U.S. patent application number 13/577579 was filed with the patent office on 2013-02-21 for therapeutic inhibition of granulocyte function in demyelinating disease.
The applicant listed for this patent is Robert C. Axtell, Lawrence Steinman. Invention is credited to Robert C. Axtell, Lawrence Steinman.
Application Number | 20130046015 13/577579 |
Document ID | / |
Family ID | 44368154 |
Filed Date | 2013-02-21 |
United States Patent
Application |
20130046015 |
Kind Code |
A1 |
Axtell; Robert C. ; et
al. |
February 21, 2013 |
Therapeutic Inhibition of Granulocyte Function in Demyelinating
Disease
Abstract
Compositions and methods are provided for the treatment of
IL-17-type inflammatory demyelinating diseases with inhibitors of
granulocyte function, e.g. elastase inhibitors. Diseases of
interest include multiple sclerosis, neuromyelitis optica, animal
models of such diseases, etc. In some embodiments pharmaceutical
formulations comprising an elastase inhibitor in an effective dose
for treatment of IL-17-type inflammatory demyelinating disease and
a pharmaceutically acceptable excipient are provided. Patients may
be classified into subtypes prior to treatment, which subtypes are
informative of the patient's need for therapy and responsiveness to
a therapy of interest.
Inventors: |
Axtell; Robert C.; (Menlo
Park, CA) ; Steinman; Lawrence; (Stanford,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Axtell; Robert C.
Steinman; Lawrence |
Menlo Park
Stanford |
CA
CA |
US
US |
|
|
Family ID: |
44368154 |
Appl. No.: |
13/577579 |
Filed: |
February 11, 2011 |
PCT Filed: |
February 11, 2011 |
PCT NO: |
PCT/US11/24557 |
371 Date: |
October 26, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61303590 |
Feb 11, 2010 |
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61315753 |
Mar 19, 2010 |
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61401045 |
Aug 6, 2010 |
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61402757 |
Sep 2, 2010 |
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Current U.S.
Class: |
514/548 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
3/04 20180101; A61P 25/16 20180101; G01N 2800/285 20130101; Y02A
90/10 20180101; G01N 33/6863 20130101; A61P 11/00 20180101; A61P
11/06 20180101; Y02A 90/26 20180101; A61P 1/16 20180101; A61K
31/225 20130101; A61P 9/10 20180101; G01N 33/6896 20130101; A61P
25/00 20180101; G01N 2333/54 20130101; A61K 31/18 20130101; A61P
29/00 20180101 |
Class at
Publication: |
514/548 |
International
Class: |
A61K 31/225 20060101
A61K031/225; A61P 25/00 20060101 A61P025/00; A61P 29/00 20060101
A61P029/00 |
Claims
1. A method for treating a patient suffering from an inflammatory
demyelinating disease having an IL-17 subtype, the method
comprising: administering to said patient an inhibitor of
granulocyte function in an amount effective to decrease disease
symptoms or progression.
2. The method of claim 1, wherein said inhibitor of granulocyte
function is an inhibitor of neutrophil elastase.
3. The method of claim 2, wherein the elastase inhibitor is
sivelestat sodium hydrate.
4. The method of claim 1, wherein said patient is a human.
5. The method of claim 4, wherein said inflammatory demyelinating
disease having an IL-17 subtype is neuromyelitis optica.
6. The method of claim 4, wherein said inflammatory demyelinating
disease having an IL-17 subtype is multiple sclerosis.
7. The method of claim 1, wherein prior to said administering step
the patient is classified for responsiveness by the method
comprising: determining circulating levels of at least one cytokine
or granulocyte marker in a patient to provide a marker signature
pattern, where the marker is indicative of the TH1/TH17 status of
the patient; comparing said marker signature pattern with a control
signature pattern; wherein a statistically significant match with a
responder pattern for said therapy of interest or a statistically
significant difference from a non-responder pattern for said
therapy of interest is indicative that said multiple sclerosis
patient has a positive prognosis for being responsive to said
therapy of interest; directing therapeutic intervention for said
patient based on said prognosis.
8. The method according to claim 7, wherein said at least one
marker includes IL-17F.
9. The method according to claim 8, wherein said at least one
marker further includes one or more of IL-17A, .beta.-IFN, IL-7,
IL-5, IL-8, IL-23, elastase, Gro-alpha, and .gamma.-IFN.
10. The method of claim 9, wherein said determining step comprises:
preparing a measurement panel comprising one or more affinity
reagents specific for markers indicative of the TH1/TH17 status of
the patient; physically contacting the panel with a patient sample;
quantifying the markers that bind to the panel.
11. The method of claim 7 wherein the patient sample is blood or a
derivative thereof.
Description
BACKGROUND OF THE INVENTION
[0001] There is a long-standing interest in manipulating cells of
the immune system to achieve control of autoimmune disease. While
targeted antigen-specific therapy remains of great interest, there
has also been considerable development of polyclonal, or
non-antigen specific therapies. In addition to general
immunosuppression, e.g. through the use of agents such as
hydrocortisone, many therapies are now being brought to the clinic
that provide for a more selective modification of the immune
system.
[0002] A downside to this promising therapy is the diversity of
responses in patient populations. While a significant proportion of
patients may respond to a particular therapy, many do not. The
clinician may therefore need to prescribe sequential expensive and
time-consuming therapies in order to determine which is effective
for the individual patient. Furthermore, it has been reported that
IFN-.beta. can exacerbate symptoms in some individuals.
[0003] The use of disease-modifying therapies in autoimmune
conditions is of great clinical interest; however these therapies
suffer from the inability to determine a priori which patients will
benefit. The present invention addresses this need.
[0004] Publications of interest include Guo et al. J Clin Invest
(2008); Nagai et al. Scand J Immunol 65, 107-17 (2007); McRae et
al. J Immunol 160, 4298-304 (1998); Martin-Saavedra et al. Mol
Immunol 45, 4008-19 (2008).
SUMMARY OF THE INVENTION
[0005] Compositions and methods are provided for the treatment of
IL-17-type inflammatory demyelinating diseases with inhibitors of
granulocyte function, e.g. elastase inhibitors. Diseases of
interest include multiple sclerosis, neuromyelitis optica, animal
models of such diseases, etc. In some embodiments pharmaceutical
formulations comprising an elastase inhibitor in an effective dose
for treatment of IL-17-type inflammatory demyelinating disease and
a pharmaceutically acceptable excipient are provided. Significant
granulocyte infiltrates are shown herein to be characteristic of
NMO and IL-17-type MS/EAE. Characteristic also of granulocytes are
the presence of elastase and Gro-.alpha..
[0006] Assessment in an inflammatory demyelinating disease patient
allows improved care, where patients classified according to
responsiveness can be treated with an appropriate agent, e.g.
patients classified as a predominantly TH17-type disease subtype
can be treated with appropriate agents, e.g. inhibitors of
granulocyte function, etc. Patients may be classified upon initial
presentation of symptoms, and may be further monitored for status
over the course of the disease to maintain appropriate therapy, or
may be classified at any appropriate stage of disease progression.
A patient having high levels of markers indicative of a TH17
subtype, e.g. IL-17F, IL-23, .beta.-IFN, and/or low levels of IL-7
etc. is classified as non-responder to TH1 subtype immunotherapy,
and may appropriately be treated with an inhibitor of granulocyte
function. Patients having an IL-17-type disease subtype are also
characterized by the presence of excessive numbers of
polymorphonuclear leukocytes (PMN or PML), a clinical factor.
Classification of a patient may alternatively, or in combination
with marker assessment, comprise determining the levels of one or
more of PMN-associated markers, including elastase and
Gro-.alpha..
[0007] Methods of determining responder status in a patient with an
immune-related disease may comprise obtaining or preparing a
cytokine measurement panel comprising one or more affinity reagents
specific for markers, including for example, anti-IL-17, e.g.
IL-17F, .beta.-IFN, IL-7, etc.; physically contacting the panel
with a patient sample such as blood, serum, cerebrospinal fluid,
etc.; identifying the markers that bind to the panel; comparing the
markers bound to the those bound with a control sample known to be
one or more of: a non-diseased individual, an individual known to
have a responder or non-responder phenotype, etc. Those patients
non-responsive to TH1-therapy, such as .beta.-IFN, may be
characterized has having a level of IL-17, e.g. IL-17F, that is
significantly higher than a non-diseased individual, while those
patients responsive to such therapy have a level of IL-17 not
significantly different than a non-diseased individual. Those
patients responsive to TH1-therapy, such as .beta.-IFN, may be
characterized has having a level of IL-7 that is significantly
higher than a non-diseased individual, while those patients
non-responsive to such therapy have a level of IL-7 not
significantly different than a non-diseased individual. The
resulting data sets provide a signature pattern from which the
prognostic classification can be determined.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1. Correlation of a) IL-17F vs IFN-.beta. levels, b)
IL-17F vs MIP1.beta. levels and c) IFN-.beta. vs
[0009] MIP1.beta. in serum from responders, non-responders and
healthy controls. R.sup.2 values close to 1 demonstrate that the
cytokines are positively correlated.
[0010] FIG. 2. Comparison of the effect of copaxone and control on
TH1 and TH17 induced disease.
[0011] FIG. 3A-3C, differential expression of IL-17A and IL-17F in
NMO and EAE.
[0012] FIG. 4A-4C, Role of granulocytes in NMO and effect of
neutrophil elastase inhibition in the treatment of TH17 EAE.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0013] Mammalian species that provide samples for analysis and that
are amenable to treatment include canines; felines; equines;
bovines; ovines; etc. and primates, particularly humans. Animal
models, particularly small mammals, e.g. murine, lagomorpha, etc.
may be used for experimental investigations. Animal models of
interest include those for models of autoimmunity, graft rejection,
and the like.
Definitions
[0014] Inflammatory demyelinating disease. The term "inflammatory"
response is the development of a humoral (antibody mediated) and/or
a cellular (mediated by antigen-specific T cells or their secretion
products) response. Inflammatory demyelinating diseases of the
central nervous system are of particular interest and include,
without limitation, multiple sclerosis (MS), neuromyelitis optica
(NO), and experimental acquired encephalitis (EAE). Demyelinating
inflammatory diseases of the peripheral nervous system include
Guillain-Barre syndrome (GBS) with its subtypes acute inflammatory
demyelinating polyradiculoneuropathy, acute motor axonal
neuropathy, acute motor and sensory axonal neuropathy, Miller
Fisher syndrome, and acute pandysautonomia; chronic inflammatory
demyelinating polyneuropathy (CIDP) with its subtypes classical
CIDP, CIDP with diabetes, CIDP/monoclonal gammopathy of
undetermined significance (MGUS), sensory CIDP, multifocal motor
neuropathy (MMN), multifocal acquired demyelinating sensory and
motor neuropathy or Lewis-Sumner syndrome, multifocal acquired
sensory and motor neuropathy, and distal acquired demyelinating
sensory neuropathy.
[0015] Multiple sclerosis is characterized by various symptoms and
signs of CNS dysfunction, with remissions and recurring
exacerbations. The most common presenting symptoms are paresthesias
in one or more extremities, in the trunk, or on one side of the
face; weakness or clumsiness of a leg or hand; or visual
disturbances, e.g. partial blindness and pain in one eye
(retrobulbar optic neuritis), dimness of vision, or scotomas. Other
common early symptoms are ocular palsy resulting in double vision
(diplopia), transient weakness of one or more extremities, slight
stiffness or unusual fatigability of a limb, minor gait
disturbances, difficulty with bladder control, vertigo, and mild
emotional disturbances; all indicate scattered CNS involvement and
often occur months or years before the disease is recognized.
Excess heat may accentuate symptoms and signs.
[0016] The course is highly varied, unpredictable, and, in most
patients, remittent. At first, months or years of remission may
separate episodes, especially when the disease begins with
retrobulbar optic neuritis. However, some patients have frequent
attacks and are rapidly incapacitated; for a few the course can be
rapidly progressive (primary progressive MS, PPMS). Relapsing
remitting MS (RR MS) is characterized clinically by relapses and
remissions that occur over months to years, with partial or full
recovery of neurological deficits between attacks. Such patients
manifest approximately 1 attack, or relapse, per year. Over 10 to
20 years, approximately 50% of RR MS patients develop secondary
progressive MS (SP MS) which is characterized by incomplete
recovery between attacks and accumulation of neurologic deficits
resulting in increasing disability.
[0017] Diagnosis is indirect, by deduction from clinical,
radiographic (brain plaques on magnetic resonance [MR] scan), and
to a lesser extent laboratory (oligoclonal bands on CSF analysis)
features. Typical cases can usually be diagnosed confidently on
clinical grounds. The diagnosis can be suspected after a first
attack. Later, a history of remissions and exacerbations and
clinical evidence of CNS lesions disseminated in more than one area
are highly suggestive.
[0018] MRI, the most sensitive diagnostic imaging technique, may
show plaques. It may also detect treatable nondemyelinating lesions
at the junction of the spinal cord and medulla (eg, subarachnoid
cyst, foramen magnum tumors) that occasionally cause a variable and
fluctuating spectrum of motor and sensory symptoms, mimicking MS.
Gadolinium-contrast enhancement can distinguish areas of active
inflammation from older brain plaques. MS lesions may also be
visible on contrast-enhanced CT scans; sensitivity may be increased
by giving twice the iodine dose and delaying scanning (double-dose
delayed CT scan).
[0019] Conventional treatments for MS include interferon .beta.
(Avonex, Betaseron, Rebif), Copaxone (Glatiramer acetate), and
anti-VLA4 (Tysabri, natalizumab), which reduce relapse rate and to
date have only exhibited a modest impact on disease progression. MS
is also treated with immunosuppressive agents including
methylprednisolone, other steroids, methotrexate, cladribine and
cyclophosphamide. Many biological agents, such as anti-IFNgamma
antibody, CTLA4-Ig (Abetacept), anti-CD20 (Rituxan), and other
anti-cytokine agents are in clinical development for MS.
[0020] Neuromyelitis optica (NMO), or Devic's disease, is an
autoimmune, inflammatory disorder of the optic nerves and spinal
cord. Although inflammation may affect the brain, the disorder is
distinct from multiple sclerosis, having a different pattern of
response to therapy, possibly a different pattern of autoantigens
and involvement of different lymphocyte subsets.
[0021] The main symptoms of Devic's disease are loss of vision and
spinal cord function. As for other etiologies of optic neuritis,
the visual impairment usually manifests as decreased visual acuity,
although visual field defects, or loss of color vision may occur in
isolation or prior to formal loss of acuity. Spinal cord
dysfunction can lead to muscle weakness, reduced sensation, or loss
of bladder and bowel control. The damage in the spinal cord can
range from inflammatory demyelination to necrotic damage of the
white and grey matter. The inflammatory lesions in Devic's disease
have been classified as type II lesions (complement mediated
demyelinization), but they differ from MS pattern II lesions in
their prominent perivascular distribution. Therefore, the pattern
of inflammation is often quite distinct from that seen in MS.
[0022] Attacks are conventionally treated with short courses of
high dosage intravenous corticosteroids such as methylprednisolone
IV. When attacks progress or do not respond to corticosteroid
treatment, plasmapheresis may be used. Commonly used
immunosuppressant treatments include azathioprine (Imuran) plus
prednisone, mycophenolate mofetil plus prednisone, Rituximab,
Mitoxantrone, intravenous immunoglobulin (IVIG), and
Cyclophosphamide. The monoclonal antibody rituximab is under
study.
[0023] The disease can be monophasic, i.e. a single episode with
permanent remission. However, at least 85% of patients have a
relapsing form of the disease with repeated attacks of transverse
myelitis and/or optic neuritis. In patients with the monophasic
form the transverse myelitis and optic neuritis occur
simultaneously or within days of each other. On the other hand,
patients with the relapsing form are more likely to have weeks or
months between the initial attacks and to have better motor
recovery after the initial transverse myelitis event. Relapses
usually occur early with about 55% of patients having a relapse in
the first year and 90% in the first 5 years. Unlike MS, Devic's
disease rarely has a secondary progressive phase in which patients
have increasing neurologic decline between attacks without
remission. Instead, disabilities arise from the acute attacks.
[0024] Polymorphonuclear leukocytes. Polymorphonuclear leukocytes
(PMN or PML) are a category of white blood cells characterized by
the presence of granules in their cytoplasm, which are also
referred to as granulocytes. There are three types of granulocytes,
distinguished by their appearance under Wright's stain: neutrophil,
eosinophil, and basophil.
[0025] Neutrophils are normally found in the bloodstream and are
the most abundant type of phagocyte, constituting 50% to 60% of the
total circulating white blood cells. Neutrophils are professional
phagocytes. Mature neutrophils are smaller than monocytes, and have
a segmented nucleus with several sections. The intra-cellular
granules of the human neutrophil have long been recognized for
their protein-destroying and bactericidal properties. Neutrophils
can secrete products that stimulate monocytes and macrophages;
these secretions increase phagocytosis and the formation of
reactive oxygen compounds involved in intracellular killing.
Neutrophils have two types of granules. Primary granules contain
cationic proteins and defensins that are used to kill bacteria,
proteolytic enzymes and cathepsin G to breakdown (bacterial)
proteins, lysozyme to break down bacterial cell walls, and
myeloperoxidase. The secondary granules contain compounds that are
involved in the formation of toxic oxygen compounds, lysozyme, and
lactoferrin.
[0026] Eosinophils also have lobed nuclei (two to four lobes).
Eosinophils play a crucial part in the killing of parasites because
their granules contain a unique, toxic basic protein and cationic
protein; IgE receptors are involved in this process.
[0027] Basophils are low abundance cells in bone marrow and blood.
Like neutrophils and eosinophils they have lobed nuclei. Basophils
express receptors for IgE, IgG, complement, and histamine. Granule
contents of basophils include histamine, heparin, chondroitin
sulfate, peroxidase, platelet activating factor. When basophils are
injured they release prostaglandin and histamine; causing dilation
and increased permeability of capillaries close to the
basophil.
[0028] Elastase. Neutrophil elastase (EC 3.4.21.37) is a serine
protease of neutrophil and monocyte granules. Its key physiologic
role is in innate host defense, but it can also participate in
tissue remodeling and possesses secretagogue actions important to
local inflammatory responses. The protein consists of 218 amino
acid residues, contains 2 asparagine-linked carbohydrate side
chains, and is joined together by 2 disulfide bonds.
[0029] Elastase inhibitors find use in the treatment of NMO and
IL-17-type MS. Inhibitors are known in the art, and include without
limitation, sivelestat sodium hydrate (Ono Pharmaceutical);
alpha1-antitrypsin; pafistatin-like protease inhibitors (de Marco
(2010) Peptides 31(7):1280-1260); Clitocybin D (Kim et al. (2009) J
Microbiol Biotechnol. 19(10):1139-41); marama bean inhibitor
(Nadaraja et al. (2010) J Enzyme Inhib Med Chem. 25(3):377-82;
AE-3763 (Inoue et al. (2009) Bioorg Med Chem. 17(21):7477-86);
Isodeoxyhelicobasidin (Xu et al. (2009) J Antibiot 62(6):333-4);
guamerin (Jo et al. (2008) Int Immunopharmacol. 8(7):959-66); elaf
in (Wang et al. (2008) Am J Respir Cell Mol Biol. 38(6):724-32);
Bornyl (3,4,5-trihydroxy)-cinnamate (Steinbrecher et al. (2008)
Bioorg Med Chem. 16(5):2385-90); and the like as known in the art.
Other inhibitors of interest include antibodies specific for
neutrophil elastase, anti-sense oligonucleotides, siRNA, shRNA, and
the like.
[0030] Gro-alpha inhibitors are also of interest, e.g.
antileukinate (see, for example Fujisawa et al. (1999) Melanoma
res. 9(2):105-114. Other inhibitors of interest include antibodies
specific for gro-alpha, anti-sense oligonucleotides, siRNA, shRNA,
and the like.
[0031] T helper 17 cells (Th17) are a subset of T helper cells,
characterized by their production of interleukin 17 (IL-17). They
are considered developmentally distinct from Th1 and Th2 cells and
excessive amounts of the cell are thought to play a key role in
autoimmune disease.
[0032] In humans, a combination of TGF-.beta., IL-1.beta. and IL-23
induces Th17 differentiation from naive T cells. Both interferon
gamma (IFN.gamma.) and IL-4, the main stimulators of Th1 and Th2
differentiation respectively, negatively regulate Th17
differentiation.
[0033] Th17 cells primarily produce two main members of the IL-17
family; IL-17A and IL-17F, which are involved in the recruitment,
activation and migration of neutrophils. These cells also secrete
IL-21 and IL-22.
[0034] T helper 1 cells (Th1). Proliferating helper T cells that
develop into effector T cells differentiate into two major subtypes
of cells known as Th1 and Th2 cells. Th1 cells primarily produce
IFN-.gamma. and TNF-.beta. cytokines. IFN-.gamma. increases the
production of interleukin-12 by dendritic cells and macrophages,
and via positive feedback, IL-12 stimulates the production of
IFN-.gamma. in helper T cells, thereby promoting the Th1 profile.
IFN-.gamma. also inhibits the production of cytokines such as IL-4.
Conditions that polarize to the TH1 type include antigen presenting
cells and IL-12.
[0035] Interleukin-17 (IL-17) refers to a group of cytokines called
the IL-17 family. IL-17 shows high homology to viral IL-17 encoded
by an open reading frame of the T lymphotropic rhadinovirus
Herpesvirus saimiri. To elicit its functions, IL-17 binds to a type
I cell surface receptor called IL-17R of which there are at least
three variants IL17RA, IL17RB, and IL17RC.
[0036] In response to .beta.-IFN, human T cells produce increased
amounts of IL-17F, but not IL-17A, which may be accounted for by
the presence of putative Type I interferon elements upstream of
IL-17F but not IL-17A. In some embodiments of the invention,
classification of .beta.-IFN responsiveness in NMO or MS relies on
a determination of IL-17F levels, which are optionally compared
with levels of IL-17A. Increased levels of II-17F and baseline
levels of IL-17A are indicative of NMO or .beta.-IFN non-responsive
MS.
[0037] Members of the IL-17 family include IL-17B, IL-17C, IL-17D,
IL-17E (also called IL-25), and IL-17F. All members of the IL-17
family have a similar protein structure, with four highly conserved
cysteine residues critical to their 3-dimensional shape, although
with no sequence similarity to any other known cytokines. IL-17A is
a 155 amino acid protein that is a disulfide linked, homodimeric,
secreted glycoprotein with a molecular mass of 35 kDa. Each subunit
of the homodimer is approximately 15-20 KDa. The structure of IL-17
consists of a signal peptide of 23 amino acids (aa) followed by a
123 aa chain region characteristic of the IL-17 family. IL-17F is
structurally similar to the cysteine knot family of proteins that
includes the neurotrophins. The cysteine knot fold is characterized
by two sets of paired .beta.-strands stabilized by three disulfide
interactions. However, in contrast to the other cysteine knot
proteins, IL-17F lacks the third disulfide bond. Instead, a serine
replaces the cysteine at this position. This unique feature is
conserved in the other IL-17 family members. IL-17F also dimerizes
in a fashion similar to nerve growth factor (NGF) and other
neurotrophins. The genetic sequence of IL-17F may be accessed at
Genbank, NM.sub.--052872. Also see Ely et al. (2009) Nat. Immunol.
10 (12), 1245-1251; Kawaguchi e al. (2002) J. Biol. Chem. 277 (18),
15229-15232; Starnes et al. (2001) J. Immunol. 167 (8), 4137-4140;
and Hymowitz et al. (2001) EMBO J. 20 (19), 5332-5341, each herein
specifically incorporated by reference.
[0038] Numerous immune regulatory functions have been reported for
the IL-17 family. Most notably, IL-17 is involved in inducing and
mediating proinflammatory and allergic responses. IL-17 induces the
production of many other cytokines and prostaglandins from various
cell types (fibroblasts, endothelial cells, epithelial cells,
keratinocytes and macrophages). Each member of the IL-17 family has
a distinct pattern of cellular expression. The expression of IL-17A
and IL-17F appear to be restricted to a small group of activated T
cells, and upregulated during inflammation.
[0039] An N-linked glycosylation site on the protein was first
identified after purification of the protein revealed two bands,
one at 15 KDa and another at 20 KDa. Comparison of different
members of the IL-17 family revealed four conserved cysteines that
form two disulfide bonds.[5] IL-17 is unique in that it bears no
resemblance to other known interleukins. Furthermore, IL-17 bears
no resemblance to any other known proteins or structural
domains.[4]
[0040] The IL-17 receptor family consists of five, broadly
distributed receptors that present with individual ligand
specificities. Within this family of receptors, IL-17R is the best
described. IL-17R binds both IL-17A and IL-17F and is expressed in
multiple tissues: vascular endothelial cells, peripheral T cells, B
cell lineages, fibroblast, lung, myelomonocytic cells and marrow
stromal cells.
[0041] "Suitable conditions" shall have a meaning dependent on the
context in which this term is used. That is, when used in
connection with an antibody, the term shall mean conditions that
permit an antibody to bind to its corresponding antigen. When used
in connection with contacting an agent to a cell, this term shall
mean conditions that permit an agent capable of doing so to enter a
cell and perform its intended function. In one embodiment, the term
"suitable conditions" as used herein means physiological
conditions.
[0042] The term "inflammatory" response is the development of a
humoral (antibody mediated) and/or a cellular (mediated by
antigen-specific T cells or their secretion products) response. An
"immunogen" is capable of inducing an immunological response
against itself on administration to a mammal or due to autoimmune
disease.
[0043] Unless otherwise apparent from the context, all elements,
steps or features of the invention can be used in any combination
with other elements, steps or features.
[0044] General methods in molecular and cellular biochemistry can
be found in such standard textbooks as Molecular Cloning: A
Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory
Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel
et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag
et al., John Wiley & Sons 1996); Nonviral Vectors for Gene
Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors
(Kaplift & Loewy eds., Academic Press 1995); Immunology Methods
Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue
Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors,
and kits for genetic manipulation referred to in this disclosure
are available from commercial vendors such as BioRad, Stratagene,
Invitrogen, Sigma-Aldrich, and ClonTech.
[0045] The present invention has been described in terms of
particular embodiments found or proposed by the present inventor to
comprise preferred modes for the practice of the invention. It will
be appreciated by those of skill in the art that, in light of the
present disclosure, numerous modifications and changes can be made
in the particular embodiments exemplified without departing from
the intended scope of the invention. Due to biological functional
equivalency considerations, changes can be made in protein
structure without affecting the biological action in kind or
amount. All such modifications are intended to be included within
the scope of the appended claims.
[0046] The subject methods are used for prophylactic or therapeutic
purposes. As used herein, the term "treating" is used to refer to
both prevention of relapses, and treatment of pre-existing
conditions. For example, the prevention of autoimmune disease may
be accomplished by administration of the agent prior to development
of a relapse. The treatment of ongoing disease, where the treatment
stabilizes or improves the clinical symptoms of the patient, is of
particular interest.
Therapeutic Agents
[0047] In one embodiment of the invention, modulators of T cell
and/or granulocyte activity are used in the treatment of
inflammatory demyelinating disease of an IL-17 subtype, including
subtypes of MS and NO. Patients may be classified according to
cytokine subtype prior to administration of a granulocyte
inhibitor, particularly MS patients. NO patients generally have an
IL-17 type disease, and may be treated with a granulocyte inhibitor
in the absence of cytokine profiling.
[0048] In some embodiments of the invention, the therapeutic agent
is an elastase inhibitor, e.g. a small molecule inhibitor which may
include without limitation, sivelestat sodium hydrate;
alpha1-antitrypsin; pafistatin-like protease inhibitors; Clitocybin
D); marama bean inhibitor; AE-3763; Isodeoxyhelicobasidin;
guamerin; elafin; Bornyl (3,4,5-trihydroxy)-cinnamate; and the like
as known in the art. Alternatively the therapeutic agent is an
inhibitor of gro-alpha, e.g. peptides, small molecules, and the
like.
[0049] In some embodiments the therapeutic agents are antibodies
specific for a granulocyte marker, e.g. elastase, gro-alpha, etc.
The term "antibody" is used in the broadest sense and specifically
covers monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments so long as they
exhibit the desired biological activity. "Antibodies" (Abs) and
"immunoglobulins" (Igs) are glycoproteins having the same
structural characteristics. While antibodies exhibit binding
specificity to a specific antigen, immunoglobulins include both
antibodies and other antibody-like molecules which lack antigen
specificity. Polypeptides of the latter kind are, for example,
produced at low levels by the lymph system and at increased levels
by myelomas.
[0050] The method also provide for combination therapy, where the
combination may provide for additive or synergistic benefits.
Combinations of agents may be obtained with a second agent selected
from one or more of the general classes of drugs commonly used in
the non-antigen specific treatment of autoimmune disease, which
include corticosteroids and disease modifying drugs; or from an
antigen-specific agent. Corticosteroids have a short onset of
action, but many disease modifying drugs take several weeks or
months to demonstrate a clinical effect. These agents include
methotrexate, leflunomide (Arava.TM.), etanercept (Enbrel.TM.),
infliximab (Remicade.TM.), adalimumab (Humira.TM.), anakinra
(Kineret.TM.), rituximab (Rituxan.TM.), CTLA4-Ig (abatacept),
antimalarials, gold salts, sulfasalazine, d-penicillamine,
cyclosporin A, cyclophosphamide azathioprine; and the like.
[0051] Antigen specific therapeutic methods include administration
of an antigen or epitope specific therapeutic agent. One method to
induce immune tolerance is tolerizing DNA vaccines (Garren et al.
(2001) Immunity, 15:15-22; Robinson et al. (2003) Nature
Biotechnology 21:1033-9). Tolerizing DNA vaccines are DNA plasmids
containing the regulatory regions necessary for expression of the
encoded cDNA in mammalian cells, and would be engineered to contain
cDNA sequence encoding all or a portion of a targeted antigen in
order to induce immune tolerance to the encoded epitopes. To
enhance the ability of such plasmids to induce immune tolerance,
the immunostimulatory CpG sequences (Krieg et al. (1998) Trends
Microbiol. 6:23-27) can be reduced in number or completely removed
from the plasmid vector. Additionally, immunoinhibitory GpG
sequences can be added to the vector (see Ho et al. (2005) J.
Immunology, 175:6226-34). Tolerizing DNA plasmids are delivered
intramuscularly to induce immune tolerance to an antigen, thereby
reducing T cell and autoantibody responses to reduce autoimmune
destruction of the myelin sheath.
[0052] As an alternative, or in addition to DNA tolerization,
specific peptides, altered peptides, or proteins may be
administered therapeutically to induce antigen-specific tolerance
to treat autoimmunity. Native peptides targeted by the autoimmune
response can be delivered to induce antigen-specific tolerance
(Science 258:1491-4). Native peptides have been delivered
intravenously to induce immune tolerance (J Neurol Sci. 152:31-8).
Delivery of peptides that are altered from the native peptide, is
also known in the art. Alteration of native peptides with selective
changes of crucial residues (altered peptide ligands or "APL") can
induce unresponsiveness or change the responsiveness of
antigen-specific autoreactive T cells. In another embodiment, whole
protein antigens targeted by the autoimmune response can be
delivered to restore immune tolerance to treat autoimmunity
(Science 263:1139).
[0053] Active ingredients in pharmaceutical compositions formulated
for the treatment of various disorders are as described above. The
active ingredient is present in a therapeutically effective amount,
i.e., an amount sufficient when administered to substantially
modulate the effect of the targeted protein or polypeptide to treat
a disease or medical condition mediated thereby. The compositions
can also include various other agents to enhance delivery and
efficacy, e.g. to enhance delivery and stability of the active
ingredients.
[0054] Thus, for example, the compositions can also include,
depending on the formulation desired, pharmaceutically-acceptable,
non-toxic carriers or diluents, which are defined as vehicles
commonly used to formulate pharmaceutical compositions for animal
or human administration. The diluent is selected so as not to
affect the biological activity of the combination. Examples of such
diluents are distilled water, buffered water, physiological saline,
PBS, Ringer's solution, dextrose solution, and Hank's solution. In
addition, the pharmaceutical composition or formulation can include
other carriers, adjuvants, or non-toxic, nontherapeutic,
nonimmunogenic stabilizers, excipients and the like. The
compositions can also include additional substances to approximate
physiological conditions, such as pH adjusting and buffering
agents, toxicity adjusting agents, wetting agents and detergents.
The composition can also include any of a variety of stabilizing
agents, such as an antioxidant.
[0055] When the pharmaceutical composition includes a polypeptide
as the active ingredient, the polypeptide can be complexed with
various well-known compounds that enhance the in vivo stability of
the polypeptide, or otherwise enhance its pharmacological
properties (e.g., increase the half-life of the polypeptide, reduce
its toxicity, enhance solubility or uptake). Examples of such
modifications or complexing agents include sulfate, gluconate,
citrate and phosphate. The polypeptides of a composition can also
be complexed with molecules that enhance their in vivo attributes.
Such molecules include, for example, carbohydrates, polyamines,
amino acids, other peptides, ions (e.g., sodium, potassium,
calcium, magnesium, manganese), and lipids.
[0056] Further guidance regarding formulations that are suitable
for various types of administration can be found in Remington's
Pharmaceutical Sciences, Mace Publishing Company, Philadelphia,
Pa., 17th ed. (1985). For a brief review of methods for drug
delivery, see, Langer, Science 249:1527-1533 (1990).
[0057] The pharmaceutical compositions can be administered for
prophylactic and/or therapeutic treatments. Toxicity and
therapeutic efficacy of the active ingredient can be determined
according to standard pharmaceutical procedures in cell cultures
and/or experimental animals, including, for example, determining
the LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD.sub.50/ED.sub.50. Compounds that exhibit large therapeutic
indices are preferred.
[0058] The data obtained from cell culture and/or animal studies
can be used in formulating a range of dosages for humans. The
dosage of the active ingredient typically lies within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage can vary within this range depending
upon the dosage form employed and the route of administration
utilized.
[0059] The pharmaceutical compositions described herein can be
administered in a variety of different ways. Examples include
administering a composition containing a pharmaceutically
acceptable carrier via oral, intranasal, rectal, topical,
intraperitoneal, intravenous, intramuscular, subcutaneous,
subdermal, transdermal, intrathecal, or intracranial method.
[0060] For oral administration, the active ingredient can be
administered in solid dosage forms, such as capsules, tablets, and
powders, or in liquid dosage forms, such as elixirs, syrups, and
suspensions. The active component(s) can be encapsulated in gelatin
capsules together with inactive ingredients and powdered carriers,
such as glucose, lactose, sucrose, mannitol, starch, cellulose or
cellulose derivatives, magnesium stearate, stearic acid, sodium
saccharin, talcum, magnesium carbonate. Examples of additional
inactive ingredients that may be added to provide desirable color,
taste, stability, buffering capacity, dispersion or other known
desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium dioxide, and edible white ink. Similar diluents
can be used to make compressed tablets. Both tablets and capsules
can be manufactured as sustained release products to provide for
continuous release of medication over a period of hours. Compressed
tablets can be sugar coated or film coated to mask any unpleasant
taste and protect the tablet from the atmosphere, or enteric-coated
for selective disintegration in the gastrointestinal tract. Liquid
dosage forms for oral administration can contain coloring and
flavoring to increase patient acceptance.
[0061] The active ingredient, alone or in combination with other
suitable components, can be made into aerosol formulations (i.e.,
they can be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen.
[0062] Suitable formulations for rectal administration include, for
example, suppositories, which are composed of the packaged active
ingredient with a suppository base. Suitable suppository bases
include natural or synthetic triglycerides or paraffin
hydrocarbons. In addition, it is also possible to use gelatin
rectal capsules, which are composed of a combination of the
packaged active ingredient with a base, including, for example,
liquid triglycerides, polyethylene glycols, and paraffin
hydrocarbons.
[0063] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives.
[0064] The components used to formulate the pharmaceutical
compositions are preferably of high purity and are substantially
free of potentially harmful contaminants (e.g., at least National
Food (NF) grade, generally at least analytical grade, and more
typically at least pharmaceutical grade). Moreover, compositions
intended for in vivo use are preferably sterile. To the extent that
a given compound must be synthesized prior to use, the resulting
product is preferably substantially free of any potentially toxic
agents, such as any endotoxins, which may be present during the
synthesis or purification process. Compositions for parental
administration are also preferably sterile, substantially isotonic
and made under GMP conditions.
[0065] The compositions may be administered in a single dose, or in
multiple doses, usually multiple doses over a period of time, e.g.
daily, every-other day, weekly, semi-weekly, monthly etc. for a
period of time sufficient to reduce severity of the inflammatory
disease, which may comprise 1, 2, 3, 4, 6, 10, or more doses.
[0066] Determining a therapeutically or prophylactically effective
amount an agent can be done based on animal data using routine
computational methods. In one embodiment, the therapeutically or
prophylactically effective amount contains between about 0.1 mg and
about 1 g of nucleic acid or protein, as applicable. In another
embodiment, the effective amount contains between about 1 mg and
about 100 mg of protein, as applicable. In a further embodiment,
the effective amount contains between about 10 mg and about 50 mg
of the nucleic acid or protein, as applicable. The effective dose
will depend at least in part on the route of administration. The
agents may be administered orally, in an aerosol spray; by
injection, e.g. i.m., s.c., i.p., i.v., etc. In some embodiments,
administration by other than i.v. may be preferred. The dose may be
from about 0.1 .mu.g/kg patient weight; about 1 .mu.g/kg; about 10
.mu.g/kg; to about 100 .mu.g/kg.
[0067] The compositions are administered in a pharmaceutically
acceptable excipient. The term "pharmaceutically acceptable" refers
to an excipient acceptable for use in the pharmaceutical and
veterinary arts, which is not toxic or otherwise inacceptable. The
concentration of compositions of the invention in the
pharmaceutical formulations can vary widely, i.e. from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0068] Treating, treatment, or therapy of a disease or disorder
shall mean slowing, stopping or reversing the disease's progression
by administration of treatment according to the present invention.
In the preferred embodiment, treating a disease means reversing the
disease's progression, ideally to the point of eliminating the
disease itself. As used herein, ameliorating a disease and treating
a disease are equivalent. Preventing, prophylaxis or prevention of
a disease or disorder as used in the context of this invention
refers to the administration of an aBC composition to prevent the
occurrence or onset of a disease or disorder or some or all of the
symptoms of a disease or disorder or to lessen the likelihood of
the onset of a disease or disorder.
Patient Classification
[0069] Cytokines are messenger molecules produced by B cells, T
cells, macrophage, dendritic cells and other immune and host cells.
Cytokines play roles in the pathogenesis of multiple sclerosis and
other autoimmune diseases. Cytokines include chemokines,
lymphokines, growth factors, angiogenesis factors, and other
secreted and cell surface molecules that transmit signals to other
cells. Cytokines of interest for the methods of the invention
include, without limitation, IL-17F, IL-17A, IL-5, IL-8,
.beta.-IFN, .gamma.-IFN and IL-23. While serum levels of IL-17F are
found to be increased in certain MS and NO patients, in some cases
serum levels of IL-17A may be normal.
[0070] In addition, greater infiltration of granulocytes (PMN) are
found in lesions of NO and IL-17-type MS or EAE, and markers of
granulocytes such as elastase and GroA find use in disease
classification, where a patient sample, e.g. CSF, is analyzed for
the presence of increased levels of granulocytes relative to a
normal control sample are indicative of NO or IL-17-type MS or EAE;
and/or the presence of markers indicative of granulocytes, where
increased levels of granulocytes markers relative to a normal
control sample are indicative of NO or IL-17-type MS or EAE.
[0071] In one embodiment of the invention, a method is provided for
determining which immunomodulatory treatment a multiple sclerosis
patient will be non-responsive to, the method comprising
determining circulating levels of at least one cytokine in a
patient, where the cytokine(s) is indicative of the TH1/TH17 status
of the patient. A patient having high levels of cytokines
indicative of a TH17 subtype, e.g. IL-17F, IL-5, IL-8, IL-23,
.beta.-IFN, etc. or granulocytes or markers thereof, or low levels
of a cytokine indicative of a TH1-subtype, e.g. IL-7 relative to a
non-diseased individual or a patient with a known responder
phenotype is classified as non-responder to TH1 subtype
immunotherapy. Such non-responder patients may be treated with
inhibitors of granulocyte function, e.g. elastase inhibitors,
gro-alpha inhibitors, and the like.
[0072] A variety of different assays can be utilized to quantitate
the presence of such markers. Many such methods are known to one of
skill in the art, including ELISA, protein arrays, eTag system,
bead based systems, tag or other array based systems etc. Examples
of such methods are set forth in the art, including, inter alia,
chip-based capillary electrophoresis: Colyer et al. (1997) J
Chromatogr A. 781(1-2):271-6; mass spectroscopy: Petricoin et al.
(2002) Lancet 359: 572-77; eTag systems: Chan-Hui et al. (2004)
Clinical Immunology 111:162-174; microparticle-enhanced
nephelometric immunoassay: Montagne et al. (1992) Eur J Clin Chem
Clin Biochem. 30(4):217-22; antigen arrays: Robinson et al. (2002)
Nature Medicine, 8:295-301; the Luminex XMAP bead array system
(www.luminexcorp.com); and the like, each of which are herein
incorporated by reference. Detection may utilize one or a panel of
specific binding members, e.g. specific for one, two, three, four,
five or more cytokines.
[0073] The signature pattern may be generated from a biological
sample using any convenient protocol, for example as described
below. The readout may be a mean, average, median or the variance
or other statistically or mathematically-derived value associated
with the measurement. The cytokine readout information may be
further refined by direct comparison with the corresponding
reference or control pattern. A binding pattern may be evaluated on
a number of points: to determine if there is a statistically
significant change at any point in the data matrix; whether the
change is an increase or decrease in the binding; whether the
change is specific for one or more physiological states, and the
like. The absolute values obtained for each cytokine under
identical conditions will display a variability that is inherent in
live biological systems and also reflects the variability inherent
between individuals.
[0074] Following obtainment of the signature pattern from the
sample being assayed, the signature pattern is compared with a
reference or control profile to make a prognosis regarding the
phenotype of the patient from which the sample was
obtained/derived. Typically a comparison is made with a sample or
set of samples from an unaffected, normal source. Additionally, a
reference or control signature pattern may be a signature pattern
that is obtained from a sample of a patient known to be responsive
or non-responsive to the therapy of interest, and therefore may be
a positive reference or control profile.
[0075] In certain embodiments, the obtained signature pattern is
compared to a single reference/control profile to obtain
information regarding the phenotype of the patient being assayed.
In yet other embodiments, the obtained signature pattern is
compared to two or more different reference/control profiles to
obtain more in depth information regarding the phenotype of the
patient. For example, the obtained signature pattern may be
compared to a positive and negative reference profile to obtain
confirmed information regarding whether the patient has the
phenotype of interest.
[0076] Samples can be obtained from the tissues or fluids of an
individual. For example, samples can be obtained from whole blood,
tissue biopsy, serum, etc. Other sources of samples are body fluids
such as lymph, cerebrospinal fluid, and the like. Also included in
the term are derivatives and fractions of such cells and fluids.
Diagnostic samples are collected any time after an individual is
suspected to have an autoimmune disease or has exhibited symptoms
that predict such a disease.
[0077] Various immunoassays designed to quantitate cytokines may be
used in screening. Measuring the concentration of the target
protein in a sample or fraction thereof may be accomplished by a
variety of specific assays. For example, a conventional sandwich
type assay may be used in an array, ELISA, RIA, etc. format.
[0078] Other immunoassays are known in the art and may find use as
diagnostics. Ouchterlony plates provide a simple determination of
antibody binding. Western blots may be performed on protein gels or
protein spots on filters, using a detection system specific for the
cytokines as desired, conveniently using a labeling method.
[0079] For multiplex analysis of cytokines, arrays containing one
or more anti-cytokine affinity reagents, e.g. antibodies can be
generated. Such an array may be constructed comprising antibodies
against cytokines.
[0080] Arrays provide a high throughput technique that can assay a
large number of polypeptides in a sample. Arrays can be created by
spotting a probe onto a substrate (e.g., glass, nitrocellulose,
etc.) in a two-dimensional matrix or array having bound probes. The
probes can be bound to the substrate by either covalent bonds or by
non-specific interactions, such as hydrophobic interactions.
Techniques for constructing arrays and methods of using these
arrays are described in, for example, Schena et al. (1996) Proc
Natl Acad Sci USA. 93(20):10614-9; Schena et al. (1995) Science
270(5235):467-70; Shalon et al. (1996) Genome Res. 6(7):639-45,
U.S. Pat. No. 5,807,522, EP 799 897; WO 97/29212; WO 97/27317; EP
785 280; WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No.
5,578,832; EP 728 520; U.S. Pat. No. 5,599,695; EP 721 016; U.S.
Pat. No. 5,556,752; WO 95/22058; and U.S. Pat. No. 5,631,734.
[0081] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, and reagents described, as such may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0082] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. All technical and scientific terms used herein have the
same meaning as commonly understood to one of ordinary skill in the
art to which this invention belongs unless clearly indicated
otherwise.
[0083] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
[0084] 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.
EXPERIMENTAL
[0085] Cytokine Profiles Before Treatment of RRMS patients
responding to IFN-.beta. versus non-responders. We analyzed the
pre-treatment levels of 26 cytokines/chemokines in the serum of
RRMS patients, 12 were classified as responders and 14 as
non-responders to IFN-.beta. treatment. The median relapse rate in
the two years following initiation of IFN-.beta. treatment in the
non-responders was 2, while it was 0 in the responder population.
Likewise, the median number of steroid interventions was 2 in the
non-responders and 0 in the responders. Cluster analysis of the
cytokine profiles grouped 5 non-responders together. This group of
non-responders had significantly elevated serum concentrations of
both IL-17F and IFN-.beta. compared to the responders. Furthermore,
we found there was a significant correlation with IL-17F and
IFN-.beta. levels found in the serum of responders, non-responders
and healthy individuals (FIG. 1a). This strong correlation was not
found in comparing IL-17F or IFN-.beta. to MIP1.beta. or other
analytes.
[0086] We analyzed cytokine and chemokine levels in serum from RRMS
patients prior to the initiation of IFN-.beta. treatment. After
treatment began, disease course was monitored for two years or
longer and the patients were classified as responders and
non-responders. All non-responders had relapses during first 2
years of IFN-.beta. treatment where the responders had none. During
relapses, the non-responders received steroids therapy which has
powerful immune suppressive activity that would attenuate disease
progression in these patients. Therefore, it is quite reasonable to
describe the clinical course of the non-responders as exacerbated
compared to the responders. Strikingly, we found a subset of
non-responders that have high serum levels of the TH17 cytokine,
IL-17F. IL-17F has been shown to be produced by TH17 cells in EAE
suggesting that this group of MS patient is skewed towards a TH17
disease. Furthermore, these patients have high levels of endogenous
IFN-.beta. in their serum compared to the responders. These data
show that there is striking correlation in the concentration IL-17F
and IFN-.beta. in serum, demonstrating that these two cytokines are
associated biologically.
[0087] Without limiting the invention, it may be hypothesized that
IFN-.beta. is pro-inflammatory during TH17 biased disease.
Therefore, not only would IFN-.beta. treatment be ineffective, it
could worsen symptoms. This is supported by observations in EAE,
where symptoms were worsened by IFN-.beta. treatment in TH17
induced EAE, and in RRMS, where patients with high IL-17F have
exacerbated disease.
[0088] In addition, neuromyelitis optica (NMO), another
demyelinating disorder closely related to MS, also provides
evidence for this hypothesis. IFN-.beta. treatment of individuals
with NMO induces severe relapses. The main cellular component of
NMO lesions are granulocytes and the function of a TH17 response is
to mobilize and attract granulocytes from the bone marrow to the
site of inflammation. It has been shown that there are high levels
of IL-17 found in the CSF of patients with NMO. The disease
processes of NMO and the group of non-responders with high levels
of IL-17F are likely similar.
[0089] Manipulating the effects of cytokines has been a popular and
at times effective strategy for the development of treatments for
MS. However, our data demonstrate that IFN-.beta. is a double-edged
sword. In the context of a TH1 response with high IFN-.gamma.
levels, IFN-.beta. is anti-inflammatory and is effective in
attenuating disease. However, in the context of a TH17 response,
with high levels of IL-17A/F, IFN-.beta. is pro-inflammatory and
IFN-.beta. can exacerbate disease.
[0090] A direct result of this work is the utility of a test for
IL-17F prior to embarking with therapy using IFN-.beta.. This will
reduce the morbidity of IFN-.beta., as it would preclude those
whose clinical symptoms are likely to worsen when it is used. And
it would select those patients who would benefit from the drug. In
an contemporary environment where economic impacts of medical
decision making is more and more important, the virtues of
excluding certain patients has further urgency. And here the
exclusion would save not only the unnecessary expenditure of funds,
it would also save individuals from undesired complications. IL-17F
is thus one of the first biomarkers that could govern whether or
not a high priced recombinant drug is used in the clinic.
Materials and Methods
[0091] MS Patients Clinical Classification and Serum Collection.
Twenty-six closely monitored RRMS patients receiving IFN.beta.
treatment for at least 12 months were identified from the
outpatient clinic as responders or non-responders to IFN.beta.
therapy at the MS Center Amsterdam, the Netherlands. The patients
were classified based on EDSS (Extended Disability Status Scale)
progression and the number of relapses and steroid interventions (3
days of 1000 mg/day i.v. methylprednisolone) in the two years
before initiation of treatment as compared to the first two years
after starting treatment (see Table 1). Two MS neurologists,
blinded to the laboratory data, independently classified the
selected patients as responder or non-responder. In case of
disagreement there was a consensus meeting afterwards. Serum
samples were obtained at a fixed time of the day just before
starting IFN.beta. therapy. The study design received approval from
the institutional Medical Ethics Board of the VU University Medical
Center, Amsterdam, the Netherlands. All patients signed written
informed consent.
[0092] ELISA and Multiplex Analysis of Human cytokines.
Supernatants from TH differentiation cultures were assayed for
IL-17, IL-10 and IFN-.gamma. by ELISA (Ebioscience). Analysis of
cytokines in the sera from MS patients and healthy controls was
performed by multiplex bead analysis (Panomics) according to the
protocol recommended by the manufacturer. Multiplex results were
analysed using Gene Cluster software to identify features with
significant differences in antibody reactivity and the patient
samples were ordered using a hierarchical clustering algorithm and
the results presented as a heat map using TreeView software.
[0093] Statistical analysis. EAE data are presented as means.+-.SEM
and statistical significance was determined using a two tailed
Mann-Whitney test with a value of P<0.05 was considered
significant. STAT1 activation and ELISA data are presented as
means.+-.1 standard deviation and statistics significance was
determined using a two tailed student T-test.
TABLE-US-00001 TABLE 1 Demographic and clinical characteristics of
patients with relapsing remitting multiple sclerosis and their
clinical response to IFN.beta. therapy Responder Non-responder
Number 12 14 Female/Male (n) 10/2 11/3 Median age at onset (yr)
27.6 [24.5; 35.8] 26.7 [19.3; 36.0] Median age at start 33.5 [30.3;
39.5] 33.0 [23.0; 37.8] IFN.beta. (yr) Median EDSS score 2.5 [2.0;
3.5] 2.5 [1.8; 4.3] around start IFN.beta. Relapse rate in 2 2
[2-3] 2 [1-3] yrs before start IFN.beta. Relapse rate in 2 0 [0; 0]
2 [1.5; 2.0] yrs after start IFN.beta. Steroid interventions 0 [0;
2] 1 [0; 3] before start IFN.beta. (n) Steroid interventions 0 [0;
0.5] 2 [1; 3] after start IFN.beta. (n) Duration of IFN.beta. 80
[46; 141] 56 [38; 104] treatment (mnths) Avonex 4 5 Rebif 2 8
Betaferon 6 1 Median values are shown with 25 and 75 percentiles.
IFNb = Interferon-beta. EDSS = Expanded Disability Status Scale
Example 2
Increased Granulocytes are Indicative of NMO and TH17 EAE
[0094] As shown in FIG. 4A, markers indicative of granulocytes are
found at increased levels in NMO and TH17EAE. In plasma, increased
levels of neutrophil elastase is found in patients with NMO (FIG.
4B).
[0095] Remarkably, an elastase inhibitor attenuated TH17 EAE, shown
in FIG. 4C, indicating the utility of this treatment for
.beta.-interferon resistant MS and NMO.
* * * * *