoimmune diseases, tissue destruction occurs
primarily as a result of the production of high levels of
autoantibodies. Such diseases include rheumatoid arthritis,
characterized by destruction of the joint cartilage and
inflammation of the synovium. Patients with rheumatoid arthritis
have an accumulation of immune complexes in their joints which are
formed by association of autoantibodies against the Fc portion of
IgG and IgG molecules. These immune complexes activate the
complement cascade which results in tissue damage. Myasthenia
gravis, a disease of progressive muscle weakness, is caused by the
production of autoantibodies reactive to acetylcholine receptors in
the motor end plates of neuromuscular junctions.
[0004] In other autoimmnune diseases, tissue destruction does not
appear to be primarily mediated by production of autoantibodies,
but rather by auto-reactive T lymphocytes. For example,
experimental allergic encephalomyelitis (EAE), an animal model for
multiple sclerosis, and characterized by demyelination in the brain
and the spinal cord, can be induced in naive animals by transfer of
CD4+T cells from diseased animals. Thus, it is generally considered
that EAE represents a T cell mediated autoimmune disease, rather
than a B cell mediated autoimmune disease (Ben-Nun, A. et al.
(1981) Eur. J. Immunol.11, 195).
[0005] Multiple sclerosis (MS) is a common demyelinating disease of
the brain and spinal cord. It is a progressive disease that is
characterized by remissions and exacerbations of neurologic
dysfunction affecting different regions of the central nervous
system. The symptoms of the disease result from a focus of
inflammatory demyelination, which later forms a scar, appearing as
a "plaque" in the white matter of the brain, brain stem or spinal
cord. Presently, there is no definitive diagnostic test available
for MS and diagnoses and treatment regimes are being formulated
based on such factors as the extent of a patient's symptoms and/or
the age of the patient at the time of onset of the exacerbations of
neurologic dysfunction.
[0006] Patients having MS typically have been treated with steroids
with a goal of either sending the patient into remission or slowing
the progression of the disease in the patient. Other drugs have
been used to treat particular symptoms of the disease, e.g. muscle
relaxants. Recent developments in treatments available for MS
include the administration of beta-interferon. Beta interferon has
shown some promise for slowing the progression of the disease.
However, effective treatments for MS are still needed.
SUMMARY OF THE INVENTION
[0007] This invention pertains to methods for treating
(therapeutically or prophylactically) a T cell mediated autoimmune
disorder, such as multiple sclerosis. The method comprises
administering to the subject a therapeutically or prophylactically
effective amount of an antagonist of a receptor on a surface of a T
cell which mediates contact dependent helper effector functions. In
a preferred embodiment the antagonist administered is an antibody
or fragment thereof which specifically binds to the T cell receptor
gp39.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 is a graphical representation of DAS units measured
daily in mice injected at day 0 with 75 .mu.g (Panel A) or 300
.mu.g (Panel B) of PLP-peptide with an anti-gp39 antibody (black
bars) or with PBS (grey bars) showing that anti-gp39 administration
prevents the development of experimental allergic encephalomyelitis
EAE. FIG.
[0009] FIG. 2 is a graphical representation of the percentage
suppression of EAE induction in mice injected with PLP-peptide at
day 0 and further injected with anti-gp39 antibodies (black bars)
or PBS (grey bars) at days 0, 2, and 6, or at days 4, 6, and 8, or
at days 7, 9, and 11, showing that administration of anti-gp39
after induction of the disease significantly prevents EAE.
[0010] FIG. 3 is a graphical representation of DAS units measured
daily in mice transplanted with donor spleen cells from mice
injected with PLP-peptide and anti-gp39 antibodies (black bars) or
with donor cells from mice injected with PLP-peptide alone (grey
bars) and injected with PLP-peptide.
DETAILED DESCRIPTION OF THE INVENTION
[0011] This invention pertains to the treatment of T cell mediated
autoimmnune disorder such as multiple sclerosis. The disease is
treated by administering an antagonist to a receptor on the surface
of T cells which mediates contact dependent T cell helper effector
function.
[0012] As defined herein, a "molecule or receptor which mediates
contact dependent helper effector functions" is one which is
expressed on a Th cell and interacts with a ligand on an effector
cell (e.g., a B cell), wherein the interaction of the receptor with
its ligand is necessary for generation of an effector cell response
(e.g., B cell activation). In addition to being involved in
effector cell responses, it has been found that such a molecule is
involved in the response of the T cell to antigen.
[0013] In a preferred embodiment, the receptor on the surface of
the T cell which mediates contact-dependent helper effector
functions is gp39. In this embodiment, the antagonist is a molecule
which inhibits the interaction of gp39 with its ligand on a cell
which presents antigen to the T cell. A particularly preferred gp39
antagonist is an anti-gp39 antibody. Alternatively, the gp39
antagonist is a soluble form of a gp39 ligand, for example soluble
CD40.
[0014] The method of the invention is based at least in part on the
observation that administration of anti-gp39 antibodies to mice
prevents induction of EAE and reverses the disease in animals
having EAE. Thus, it has been found that an agent which inhibits
the interaction of gp39 on a T cell with its ligands(s) on other
cells is effective, both prophylactically and therapeutically, in
treating a typical T cell mediated autoimmune disease. This result
is surprising in view of previous studies which have attributed to
gp39 a primary role in regulating B cell responses. The finding
that anti-gp39 antibodies are effective in treating T cell mediated
autoimmune disease forms the basis of the present invention.
According to the invention, subjects having a T cell mediated
autoimmune disease, such as multiple sclerosis, are treated by
administration of agents that mimic the effect of anti-gp39
antibodies.
T Cell Mediated Autoimmune Diseases
[0015] The language "autoimmune disorder" is intended to include
disorders in which the immune system of a subject reacts to
autoantigens, such that significant tissue or cell destruction
occurs in the subject. The term "autoantigen" is intended to
include any antigen of a subject that is recognized by the immune
system of the subject. The terms "autoantigen" and "self-antigen"
are used interchangeably herein. The term "self" as used herein is
intended to mean any component of a subject and includes molecules,
cells, and organs. Autoantigens may be peptides, nucleic acids, or
other biological substances. The language "T cell mediated
autoimmune disorder" is intended to include autoimmune disorders in
which the reaction to self primarily involves cell-mediated immune
mechanisms, as opposed to humoral immune mechanisms. Thus, the
methods of the invention pertain to treatments of autoimmune
disorders in which tissue destruction is primarily mediated through
activated T cells and immune cells other than B lymphocytes.
However, even though the methods of the invention are intended for
treatment of autoimmune disorders in which reaction to self is
primarily mediated by cells other than B cells, the autoimmune
disorders may be characterized by the presence of autoantibodies.
For example, EAE, a T cell mediated autoimmune disorder, which can
be treated by a method of the invention, is frequently associated
with the presence of autoantibodies to components of the central
nervous system, such as myelin basic protein. Non limiting examples
of T cell mediated autoimmune disorders that can be treated by the
methods of the invention include multiple sclerosis, EAE, diabetes
type I, oophoritis, and thyroiditis.
gp39 Antagonists
[0016] According to the methods of the invention, a gp39 antagonist
is administered to a subject to interfere with the interaction of
gp39 on T cells with a gp39 ligand on antigen presenting cells,
such as B cells and thereby to prevent, alleviate or ameliorate the
disorder. A gp39 antagonist is defined as a molecule which
interferes with this interaction. As described more fully below,
the gp39 antagonist can be an antibody directed against gp39 (e.g.,
a monoclonal antibody against gp39), a fragment or derivative of an
antibody directed against gp39 (e.g., Fab or F(ab)'2 fragments,
chimeric antibodies or humanized antibodies), soluble forms of a
gp39 ligand (e.g., soluble CD40), soluble forms of a fusion protein
of a gp39 ligand (e.g., soluble CD40Ig), or pharmaceutical agents
which disrupt or interfere with the gp39-CD40 interaction.
A. Antibodies
[0017] To prepare anti-gp39 antibodies, a mammal (e.g., a mouse,
hamster, or rabbit) can be immunized with an immunogenic form of
gp39 protein or protein fragment (e.g., peptide fragment) which
elicits an antibody response in the mammal. A cell which expresses
gp39 on its surface can also be used as the immunogen. Alternative
immunogens include purified gp39 protein or protein fragments. gp39
can be purified from a gp39-expressing cell by standard
purification techniques, e.g., gp39 cDNA (Armitage et al., Nature,
357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101
(1992); Hollenbaugh et al., EMBO J., 11:4313-4319 (1992)) can be
expressed in a host cell, e.g., bacteria or a mammalian cell line,
and gp39 protein purified from the cell culture by standard
techniques. gp39 peptides can be synthesized based upon the amino
acid sequence of gp39 (disclosed in Armitage et al., Nature,
357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101
(1992); Hollenbaugh et al., EMBO J., 11:4313-4319 (1992)) using
known techniques (e.g. F-moc or T-boc chemical synthesis).
Techniques for conferring inmmunogenicity on a protein include
conjugation to carriers or other techniques well known in the art.
For example, the protein can be administered in the presence of
adjuvant. The progress of immunization can be monitored by
detection of antibody titers in plasma or serum. Standard ELISA or
other immunoassay can be used with the immunogen as antigen to
assess the levels of antibodies.
[0018] Following immunization, antisera can be obtained and, if
desired, polyclonal antibodies isolated from the sera. To produce
monoclonal antibodies, antibody producing cells (lymphocytes) can
be harvested from an immunized animal and fused with myeloma cells
by standard somatic cell fusion procedures thus immortalizing these
cells and yielding hybridoma cells. Such techniques are well known
in the art. For example, the hybridoma technique originally
developed by Kohler and Milstein (Nature (1975) 256:495-497) as
well as other techniques such as the human B-cell hybridoma
technique (Kozbar et al., Immunol. Today (1983) 4:72), the
EBV-hybridoma technique to produce human monoclonal antibodies
(Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) (Allen
R. Bliss, Inc., pages 77-96), and screening of combinatorial
antibody libraries (Huse et al., Science (1989) 246:1275).
Hybridoma cells can be screened immunochemically for production of
antibodies specifically reactive with the protein or peptide and
monoclonal antibodies isolated.
[0019] The term antibody as used herein is intended to include
fragments thereof which are specifically reactive with a gp39
protein or peptide thereof or gp39 fusion protein. Antibodies can
be fragmented using conventional techniques and the fragments
screened for utility in the same manner as described above for
whole antibodies. For example, F(ab').sub.2 fragments can be
generated by treating antibody with pepsin. The resulting
F(ab').sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab' fragments. The antibody of the present invention is
further intended to include bispecific and chimeric molecules
having an anti-gp39 portion.
[0020] When antibodies produced in non-human subjects are used
therapeutically in humans, they are recognized to varying degrees
as foreign and an immune response may be generated in the patient
One approach for minimizing or eliminating this problem, which is
preferable to general immunosuppression, is to produce chimeric
antibody derivatives, i.e., antibody molecules that combine a
non-human animal variable region and a human constant region.
Chimeric antibody molecules can include, for example, the antigen
binding domain from an antibody of a mouse, rat, or other species,
with human constant regions. A variety of approaches for making
chimeric antibodies have been described and can be used to make
chimeric antibodies containing the immunoglobulin variable region
which recognizes gp39. See, for example, Morrison et al., Proc.
Natl. Acad. Sci U.S.A. 81:6851 (1985); Takeda et al., Nature
314:452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et
al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent
Publication EP171496; European Patent Publication 0173494, United
Kingdom Patent GB 2177096B. It is expected that such chimeric
antibodies would be less immunogenic in a human subject than the
corresponding non-chimeric antibody.
[0021] For human therapeutic purposes the monoclonal or chimeric
antibodies specifically reactive with a gp39 protein or peptide can
be further humanized by producing human variable region chimeras,
in which parts of the variable regions, especially the conserved
framework regions of the antigen-binding domain, are of human
origin and only the hypervariable regions are of non-human origin.
Such altered immunoglobulin molecules may be made by any of several
techniques known in the art, (e.g., Teng et al., Proc. Natl Acad.
Sci. U.S.A., 80:7308-7312 (1983); Kozbor et al., Immunology Today,
4:7279 (1983); Olsson et al., Meth Enzymol., 92:3-16 (1982)), and
are preferably made according to the teachings of PCT Publication
WO92/06193 or EP 0239400. Humanized antibodies can be commercially
produced by, for example, Scotgen Limited, 2 Holly Road,
Twickenham, Middlesex, Great Britain.
[0022] Another method of generating specific antibodies, or
antibody fragments, reactive against a gp39 protein or peptide is
to screen expression libraries encoding immunoglobulin genes, or
portions thereof, expressed in bacteria with a gp39 protein or
peptide. For example, complete Fab fragments, VH regions and FV
regions can be expressed in bacteria using phage expression
libraries. See for example Ward et al., Nature, 341: 544-546:
(1989); Huse et al., Science, 246: 1275-1281 (1989); and McCafferty
et al., Nature, 348: 552-554 (1990). Screening such libraries with,
for example, a gp39 peptide can identify immunoglobulin fragments
reactive with gp39. Alternatively, the SCID-hu mouse (available
from Genpharm) can be used to produce antibodies, or fragments
thereof.
[0023] Methodologies for producing monoclonal antibodies directed
against gp39, including human gp39 and mouse gp39, and suitable
monoclonal antibodies for use in the methods of the invention, are
described in PCT Patent Application No. WO 95/06666 entitled
"Anti-gp39 Antibodies and Uses Therefor", the teachings of which
are incorporated by reference. Particularly preferred anti-human
gp39 antibodies of the invention are mAbs 24-31 and 89-76, produced
respectively by hybridomas 24-31 and 89-76. The 89-76 and 24-31
hybridomas, producing the 89-76 and 24-31 antibodies, respectively,
were deposited under the provisions of the Budapest Treaty with the
American Type Culture Collection, Parklawn Drive, Rockville, Md.,
on Sep. 2, 1994. The 89-76 hybridoma was assigned ATCC Accession
Number HB11713 and the 24-31 hybridoma was assigned ATCC Accession
Number HB11712.
[0024] Recombinant anti-gp39 antibodies, such as chimeric and
humanized antibodies, can be produced by manipulating nucleic acid
(e.g., DNA) encoding an anti-gp39 antibody according to standard
recombinant DNA techniques. Accordingly, another aspect of this
invention pertains to isolated nucleic acid molecules encoding
immunoglobulin heavy or light chains, or portions thereof, reactive
with gp39, particularly human gp39. The immunoglobulin-encoding
nucleic acid can encode an immunoglobulin light or heavy chain
variable region, with or without a linked heavy or light chain
constant region (or portion thereof). Such nucleic acid can be
isolated from a cell (e.g., hybridoma) producing an anti-human gp39
mAb by standard techniques. For example, nucleic acid encoding the
24-31 or 89-76 mAb can be isolated from the 24-31 or 89-76
hybridoma, respectively, by cDNA library screening, PCR
amplification or other standard technique. Following isolation of,
and possible further manipulation of, Moreover, nucleic acid
encoding an anti-human gp39 mAb can be incorporated into an
expression vector and introduced into a host cell to facilitate
expression and production of recombinant forms of anti-human gp39
antibodies.
B. Soluble Ligands for gp39
[0025] Other gp39 antagonists which can be used to induce T cell
tolerance are soluble forms of a gp39 ligand. A monovalent soluble
ligand of gp39, such as soluble CD40 can bind gp39, thereby
inhibiting the interaction of gp39 with CD40 on B cells. The term
"soluble" indicates that the ligand is not permanently associated
with a cell membrane. A soluble gp39 ligand can be prepared by
chemical synthesis, or, preferably by recombinant DNA techniques,
for example by expressing only the extracellular domain (absent the
transmembrane and cytoplasmic domains) of the ligand. A preferred
soluble gp39 ligand is soluble CD40. Alternatively, a soluble gp39
ligand can be in the form of a fusion protein. Such a fusion
protein comprises at least a portion of the gp39 ligand attached to
a second molecule. For example, CD40 can be expressed as a fusion
protein with immunoglobulin (i.e., a CD40Ig fusion protein). In one
embodiment, a fusion protein is produced comprising amino acid
residues of an extracellular domain portion of the CD40 molecule
joined to amino acid residues of a sequence corresponding to the
hinge, CH2 and CH3 regions of an immunoglobulin heavy chain, e.g.,
C.gamma.1, to form a CD40Ig fusion protein (see e.g., Linsley et
al. (1991) J. Exp. Med. 1783:721-730; Capon et al. (1989) Nature
337, 525-531; and Capon U.S. Pat. No. 5,116,964). The fusion
protein can be produced by chemical synthesis, or, preferably by
recombinant DNA techniques based on the cDNA of CD40 (Stamenkovic
et al., EMBO J., 8:1403-1410 (1989)).
[0026] An antagonist of the invention is administered to subjects
in a biologically compatible form suitable for pharmaceutical
administration in vivo. By "biologically compatible form suitable
for administration in vivo" is meant a form of the antagonist to be
administered in which any toxic effects are outweighed by the
therapeutic effects of the protein. The term subject is intended to
include living organisms in which an immune response can be
elicited, e.g., mammals. Examples of subjects include humans, dogs,
cats, mice, rats, and transgenic species thereof. A gp39 antagonist
can be administered in any pharmacological form, optionally in a
pharmaceutically acceptable carrier. Administration of a
therapeutically active amount of the antagonist is defined as an
amount effective, at dosages and for periods of time necessary to
achieve the desired result. For example, a therapeutically active
amount of an antagonist of gp39 may vary according to factors such
as the disease state, age, sex, and weight of the individual, and
the ability of the antagonist to elicit a desired response in the
individual. Dosage regima may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
[0027] The active compound (e.g., antagonist) may be administered
in a convenient manner such as by injection (subcutaneous,
intravenous, etc.), oral administration, inhalation, transdermal
application, or rectal administration. Depending on the route of
administration, the active compound may be coated in a material to
protect the compound from the action of enzymes, acids and other
natural conditions which may inactivate the compound. A preferred
route of administration is by intravenous injection.
[0028] To administer an antagonist of gp39 by other than parenteral
administration, it may be necessary to coat the antagonist with, or
co-administer the antagonist with, a material to prevent its
inactivation. For example, an antagonist can be administered to an
individual in an appropriate carrier or diluent, co-administered
with enzyme inhibitors or in an appropriate carrier such as
liposomes. Pharmaceutically acceptable diluents include saline and
aqueous buffer solutions. Enzyme inhibitors include pancreatic
trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
Liposomes include water-in-oil-in-water emulsions as well as
conventional liposomes (Strejan et al., (1984) J. Neuroimmunol
7:27).
[0029] The active compound may also be administered parenterally or
intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
may contain a preservative to prevent the growth of
microorganisms.
[0030] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. In all cases, the
composition 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 (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can 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. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
asorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0031] Sterile injectable solutions can be prepared by
incorporating active compound (e.g., an antagonist of gp39) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient (e.g., antagonist) plus any
additional desired ingredient from a previously sterile-filtered
solution thereof.
[0032] When the active compound is suitably protected, as described
above, the protein may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. As used herein
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use thereof in
the therapeutic compositions is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0033] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0034] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
EXAMPLE 1
EAE Prevention by Anti gp39 Antibody Administration
[0035] This example demonstrates that administration of anti-gp39
antibodies to mice prevents induction of experimental allergic
encephalomyelitis (EAE), an animal model for multiple
sclerosis.
[0036] EAE is a well characterized model of a T cell mediated
autoimmune disease and is an instructive model for the human
autoimmune disease multiple sclerosis. EAE can be induced in
susceptible animals, such as mice, by immunizing the animals with
myelin basic protein (MBP), proteolipid protein (PLP), myelin
oliogodendrocyte protein (MOG), or synthetic peptides based on the
sequences of these myelin associated proteins together with an
adjuvant containing pertussis bacteria. One to two weeks after
immunization, the animals develop encephalomyelitis, characterized
by perivascular infiltrates containing lymphocytes and macrophages
and the development of demyelination in the brain and the spinal
cord. The animals show acute, chronic or chronic relapsing
paralysis. In this example, the effect of administration of
anti-gp39 antibodies on development of EAE in susceptible mice was
analyzed.
[0037] EAE was induced in susceptible mice by subcutaneous
injections (day 0) of an emulsion containing 70 .mu.g or 300 .mu.g
PLP-peptide in 50 .mu.l PBS and 25 .mu.g Mycobacteria tuberculosis
(H37RA, Difco) in 50 .mu.l of complete Freuds adjuvant at two sites
in the abdominal flanks of the mice. 200 .mu.l Bordetella pertussis
suspension (10.10.sup.10 in 1 ml PBS) was given intravenously at
the same time as the peptide and two days later. The PLP-peptide
injected in the mice has an amino acid sequence corresponding to
amino acid residues 139 to 151 of rat PLP (Dautigny et al., FEBS
Lett. 188:33, 1985). PLP-peptide was synthesized with f-moc
protected aminoacids according to the solid phase synthesis method
(Merrifield, J. Am. Chem. Soc. 85:2149, 1963). Immunization with
this peptide results in the development of acute EAE, which is
clinically and pathologically identical to that induced by
sensitization with whole central nervous system (CNS) myelin or
with MBP (Tuohy et al., J. Immunol. 142:1523, 1989; Sobel et al.,
J. Neuropathol Exp. Neurol. 49:468, 1990).
[0038] To determine the effect of anti-gp39 on the course of the
disease, mice were injected on day 0 with PLP-peptide, as described
above, and further injected intraperitoneally with 125 .mu.g
hamster anti-gp39 Mabs (Noelle et al., Proc. Natl. Acad Sci. USA
89:6550, 1992) in 200 .mu.l PBS or with 125 .mu.g normal hamster
antibodies (Serva Feinbiochemica) in 200 .mu.l PBS (control
animals) on days 0, 2, and 4. The severity of EAE clinical signs
was evaluated each day and graded according to average disability
scale (DAS) scores: grade 0=no clinical signs, grade 1=tail
weakness, grade 2=mild paraparesis and ataxia of the hind legs,
grade 3=severe paraparesis or ataxia of the hind legs, grade
4=moribund, grade 5=dead due to EAE.
[0039] FIG. 1 represents the course of the disease in mice injected
with 70 .mu.g of PLP-peptide (Panel A) or with 300 .mu.g of
PLP-peptide (Panel B) and treated with anti-gp39 or control
antibody. DAS scores, which reflect the severity of the disease, of
control mice and anti-gp39 treated mice are depicted in grey and
black bars respectively.
[0040] The results indicate that animals having received the
control antibody developed EAE, whereas animals having received
anti-gp39 antibody were protected from induction of the disease.
For animals having received control antibody, the first clinical
signs of EAE became apparent on day eleven. In these animals, the
highest DAS score was 2.33, which was observed on days 15-22 in
animals injected with 75 .mu.g of PLP-peptide (FIG. 1, Panel A,
grey bars) and 3.6, observed on days 16-23 in animals injected with
300 .mu.g PLP-peptide (FIG. 1, Panel B, grey bars). In contrast,
animals which received the anti-gp39 monoclonal antibodies showed
no clinical signs after induction of EAE with 75 .mu.g of
PLP-peptide (FIG. 1, Panel A, black bars) and only minor clinical
signs, which completely disappeared at day 31, after induction of
the disease with 300 .mu.g of PLP-peptide (FIG. 1, Panel B, black
bars).
[0041] Thus, administration of anti-gp39 antibodies to mice
completely inhibited induction of EAE in these mice.
[0042] Induction of EAE by adoptive transfer of myelin reactive
T-cells isolated from animals immunized with myelin components or
obtained after in vitro activation with myelin components indicates
that especially activated T-cells are responsible for the
development of clinical characteristics after the inductive phase
(Pettinelli and McFarlin, J. Immunol. 127:1420, 1979; Mokhtarion et
al., Nature 309:356, 1984; Veen et al., J. Neuroimmunol. 21:183,
1989). However, the experiments described herein show that
administration of anti-gp39 monoclonal antibodies prevents the
development of EAE. In control groups, significant anti-PLP-peptide
antibody responses were observed on day 14 (absorbance 1.92) and 21
(absorbance 2.15) in animals in which EAE was induced with low and
high PLP-peptide dose, respectively. In contrast, significant
anti-PLP-peptide antibody responses in the gp39 treated animals
were observed first on day 14, and reached plateau levels on day 31
(absorbance 0.928) and 40 (absorbance 1.54) in animals which were
injected with low and high PLP-peptide dose, respectively. The
generation of significant anti-PLP-peptide antibody responses in
anti-gp39 monoclonal antibodies treated mice, was postponed until
day 14, which indicates that the anti-gp39 monoclonal antibodies
had some effect on antibody production.
[0043] Thus, this example demonstrates that anti-gp39 prevent
development of EAE and indicate that anti-gp39 antibodies can be
used for treating T cell mediated autoimmune diseases, such as
multiple sclerosis.
EXAMPLE 2
Reversal of EAE by Anti-gp39 Antibody Administration
[0044] Example 1 showed the inhibitory effect of anti-gp39 antibody
on induction of EAE. Thus, it was demonstrated that immunization of
the mice at the time of induction of the disease prevented
development of the disease. This example shows that administration
of the antibody after induction of the disease leads to regression
of the disease.
[0045] In this example, EAE was induced in female SJL/j mice (10-12
weeks old) by injection of an emulsion containing 150 .mu.g
PLP-peptide prepared as described above. For determining the effect
of the anti-gp39 antibodies when administered to the mice after
induction of the disease, mice were injected intraperitoneally with
125 .mu.g anti-gp39 monoclonal antibodies (Noelle et al., Proc.
Natl. Acad. Sci. USA 89:6550, 1992) in 200 .mu.l of PBS (anti-gp39
treated mice) or with 200 .mu.l PBS alone (control mice) on days 0,
2 and 4, on days 4, 6 and 8, or on days 7, 9 and 11. The results
are presented as percentage suppression, and are a comparison of
the total of daily DAS scores (from day 12 to 28) in anti-gp39
treated animals and in control animals.
[0046] The results, which are presented in FIG. 2, indicate that
administration of the first dose of anti-gp39 antibodies as late as
7 days after injection into the mice of PLP-peptide results in more
than 60% suppression of the disease. Thus, anti-gp39 antibodies are
capable of reversing or suppressing EAE.
[0047] The results further indicate that even though administration
of a first dose of antibody only 7 days after induction of the
disease in mice results in significant suppression of development
of the disease, anti-gp39 treatment is somewhat more efficient when
the first dose of antibody is administered sooner after induction
of the disease.
[0048] Thus, administration of anti-gp39 antibodies to mice
protects these mice from developing EAE upon induction of the
disease and suppresses the disease in mice having EAE.
EXAMPLE 3
Suppression of EAE After Spleen Cell Transfer of gp39 Treated
Mice
[0049] Regulatory suppessor T-cells have been detected in Lewis
rat, which have been recovered from EAE (Pesoa et al., J.
Neuroimmunol. 7:131, 1984) and after oral administration of myelin
components (Lider et al., J. Immunol. 142:748, 1989; Hafler et al.,
Ann NY Acad Sci. 636:251, 1991). It was postulated by Karpus and
Swanborg (J. Immunol 143:3492, 1989) that CD4+suppressor T-cells
isolated from rats which have recovered from EAE, can down regulate
EAE T-effector cells by differential inhibition of lymphokine
production. In contrast, suppression of EAE in Lewis rats by oral
administration of MBP is mediated by CD8+T-cells (Miller et al., J.
Exp. Med. 174:791, 1991). To determine whether T cells of mice
having been protected from EAE by administration of anti-gp39
antibody, are capable of protecting naive animals from EAE, the
following example was undertaken.
[0050] In this example, a first set of mice were injected with 150
.mu.g of PLP-peptide and a second set of mice were injected with
150 .mu.g of PLP-peptide and anti-gp39 antibody according to the
protocol described in Example 1. Four months later the mice were
sacrificed by CO.sub.2 euthanasia and the spleens were removed.
Erythrocytes were removed by standard ammoniumchloride treatment
(Mishell and Shiigi, Selected Methods in Cellular Immunology, W. H.
Freeman and Company, 1980). Cells of individual spleens (500 .mu.l)
were injected i.v. in naive 5 Gy irradiated recipient female SLJ/j
mice (10-12 weeks old). Two days after cell transfer, mice were
challenged by intraperitoneal injection with 150 .mu.g PLP-peptide
following the procedure described in Example 1 and DAS scores
determined.
[0051] FIG. 3 represents the DAS scores of the animals. The results
demonstrate that mice that have been transplanted with spleen cells
from animals initially injected with PLP-peptide and anti-gp39
antibodies are protected from development of the disease, whereas
mice transplanted with spleen cells from animals initially injected
with PLP-peptide only develop EAE. Moreover, considering that the
estimated half-life of antibodies is 12 days, it may be expected
that no antibodies were present in the spleen cells transplanted in
the mice. Therefore, the protective effect conferred by donor
spleen cells from animals having received PLP-peptide and anti-gp39
antibodies cannot be explained by the presence of anti-gp39
antibodies. DAS scores of these mice indicates that suppression of
EAE in the recipient mice is most likely due to the presence of a
T-suppressor cell population in the transferred spleen cell
suspension and that this T-suppressor cell population overrules the
T-effector cell population efficiently.
EXAMPLE 4
Detection of gp39 Positive Th-cells
[0052] This example demonstrates the presence of gp39 positive
cells in the central nervous system of human subjects having
multiple sclerosis.
[0053] Human autopsy central nervous system (CNS) tissues were
obtained from the Netherlands brain bank, Amsterdam, the
Netherlands. Gp39 positive cells were detected with an CD40-Ig
fusion protein according to methods known in the art. CNS tissue
sections of an MS patient and an Alzheimer patient were stained
with CD40 g. In this example, only CNS tissues of MS-patients in
which anti-MBP antibody forming cells were detected previously were
used. The results of the staining show the presence of gp39
positive cells in a 8 .mu.m coronal cerebrum section of an MS
patient, but no gp39 positive cells were detected in coronal
cerebrum sections of Alzheimer patients. Thus, gp39 positive cells
were detected only in CNS tissue sections of MS patients. The
presence of gp39 positive cells in MS-patient CNS tissue together
with the detection of anti-MBP antibody forming cells in CNS
tissues of MS-patients only and not in control CNS tissues
indicates that such cells play an role in the pathological affected
CNS tissues of MS patients.
[0054] In this study we have shown that suppression of B-cell
activation by the administration of anti-gp39 Mabs, can result in
the complete prevention of EAE development, dependent on the dose
of antigen by which EAE was induced and dependent on the time
period between EAE induction and administration of anti-gp39 Mabs.
Although the exact mechanisms responsible for EAE induction and
development are not clear, these data indicate that anti-gp39
antibody can be used for treatment of autoimmune diseases.
Equivalents
[0055] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *