U.S. patent application number 13/960441 was filed with the patent office on 2013-12-05 for use of il-23 and il-17 antagonists to treat autoimmune ocular inflammatory disease.
This patent application is currently assigned to The Government of the United States of America as represented by the Secretary of the Dep. of H.H.S.. The applicant listed for this patent is The Government of the United States of America as represented by the Secretary of the Dep. of H.H.S., Merck Sharp & Dohme Corp., The Government of the United States of America as represented by the Secretary of the Dep. of H.H.S.. Invention is credited to Rachel Caspi, Daniel J. Cua, Robert A. Kastelein, Dror Luger, Phyllis Silver, Van T. Tsai.
Application Number | 20130323251 13/960441 |
Document ID | / |
Family ID | 37686002 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323251 |
Kind Code |
A1 |
Cua; Daniel J. ; et
al. |
December 5, 2013 |
USE OF IL-23 AND IL-17 ANTAGONISTS TO TREAT AUTOIMMUNE OCULAR
INFLAMMATORY DISEASE
Abstract
Novel methods and drug products for treating autoimmune ocular
inflammatory disease are disclosed, which involve administration of
agents that antagonize one or both of IL-17 and IL-23 activity.
Inventors: |
Cua; Daniel J.; (Boulder
Creek, CA) ; Kastelein; Robert A.; (Redwood City,
CA) ; Tsai; Van T.; (San Diego, CA) ; Caspi;
Rachel; (Bethesda, MD) ; Silver; Phyllis;
(Silver Spring, MD) ; Luger; Dror; (Rockville,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America as represented by
the Secretary of the Dep. of H.H.S.
Merck Sharp & Dohme Corp. |
Rockville
Rahway |
MD
NJ |
US
US |
|
|
Assignee: |
The Government of the United States
of America as represented by the Secretary of the Dep. of
H.H.S.
Rockville
MD
Merck Sharp & Dohme Corp.
Rahway
NJ
|
Family ID: |
37686002 |
Appl. No.: |
13/960441 |
Filed: |
August 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12643152 |
Dec 21, 2009 |
8524230 |
|
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13960441 |
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11512622 |
Aug 30, 2006 |
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12643152 |
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60713792 |
Sep 1, 2005 |
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60837312 |
Aug 11, 2006 |
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Current U.S.
Class: |
424/136.1 ;
424/145.1 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 16/244 20130101; C07K 2317/76 20130101; A61P 37/06 20180101;
A61P 29/00 20180101; A61P 43/00 20180101; A61P 27/02 20180101; A61P
31/06 20180101; A61K 39/3955 20130101; A61P 1/02 20180101; A61K
2039/505 20130101; A61P 19/02 20180101; A61P 27/00 20180101 |
Class at
Publication: |
424/136.1 ;
424/145.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made in part with Government support
under Cooperative Research and Development Agreement (CRADA) Number
M-01969-04, and amendments thereto, executed between
Schering-Plough Biopharma and the National Eye Institute, National
Institutes of Health. The Government of the United States of
America has certain rights in this invention.
Claims
1. A method of treating a patient with an autoimmune ocular
inflammatory disease (AOID), comprising administering to the
patient an IL-17 antagonist, wherein the AOID is uveitis and the
IL-17 antagonist is a monoclonal antibody or a monoclonal antibody
fragment that binds to and inhibits the activity of IL-17.
2. The method of claim 1, wherein the patient has been diagnosed as
having an ocular inflammation of putative autoimmune etiology.
3. The method of claim 1, wherein a specified dose of the IL-17
antagonist is administered at a specified interval during a first
treatment period.
4. The method of claim 3, wherein the first treatment period ends
after disappearance of one or more symptoms of the AOID.
5. The method of claim 4, wherein the first treatment period ends
within 30 days after disappearance of all symptoms of the AOID.
6. The method of claim 4, wherein the dose of the IL-17 antagonist
administered is gradually reduced during a second treatment period
that begins upon the end of the first treatment period.
7. The method of claim 6, wherein the duration of the second
treatment period is at least one year.
8. The method of claim 3, further comprising administering an IL-23
antagonist to the patient during the first treatment period,
wherein the IL-23 antagonist is a monoclonal antibody or a
monoclonal antibody fragment that binds to and inhibits the
activity of IL-23p19.
9. The method of claim 8, wherein a specified dose of the IL-23
antagonist is administered at a specified interval during the first
treatment period.
10. The method of claim 9, wherein the first treatment period ends
after disappearance of one or more symptoms of the AOID.
11. The method of claim 9, wherein the first treatment period ends
within 30 days after disappearance of all symptoms of the AOID.
12. The method of claim 10, wherein the dose of each of the IL-17
antagonist and the IL-23 antagonist is gradually reduced during a
second treatment period that begins upon the end of the first
treatment period.
13. The method of claim 10, wherein the dose of the IL-17
antagonist is gradually reduced during a second treatment period
that begins upon the end of the first treatment period, and wherein
the dose of the IL-23 antagonist administered during the second
treatment period is the same as the dose administered in the first
treatment period, and wherein the second treatment period ends when
therapy with the IL-17 antagonist is stopped.
14. The method of claim 13, wherein the duration of the second
treatment period is between one month and three months.
15. The method of claim 13, further comprising administering the
IL-23 antagonist during a third treatment period that begins upon
the end of the second treatment period.
16. The method of claim 15, wherein the duration of the third
treatment period is between six months and twelve months.
17. The method of claim 15, wherein the dose of the IL-23
antagonist is gradually reduced during the third treatment
period.
18. A method of prophylactically treating a patient who is
diagnosed as being susceptible for an autoimmune ocular
inflammatory disease (AOID), the method comprising administering to
the patient an antagonist selected from the group consisting of an
IL-23 antagonist, an IL-17 antagonist and an antagonist of both
IL-17 and IL-23, wherein the AOID is uveitis and the antagonist is
a monoclonal antibody or a monoclonal antibody fragment.
19. The method of claim 18, wherein the susceptibility diagnosis is
based on the patient having a previous incident of ocular
inflammation.
20. The method of claim 18, wherein the susceptibility diagnosis is
based on the patient having a systemic autoimmune disease.
21. The method of claim 18, wherein the monoclonal antibody or
monoclonal antibody fragment binds to and inhibits the activity of
IL-23p19.
22. The method of claim 18, wherein the monoclonal antibody or
monoclonal antibody fragment binds to and inhibits the activity of
IL-17.
23. The method of claim 18, wherein a specified dose of the
antagonist is administered at a specified interval during a first
treatment period.
24. The method of claim 23, wherein the duration of the first
treatment period is between three months and two years.
25. The method of claim 24, wherein the duration of the first
treatment period is between six months and one year.
26. The method of claim 23, wherein the dose of the antagonist is
gradually reduced during a second treatment period that begins upon
the end of the first treatment period.
27. The method of claim 26, wherein the duration of the second
treatment period is between one month and six months.
28. A method of treating a patient for an autoimmune ocular
inflammatory disease (AOID), comprising administering to the
patient an IL-23 antagonist, wherein the AOID is uveitis and the
IL-23 antagonist is a monoclonal antibody or a monoclonal antibody
fragment that binds to and inhibits the activity of IL-23p19.
29. The method of claim 28, wherein a specified dose of the IL-23
antagonist is administered at a specified interval during a first
treatment period.
30. The method of claim 29, wherein the duration of the first
treatment period is between three months and two years.
31. The method of claim 30, wherein the duration of the first
treatment period is between six months and one year.
32. The method of claim 28, wherein the dose of the IL-23
antagonist is gradually reduced during a second treatment period
that begins upon the end of the first treatment period.
Description
[0001] The present application is a divisional of Ser. No.
12/643,152, filed Dec. 21, 2009, which is a divisional of Ser. No.
11/512,622, filed Aug. 30, 2006, now abandoned, which claims
benefit of U.S. provisional application No. 60/713,792, filed Sep.
1, 2005 and U.S. provisional application No. 60/837,312, filed Aug.
11, 2006.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the modulation of
immune responses in the eye. More specifically, the invention
relates to the use of antagonists of interleukin-23 (IL-23) and
interleukin-17 (IL-17) to treat autoimmune ocular inflammatory
disease.
BACKGROUND OF THE INVENTION
[0004] Ocular inflammatory disease (OID) is a general term
embracing a number of diseases and conditions in which inflammation
affects the eye or surrounding tissues. The diagnostic name given
to an OID is typically based on the location of the ocular
inflammation. For example, uveitis is inflammation in the uveal
tract; scleritis is inflammation of the sclera, pars planitis is
inflammation of the pars plana, and so forth. OIDs cause pain,
irritation, and watering, and may result in loss of visual
function. For example, uveitis is the third leading cause of
blindness in the developed world. OIDs can be caused by infections,
malignancy, exposure to toxins, response to surgery or injury, and
autoimmune disorders.
[0005] A number of autoimmune diseases exist in which the eye or
various parts of the eye becomes a target for an immune-mediated
inflammatory attack. Patients with an autoimmune-mediated OID
(AOID) often exhibit cellular and humoral responses to retinal
antigens such as retinal arrestin (retinal soluble antigen, S-Ag),
interphotoreceptor retinoid binding protein (IRB), and antigens
related to melanin and its metabolism, including GP100, MARTI, TRP1
and TRP2 (Pennesi, G. et al. (2003) J. Clin. Invest. 111:1171-1180;
Gocho, K. et al. (2001) Invest. Ophthalmol. Vis. Sci. 42:2004-2009;
Sugita S. et al., (1996) Int. Immunol. 8:799-803; Yamake, K. et al.
(2000) J. Immunol. 165: 7323-7329. However, in many cases of AOID,
the target antigen(s) are not known.
[0006] Often, OID is a manifestation of a systemic autoimmune
disease, and the eye is one of a variety of organs throughout the
body that are being attacked. Examples of such systemic autoimmune
diseases include rheumatoid arthritis, systemic lupus
erythematosus, polyarteritis nodosa, relapsing polychondritis,
Wegener's granulomatosis, scleroderma, Behcet's disease, Reiter's
disease, inflammatory bowel disease (ulcerative colitis and Crohn's
disease) and ankylosing spondylitis. However, the eye may be the
specific and only target affected in autoimmune diseases such as
ocular cicatricial pemphigoid, Mooren's corneal ulcer, and various
forms of uveitis.
[0007] AOIDs such as uveitis have been treated by various classes
of compounds including steroids and nonsteroidal anti-inflammatory
agents such as dexamethasone, fluorometholone, prednisolone,
indomethacin, aspirin, flubiprofen and diclofenac. However, a
number of uveitis cases are not responsive to or become refractory
to these drugs (see, e.g., Kulkarni, P. (2001) Journal of Ocular
Pharmacology And Therapeutics 17:181-187). Also, these drugs are
associated with serious side effects such as cataracts, glaucoma,
delayed wound healing, altered prostaglandin production, corneal
complications, increased ocular pressure, superinfections, and
reduced immunity to infection (see, e.g., Id., at 181; Guidera, A.
C., et al. (2001) Ophthalmology 108:936-944; Olsen, E. G. &
Davanger M. (1984) Acta Ophtalmol. 62:893-899).
[0008] Because the existing therapies for AOID have less than
optimal efficacy or undesirable side effects, new treatment
regimens are needed. It has been suggested that it may be
clinically beneficial to modulate the immunoregulatory mechanisms
involved in the pathogenesis of AOID (Caspi, R. R. (2002) Int Rev
Immunol 21:197-208).
[0009] These pathogenic mechanisms have been investigated using
experimental autoimmune uveitis (EAU), which is an animal model of
human autoimmune uveitis. EAU is induced in experimental animals
such as mouse, rat, guinea pig, rabbit, and monkey by immunization
with a retinal antigen shown to be reactive in uveitis patients
(e.g., arrestin, IRBP, rhodopsin/opsin, phosducin, recoverin) or by
infusion of T cells specific for these antigens. Studies using the
EAU model provided apparently contradictory evidence about the
mechanisms for induction and progression of this disease. The
results of some experiments indicated that the main pathogenic
pathway in EAU was due to the role of interleukin-12 (IL-12) in
promoting the generation of IFN-.gamma. producing Th1 effector
cells (Caspi, R. R. (2002) Int Rev Immunol 21:197-208; Tarrant, T.
K. et al., (1998) J. Immunol. 161:122-127; Caspi, R. R. (1998) Clin
Immunol Immunopathol 88:4-13; Xu, H. et al. (1997) Cell Immunol
178:69-78. However other experiments showed that IFN-.gamma.
deficient knock-out mice were susceptible for EAU, that EAU is
exacerbated by neutralization of endogenous IFN-.gamma., and that
elevated levels of IFN-.gamma. were protective against EAU in
wild-type mice (Caspi, R. R. et al. (1994) J. Immunol. 152:890-899;
Jones e tal., J. Immunol. 158:5997-6005; Tarrant, T. K., et al.
(1999) J. Exp. Med. 189:219-230.
[0010] Thus, prior to the present invention, it was not clear which
immune pathways should be targeted in developing therapies for
preventing or treating autoimmune ocular inflammatory disease.
SUMMARY OF THE INVENTION
[0011] The present invention is based on the discoveries that (1)
blocking interleukin-23 (IL-23) or interleukin-17 (IL-17) activity
prevents induction of EAU; (2) after induction, neutralization of
IL-17 activity inhibits or reverses progression of EAU, but
neutralization of IL-23 activity has little to no effect; and (3)
IL-17 activity is not necessary for induction of EAU. The present
invention uses IL-23 and/or IL-17 antagonists in methods and
compositions for treating or preventing autoimmune ocular
inflammatory disease. These antagonists antagonize either the
target cytokine itself or a functional receptor for the target
cytokine.
[0012] IL-23 is a heterodimeric cytokine comprised of two subunits:
p19, which is unique to IL-23; and p40, which is shared with IL-12.
IL-23 mediates signaling by binding to a heterodimeric receptor,
comprised of IL-23R and IL-12Rbeta1 (IL12RB1), which is shared by
the IL-12 receptor. A recent paper reported that IL-23 promotes a T
cell population characterized by the production of IL-17, IL-17F,
TNF, IL-6 and other factors, and named these cells "Th.sub.17
cells" (Langrish et al. (2005) J. Exp. Med. 201:233-240)).
[0013] IL-17, which was originally named cytotoxic
T-Lymphocyte-associated antigen 8 (CTLA8) is a homodimeric cytokine
that binds to IL-17RA (also known as IL17R) and IL-17C. The
functional receptor for IL-17 is believed to be a multimeric
receptor complex comprising one or both of IL-17RA and IL-17RC
(e.g., an IL-17RA homodimer, an IL-17RC homodimer, or an
IL-17RA/IL-17RC heterodimer) and possibly a third, as yet unknown,
protein (Toy, D. et al., (2006) J. of Immunol. 177(1):36-39;
unpublished data).
[0014] In one aspect, the invention provides a method of treating a
patient with an autoimmune ocular inflammatory disease, comprising
administering to the patient an IL-17 antagonist. The presence of
an AOID need not be directly diagnosed, but may be inferred by a
diagnosis that the patient has an ocular inflammation that is of
putative autoimmune etiology and/or that exhibits one or more
characteristics of an autoimmune response. A particularly preferred
AOID is autoimmune uveitis, e.g., uveitis without an infectious
etiology.
[0015] The IL-17 antagonist may inhibit the expression of IL-17 or
IL-17R or IL-17RC or may inhibit IL-17 signaling by directly or
indirectly interacting with one or more of these polypeptides to
prevent a functional ligand-receptor interaction. In some preferred
embodiments, the IL-17 antagonist is an antibody or antibody
fragment that binds to and inhibits the activity of either IL-17,
IL17R or IL17C. In one particularly preferred embodiment, the IL-17
antagonist is a monoclonal antibody that specifically binds to
IL-17. In other preferred embodiments, the IL-17 antagonist is a
bispecific antibody that binds to and inhibits the activity of
IL-23p19 and IL-17; IL-23p19 and IL-17RA; IL-23R and IL-17; or
IL-23R and IL-17RA. In another particularly preferred embodiment,
the IL-17 antagonist is a bispecific antibody that binds to and
inhibits the activity of IL-23p19 and IL-17.
[0016] In some embodiments, the IL-17 antagonist is administered
according to a specified treatment regimen. For example, in one
embodiment, a specified dose of the antagonist is administered at a
specified interval during a first treatment period, which may end
after disappearance of one or more symptoms of the AOID, or within
a specified period of time. In a preferred embodiment, the
treatment regimen further comprises gradually reducing the dose of
the IL-17 antagonist during a second treatment period that begins
upon the end of the first treatment period and ends when therapy
with the IL-17 antagonist is stopped. The duration of the second
treatment period is typically between one and twelve months, one
and nine months, one and six months, or one and three months.
[0017] In some preferred embodiments, the specified treatment
regimen also comprises administration of an IL-23 antagonist to the
patient during each of the first and second treatment periods, or
during only the second treatment period. The IL-23 antagonist may
inhibit the expression of either subunit of the cytokine (IL-23p19
or p40), either subunit of the functional receptor (IL-23R or
IL-12beta1), or may inhibit IL-23 signaling by directly or
indirectly interacting with one or more of these polypeptides to
prevent a functional ligand-receptor interaction. In some preferred
embodiments, the IL-23 antagonist is an antibody or antibody
fragment that binds to and inhibits the activity of either IL-23p19
or IL-23R. In one particularly preferred embodiment, the IL-23
antagonist is a monoclonal antibody that specifically binds to
IL-23p19.
[0018] The IL-23 antagonist may be administered at a specified dose
at a specified interval during one or both of the first and second
treatment periods. The dose of the IL-23 antagonist administered in
the second treatment period may be lower than the dose administered
in the first period. Also, in any or both of the treatment periods,
the doses of the IL-17 and IL-23 antagonists may be the same or
different from each other. Similarly, the two antagonists may be
administered at the same or different intervals during each
treatment period. During the second treatment period, the dose of
the IL-17 antagonist may be reduced while the dose of the IL-23
antagonist is held constant, or the dose of each antagonist may be
gradually reduced.
[0019] In other preferred embodiments, the dose of the IL-23
antagonist is held constant during the second treatment regimen and
therapy with the IL-23 antagonist is continued during a third
treatment period that begins upon the end of the second treatment
period (i.e., when therapy with the IL-17 antagonist is stopped).
During the third treatment period, the IL-23 antagonist may be
administered at the same dose and interval as in the second
treatment period or may be administered at a lower dose and/or less
frequent interval than used in the previous period. The dose of the
IL-23 antagonist may also be gradually reduced during the third
treatment period. The duration of the third treatment period is
typically between one and twelve months, one and nine months, one
and six months, or one and three months.
[0020] In still other embodiments, the specified treatment regimen
also comprises administering a therapeutic agent that does not
antagonize IL-17 or IL-23 activity but is capable of alleviating at
least one symptom of the AOID or at least one side effect of the
IL-17 or IL-23 antagonists during any or all of the treatment
periods. In some preferred embodiments, the therapeutic agent is a
steroid or a nonsteroidal anti-inflammatory agent (e.g., NSAID)
that is known to have efficacy in treating uveitis. In other
preferred embodiments, the therapeutic agent targets a cytokine
that promotes the Th1 response.
[0021] Another aspect of the invention provides a method of
prophylactically treating a patient who is diagnosed as being
susceptible for an autoimmune ocular inflammatory disease, which
comprises administering to the patient an antagonist of one or both
of IL-23 and IL-17. In some preferred embodiments of this
prophylactic method, the susceptibility diagnosis is based on the
patient having a previous incidence of ocular inflammation. In
other preferred embodiments, the susceptibility diagnosis is based
on the patient having a systemic autoimmune disease. The antagonist
may be administered in a specified dose at a specified interval
during a first treatment period, which typically ends after three
months, six months, nine months or after two years of therapy with
the antagonist. In some preferred embodiments, the dose of the
antagonist is gradually reduced during a second treatment period
that begins upon the end of the first treatment period, and
typically has a duration of between one and three months.
[0022] In a still further aspect, the invention provides a method
of treating a patient for an autoimmune ocular inflammatory
disease, comprising administering to the patient an IL-23
antagonist. The IL-23 antagonist may be administered at a specified
interval during a first treatment period, which is followed by a
second treatment period in which the IL-23 antagonist is
administered at a lower dose or at less frequent intervals, or at
gradually reduced doses. Therapy with the Il-23 antagonist will
typically continue for at least three to six months and may
continue for as many as 12 months, 18 months or 24 months.
[0023] Another aspect of the invention is the use of an IL-17
antagonist or an IL-23 antagonist for the preparation of a
pharmaceutical composition for the treatment or prevention of an
autoimmune ocular inflammatory disease (AOID) in a patient. In
preferred embodiments, the pharmaceutical composition is for
administering the antagonist according to any of the treatment
regimens described herein.
[0024] In a still further aspect, the invention provides a
manufactured drug product for treating an autoimmune ocular
inflammatory disease. The drug product comprises (i) a first
pharmaceutical formulation comprising an IL-17 antagonist; and (ii)
a second pharmaceutical formulation comprising an IL-23 antagonist.
In preferred embodiments, the drug product includes product
information which comprises instructions for administering the
pharmaceutical formulations according to any of the treatment
regimens described herein.
DETAILED DESCRIPTION
I. Definitions
[0025] So that the invention may be more readily understood,
certain technical and scientific terms are specifically defined
below. Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
that would be commonly understood by one of ordinary skill in the
art to which this invention belongs when used in similar contexts
as used herein.
[0026] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise.
[0027] "Antagonist" means any molecule that can prevent,
neutralize, inhibit or reduce a targeted activity, i.e., the
activity of a cytokine such as IL-17 or IL-23, either in vitro or
in vivo. Cytokine antagonists include, but are not limited to,
antagonistic antibodies, peptides, peptide-mimetics, polypeptides,
and small molecules that bind to a cytokine (or any of its
subunits) or its functional receptor (or any of its subunits) in a
manner that interferes with cytokine signal transduction and
downstream activity. Examples of peptide and polypeptide
antagonists include truncated versions or fragments of the cytokine
receptor (e.g., soluble extracellular domains) that bind to the
cytokine in a manner that either reduces the amount of cytokine
available to bind to its functional receptor or otherwise prevents
the cytokine from binding to its functional receptor. Antagonists
also include molecules that prevent expression of any subunit that
comprises the cytokine or its receptor, such as, for example,
antisense oligonucleotides which target mRNA, and interfering
messenger RNA, (see, e.g., Arenz and Schepers (2003)
Naturwissenschaften 90:345-359; Sazani and Kole (2003) J. Clin.
Invest. 112:481-486; Pirollo, et al. (2003) Pharmacol. Therapeutics
99:55-77; Wang, et al. (2003) Antisense Nucl. Acid Drug Devel.
13:169-189). The inhibitory effect of an antagonist can be measured
by routine techniques. For example, to assess the inhibitory effect
on cytokine-induced activity, human cells expressing a functional
receptor for a cytokine are treated with the cytokine and the
expression of genes known to be activated or inhibited by that
cytokine is measured in the presence or absence of a potential
antagonist. Antagonists useful in the present invention inhibit the
targeted activity by at least 25%, preferably by at least 50%, more
preferably by at least 75%, and most preferably by at least 90%,
when compared to a suitable control.
[0028] "Antibody" refers to any form of antibody that exhibits the
desired biological activity, such as inhibiting binding of a ligand
to its receptor, or by inhibiting ligand-induced signaling of a
receptor. Thus, "antibody" is used in the broadest sense and
specifically covers, but is not limited to, monoclonal antibodies
(including full length monoclonal antibodies), polyclonal
antibodies, and multispecific antibodies (e.g., bispecific
antibodies).
[0029] "Antibody fragment" and "antibody binding fragment" mean
antigen-binding fragments and analogues of an antibody, typically
including at least a portion of the antigen binding or variable
regions (e.g. one or more CDRs) of the parental antibody. An
antibody fragment retains at least some of the binding specificity
of the parental antibody. Typically, an antibody fragment retains
at least 10% of the parental binding activity when that activity is
expressed on a molar basis. Preferably, an antibody fragment
retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of
the parental antibody's binding affinity for the target. Examples
of antibody fragments include, but are not limited to, Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules, e.g., sc-Fv; and multispecific
antibodies formed from antibody fragments. Engineered antibody
variants are reviewed in Holliger and Hudson (2005) Nat.
Biotechnol. 23:1126-1136.
[0030] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0031] An "Fc" region contains two heavy chain fragments comprising
the C.sub.H1 and C.sub.R2 domains of an antibody. The two heavy
chain fragments are held together by two or more disulfide bonds
and by hydrophobic interactions of the CH3 domains.
[0032] A "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H.sup.2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
[0033] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H.sup.2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0034] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0035] A "single-chain Fv antibody (or "scFv antibody") refers to
antibody fragments comprising the V.sub.H and V.sub.L domains of an
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF
MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds.
Springer-Verlag, New York, pp. 269-315. See also, International
Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos.
4,946,778 and 5,260,203.
[0036] A "diabody" is a small antibody fragment with two
antigen-binding sites. The fragments comprises a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or
V.sub.L-V.sub.H). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are
forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are described more
fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993)
Proc. Natl. Acad. Sci. USA 90: 6444-6448.
[0037] A "domain antibody fragment" is an immunologically
functional immunoglobulin fragment containing only the variable
region of a heavy chain or the variable region of a light chain. In
some instances, two or more V.sub.H regions are covalently joined
with a peptide linker to create a bivalent domain antibody
fragment. The two V.sub.H regions of a bivalent domain antibody
fragment may target the same or different antigens.
[0038] Autoimmune-mediated ocular inflammatory disease (AOID) means
any disease or condition in which (a) inflammation is present in
any part of the eye or surrounding tissues (including the optic
nerve, blood vessels, muscles) and (b) the inflammation is part of
an immune response that requires or is promoted by one or both of
IL-23 and IL-17. Intraocular inflammation without an infectious
etiology is typically considered an AOID. Nonlimiting examples of
AOIDs are listed below.
[0039] Birdshot retinochoriodopathy (BSRC): A chronic intraocular
inflammatory disease affecting mainly the back (posterior) part of
the eye. BSRC is distinct from other forms of posterior uveitis
that have a strong association with the HLA-A29.2 antigen. Its
etiology remains unknown. An autoimmune mechanism is likely to play
an important pathogenic role.
[0040] Ocular cicatricial pemphigoid (OCP): A systemic autoimmune
disease. Mounting evidence supports the concept of immunoregulatory
dysfunction: antibodies are directed against the basement membrane
zone (BMZ) of the conjunctiva and other mucous membranes derived
from stratified squamous epithelia and occasionally the skin. OCP
is a vision threatening illness that usually requires treatment
with immunosuppression.
[0041] Keratitis, peripheral ulcerative Keratitis: Keratitis is
inflammation of the cornea, the outer, transparent, dome-like
structure that forms the anterior most part of the outer coat of
the eye. If ulcers develop in the peripheral cornea, it is referred
to as peripheral ulcerative Keratitis.
[0042] "Sympathetic ophtahlmia" is an AOID in which a trauma to one
eye precipitates at a later time a destructive inflammation in the
other ("sympathizing") eye, apparently due to an autoimmune
response to antigens released from the injured eye.
[0043] Vogt-Koyanagi Harada (VKH): Vogt-Koyanagi-Harada syndrome
(VKH), formerly known as uveomenigitic syndrome is a systemic
disorder involving multiple organ systems, including the ocular,
auditory, nervous, and integumentary (skin) systems. Severe
bilateral panuveitis associated with subretinal fluid accumulation
is the hallmark of ocular VKH.
[0044] Fuchs' heterochromic iridocyclitis: A chronic, unilateral
anterior uveitis characterized by iris heterochromia, a condition
in which one eye is a different color from the other. The uveitis
typically occurs in the lighter colored eye of a young adult.
[0045] "Binding compound" refers to a molecule, small molecule,
macromolecule, antibody, a fragment or analogue thereof, or soluble
receptor, capable of binding to a specified target.
[0046] "Binding compound" also may refer to any of the following
that are capable of binding to the specified target: a complex of
molecules (e.g., a non-covalent molecular complex); an ionized
molecule; and a covalently or non-covalently modified molecule
(e.g., modified by phosphorylation, acylation, cross-linking,
cyclization, or limited cleavage). In cases where the binding
compound can be dissolved or suspended in solution, "binding" may
be defined as an association of the binding compound with a target
where the association results in reduction in the normal Brownian
motion of the binding compound.
[0047] "Binding composition" refers to a binding compound in
combination with at least one other substance, such as a
stabilizer, excipient, salt, buffer, solvent, or additive.
[0048] "Bispecific antibody" means an antibody that has two antigen
binding sites having specificities for two different epitopes,
which may be on the same antigen, or on two different antigens.
Bispecific antibodies include bispecific antibody fragments. See,
e.g., Hollinger, et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:
6444-48, Gruber, et al., J. Immunol. 152: 5368 (1994).
[0049] "Consists essentially of" and variations such as "consist
essentially of" or "consisting essentially of" as used throughout
the specification and claims, indicate the inclusion of any recited
elements or group of elements, and the optional inclusion of other
elements, of similar or different nature than the recited elements,
which do not materially change the basic or novel properties of the
specified dosage regimen, method, or composition. As a nonlimiting
example, a cytokine which consists essentially of a recited amino
acid sequence may also include one or more amino acids that do not
materially affect the properties of the cytokine.
[0050] "Interleukin-12R beta1" or "IL12RB1" means a single
polypeptide chain consisting essentially of the sequence of the
mature form of human IL12RB1 as described in NCBI Protein Sequence
Database Accession Numbers NP714912, NP005526 or naturally
occurring variants thereof.
[0051] "Interleukin-17" (or "IL-17") means a protein consisting of
one or two polypeptide chains, with each chain consisting
essentially of the sequence of the mature form of human IL17A as
described in any of NCBI Protein Sequence Database Accession
Numbers NP002181, AAH67505, AAH67503, AAH67504, AAH66251, AAH66252
or naturally occurring variants thereof.
[0052] "IL-17R" or "IL-17RA" means a single polypeptide chain
consisting essentially of the sequence of the mature form of human
IL-17RA as described in WO 96/29408 or in any of NCBI Protein
Sequence Database Accession Numbers: NP 055154, Q96F46, CAJ86450,
or naturally occurring variants of these sequences.
[0053] "IL-17RC" means a single polypeptide chain consisting
essentially of the sequence of the mature form of human IL-17RC as
described in WO 238764A2 or in any of NCBI Protein Sequence
Database Accession Numbers NP703191, NP703190 and NP116121, or
naturally occurring variants of these sequences.
[0054] "Interleukin-23 (or "IL-23) means a protein consisting of
two polypeptide chains. One chain consists essentially of the
sequence of the mature form of human IL23, subunit p19 (also known
as IL23A) as described in any of NCBI Protein Sequence Database
Accession Numbers NPO.sub.57668, AAH67511, AAH66267, AAH66268,
AAH66269, AAH667512, AAH67513 or naturally occurring variants of
these sequences. The other chain consists essentially of the
sequence of the mature form of human IL12, subunit p40 (also known
as IL12B and IL23, subunit p40) as described in any of NCBI Protein
Sequence Database Accession Numbers NP002178, P29460, AAG32620,
AAH74723, AAH67502, AAH67499, AAH67498, AAH67501 or naturally
occurring variants of these sequences.
[0055] "Interleukin-23R" or "IL-23R" means a single polypeptide
chain consisting essentially of the sequence of the mature foam of
human IL23R as described in NCBI Protein Sequence Database
Accession Number NP653302 or naturally occurring variants
thereof.
[0056] "Monoclonal antibody" or "mAb" means an antibody obtained
from a substantially homogeneous population of antibodies, and is
not to be construed as requiring production of the antibody by any
particular method.
[0057] "Parenteral administration" means an intravenous,
subcutaneous, or intramuscular injection.
[0058] "Small molecule" means a molecule with a molecular weight
that is less than 10 kD, typically less than 2 kD, and preferably
less than 1 kD. Small molecules include, but are not limited to,
inorganic molecules, organic molecules, organic molecules
containing an inorganic component, molecules comprising a
radioactive atom, synthetic molecules, peptide mimetics, and
antibody mimetics. Peptide mimetics of antibodies and cytokines are
known in the art. See, e.g., Casset, et al. (2003) Biochem.
Biophys. Res. Commun. 307:198-205; Muyldermans (2001) J.
Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol. 18:1251-1256;
Apostolopoulos, et al. (2002) Curr. Med. Chem. 9:411-420;
Monfardini, et al. (2002) Curr. Pharm. Des. 8:2185-2199; Domingues,
et al. (1999) Nat. Struct. Biol. 6:652-656; Sato and Sone (2003)
Biochem. J. 371:603-608; U.S. Pat. No. 6,326,482 issued to Stewart,
et al.
[0059] "Specific" or "specifically", when referring to the binding
interaction between the members of a binding pair, such as a
cytokine and its receptor, and antibody and its antigen or epitope,
indicates a binding reaction which is determinative of the presence
of one member of the binding pair in a heterogeneous population of
proteins and other biologics. Thus, under designated conditions,
one member of a binding pair has a significantly greater affinity
for the other member of the binding pair than for irrelevant
proteins. For example, an antibody is considered to be specific for
a particular protein if it binds to that protein with an affinity
that is at least 10-fold, and preferably 50-fold higher than its
affinity for a different protein. An antibody that "specifically
binds" to a protein comprising a particular epitope does not bind
to any measurable degree to proteins that do not comprise that
epitope. Preferably, an antibody that is specific for a target
protein will have an affinity toward the target protein that is
greater than about 10.sup.9 liters/mol, as determined, e.g., by
Scatchard analysis (Munsen, et al. (1980) Analyt. Biochem.
107:220-239).
[0060] "Treat" or "Treating" means to administer a therapeutic
agent, such as a composition containing any of the IL-17 and IL-23
antagonists described herein, internally or externally to a patient
in need of the therapeutic agent. Typically, the agent is
administered in an amount effective to prevent or alleviate one or
more disease symptoms, or one or more adverse effects of treatment
with a different therapeutic agent, whether by preventing the
development of, inducing the regression of, or inhibiting the
progression of such symptom(s) or adverse effect(s) by any
clinically measurable degree. The amount of a therapeutic agent
that is effective to alleviate any particular disease symptom or
adverse effect (also referred to as the "therapeutically effective
amount") may vary according to factors such as the disease state,
age, and weight of the patient, and the ability of the therapeutic
agent to elicit a desired response in the patient. Whether a
disease symptom or adverse effect has been alleviated can be
assessed by any clinical measurement typically used by physicians
or other skilled healthcare providers to assess the severity or
progression status of that symptom or adverse effect. When a
therapeutic agent is administered to a patient who has active
disease, a therapeutically effective amount will typically result
in a reduction of the measured symptom by at least 5%, usually by
at least 10%, more usually at least 20%, most usually at least 30%,
preferably at least 40%, more preferably at least 50%, most
preferably at least 60%, ideally at least 70%, more ideally at
least 80%, and most ideally at least 90%. While an embodiment of
the present invention (e.g., a treatment method or article of
manufacture) may not be effective in preventing or alleviating the
target disease symptom(s) or adverse effect(s) in every patient, it
should alleviate such symptom(s) or effect(s) in a statistically
significant number of patients as determined by any statistical
test known in the art such as the Student's t-test, the
chi.sup.2-test, the U-test according to Mann and Whitney, the
Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test and the
Wilcoxon-test.
[0061] Uveitis means inflammation affecting one or more of the
three parts of the eye that make up the uvea: the iris (the colored
part of the eye), the ciliary body (behind the iris, responsible
for manufacturing the fluid inside the eye) and the choroid (the
vascular lining tissue underneath the retina). Panuveitis denotes
the presence of inflammation in multiple parts of the same eye
(anterior, intermediate, and posterior sections).
[0062] Uveitis can be either acute or chronic. The chronic form is
more often associated with systemic disorders including ankylosing
spondylitis, Behcet's syndrome, inflammatory bowel disease,
juvenile rheumatoid arthritis, Reiter's syndrome, sarcoidosis,
syphilis, tuberculosis, and Lyme disease.
[0063] Anterior uveitis, which involves inflammation in the front
part of the eye, is the most common form of uveitis. The
inflammation is usually isolated to the iris; thus, anterior
uveitis is often called iritis. In some patients, anterior uveitis
may be associated with the presence of an autoimmune disease such
as rheumatoid arthritis or ankylosing spondylitis, but most cases
of anterior uveitis occur in otherwise healthy people and do not
indicate an underlying systemic disease. This OID may affect only
one eye and is most common in young and middle-aged people. A
history of an autoimmune disease is a risk factor. Most attacks of
anterior uveitis last from a few days to weeks with treatment, but
relapses are common.
[0064] Intermediate uveitis denotes an idiopathic inflammatory
syndrome mainly involving the anterior vitreous, peripheral retina,
and ciliary body, with minimal or no anterior segment or
chorioretinal inflammatory signs.
[0065] Pars planitis is inflammation of the pars plana, a narrow
area between the iris and the choroid. Pars planitis usually occurs
in young men and is generally not associated with any other
disease. However, there have been a few case reports of an
association with Crohn's disease and some experts suggest a
possible association with multiple sclerosis. For this reason,
these experts recommend tliat patients over 25 years old diagnosed
with pars planitis receive an MRI of their brain and spine.
[0066] Posterior uveitis affects the back portion of the uveal
tract and involves primarily the choroid. This is called
choroiditis. Posterior uveitis is characterized by inflammation of
the layer of blood vessels underlying the retina, and usually of
the retina as well. If the adjacent retina is also involved, the
condition is typically called chorioretinitis. Posterior uveitis
may follow a systemic infection or occur in association with an
autoimmune disease. In posterior uveitis, the inflammation may last
from months to years and may cause permanent vision damage, even
with treatment.
II. General
[0067] The present invention provides methods of using antagonists
of IL-17 and IL-23 activity to treat autoimmune ocular inflammatory
disease.
[0068] IL17 activity, which is reviewed in Kolls, J. et al. (2004)
Immunity Vol. 21, 467-476, includes promoting accumulation of
neutrophils in a localized area and the activation of neutrophils.
IL17 can induce or promote the production of any of the following
proinflammatory and neutrophil-mobilizing cytokines, depending on
the cell type: IL-6, MCP-1, CXCL8 (IL-8), CXCL1, CXCL6, TNF.alpha.,
G-CSF, GM-CSF, MMP-1, and MMP-13.
[0069] IL-23 activity includes inducing the proliferation of memory
T cells, PHA blasts, CD45RO T cells, CD45RO T cells; and enhance
production of interferon-gamma (IFN.gamma.) by PHA blasts or CD45RO
T cells. In contrast to IL-12, IL-23 preferentially stimulates
memory as opposed to naive T cell populations in both human and
mouse. IL-23 activates a number of intracellular cell-signaling
molecules, e.g., Jak2, Tyk2, Stat1, Stat2, Stat3, and Stat4. IL-12
activates this same group of molecules, but Stat4 response to IL-23
is relatively weak, while Stat4 response to IL-12 is strong
(Oppmann, et al., supra; Parham, et al. (2002) J. Immunol.
168:5699-5708). IL-23 has also been implicated in the maintenance
and proliferation of IL-17 producing cells, also known as Th.sub.17
cells (see, Cua and Kastelein (2006) Nature Immunology
7:557-559).
[0070] Antagonists useful in the present invention include a
soluble receptor comprising the extracellular domain of a
functional receptor for IL-17 or IL-23. Soluble receptors can be
prepared and used according to standard methods (see, e.g., Jones,
et al. (2002) Biochim. Biophys. Acta 1592:251-263; Prudhomme, et
al. (2001) Expert Opinion Biol. Ther. 1:359-373; Fernandez-Botran
(1999) Crit. Rev. ClM. Lab Sci. 36:165-224).
[0071] Preferred IL-17 antagonists for use in the present invention
are antibodies that specifically bind to, and inhibit the activity
of, any of IL-17, IL-17RA, IL-17RC, and a heteromeric complex
comprising IL-17RA and IL-17RC. More preferably, the target of the
IL-17 antagonist is IL-17 or IL-17RA. Particularly preferred IL-17
antagonists specifically bind to, and inhibit the activity of
IL-17.
[0072] Another preferred IL-17 antagonist for use in the present
invention is a bispecific antibody, or bispecific antibody
fragment, which also antagonizes IL-23 activity. Such bispecific
antagonists specifically bind to, and inhibits the activity of,
each member in any of the following combinations: IL-17 and IL-23;
IL-17 and IL-23p19; IL-17 and IL-12p40; IL-17 and an IL-23R/IL12RB1
complex; IL-17 and IL-23R; IL-17 and IL12RB1; IL17RA and IL-23;
IL-17RA and IL-23p19; IL-17RA and IL-12p40; IL-17RA and an
IL-23R/IL12RB1 complex; IL-17RA and IL-23R; IL-17RA and IL12RB1;
IL17RC and IL-23; IL-17RC and IL-23p19; IL-17RC and IL-12p40;
IL-17RC and an IL-23R/IL12RB1 complex; IL-17RC and IL-23R; IL-17RC
and IL12RB1; an IL-17RA/IL-17RC complex and IL-23; an
IL-17RA/IL-17RC complex and IL-23p19; an IL-17RA/IL-17RC complex
and IL-12p40; an IL-17RA/IL-17RC complex and an IL-23R/IL12RB1
complex; an IL-17RA/IL-17RC complex and IL-23R; and an
IL-17RA/IL-17RC complex and IL12RB1. Preferred combinations
targeted by bispecific antibodies used in the present invention
are: IL-17 and IL-23; IL-17 and IL-23p19; IL17RA and IL-23; and
IL-17RA and IL-23p19. A particularly preferred bispecific antibody
specifically binds to, and inhibits the activity of, each of IL-17
and IL-23p19.
[0073] Preferred IL-23 antagonists are antibodies that bind to, and
inhibit the activity of, any of IL-23, IL-23p19, IL-12p40, IL23R,
IL12RB1, and an IL-23R/IL12RB1 complex. Another preferred IL-23
antagonist is an IL-23 binding polypeptide which consists
essentially of the extracellular domain of IL-23R, e.g., amino
acids 1-353 of GenBankAAM44229, or a fragment thereof.
[0074] Antibody antagonists for use in the invention may be
prepared by any method known in the art for preparing antibodies.
The preparation of monoclonal, polyclonal, and humanized antibodies
is described in Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al.
(1998) J. Immunol. 160:1029; Tang, et al. (1999) J. Biol. Chem.
274:27371-27378; Baca, et al. (1997) J. Biol. Chem.
272:10678-10684; Chothia, et al. (1989) Nature 342:877-883; Foote
and Winter (1992) J. Mol. Biol. 224:487-499; and U.S. Pat. No.
6,329,511 issued to Vasquez, et al.
[0075] Any antigenic form of the desired target can be used to
generate antibodies, which can be screened for those having the
desired antagonizing activity. Thus, the eliciting antigen may be a
peptide containing a single epitope or multiple epitopes, or it may
be the entire protein alone or in combination with one or more
immunogenicity enhancing agents known in the art. To improve the
immunogenicity of an antigenic peptide, the peptide may be
conjugated to a carrier protein. The antigen may also be an
isolated full-length protein, a cell surface protein (e.g.,
immunizing with cells transfected with at least a portion of the
antigen), or a soluble protein (e.g., immunizing with only the
extracellular domain portion of the protein). The antigen may be
expressed by a genetically modified cell, in which the DNA encoding
the antigen is genomic or non-genomic (e.g., on a plasmid).
[0076] A peptide consisting essentially of a region of predicted
high antigenicity can be used for antibody generation. For example,
regions of high antigenicity of human p19 occur at amino acids
16-28; 57-87; 110-114; 136-154; and 182-186 of GenBank AAQ89442
(gi:37183284) and regions of high antigenicity of human IL-23R
occur at amino acids 22-33; 57-63; 68-74; 101-112; 117-133;
164-177; 244-264; 294-302; 315-326; 347-354; 444-473; 510-530; and
554-558 of GenBank AAM44229 (gi: 21239252), as determined by
analysis with a Parker plot using Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.).
[0077] Any suitable method of immunization can be used. Such
methods can include use of adjuvants, other immunostimulants,
repeated booster immunizations, and the use of one or more
immunization routes. Immunization can also be performed by DNA
vector immunization, see, e.g., Wang, et al. (1997) Virology
228:278-284. Alternatively, animals can be immunized with cells
bearing the antigen of interest, which may provide superior
antibody generation than immunization with purified antigen
(Kaithamana, et al. (1999) J. Immunol. 163: 5157-5164).
[0078] Preferred antibody antagonists are monoclonal antibodies,
which may be obtained by a variety of techniques familiar to
skilled artisans. Methods for generating monoclonal antibodies are
generally described in Stites, et al. (eds.) BASIC AND CLINICAL
IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos, Calif.,
and references cited therein; Harlow and Lane (1988) ANTIBODIES: A
LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES:
PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y.
Typically, splenocytes isolated from an immunized mammalian host
are immortalized, commonly by fusion with a myeloma cell to produce
a hybridoma. See Kohler and Milstein (1976) Eur. J. Immunol.
6:511-519; Meyaard, et al. (1997) Immunity 7:283-290; Wright, et
al. (2000) Immunity 13:233-242; Preston, et al. (1997) Eur. J.
Immunol. 27:1911-1918. Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods known in the art. See, e.g., Doyle,
et al. (eds. 1994 and periodic supplements) CELL AND TISSUE
CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity, affinity and
inhibiting activity using suitable binding and biological assays.
For example, antibody to target binding properties can be measured,
e.g., by surface plasmon resonance (Karlsson, et al. (1991) J.
Immunol. Methods 145:229-240; Neri, et al. (1997) Nat. Biotechnol.
15:1271-1275; Jonsson, et al. (1991) Biotechniques 11:620-627) or
by competition ELISA (Friguet, et al. (1985) J. Immunol. Methods
77:305-319; Hubble (1997) Immunol. Today 18:305-306).
[0079] Alternatively, one may isolate DNA sequences which encode a
monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells, see e.g., Huse, et al. (1989)
Science 246:1275-1281. Other suitable techniques involve screening
phage antibody display libraries. See, e.g., Huse et al., Science
246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989);
Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991)
J. Mol. Biol. 222: 581-597; Presta (2005) J. Allergy Clin. Immunol.
116:731.
[0080] Preferred monoclonal antibodies for use in the present
invention are "chimeric" antibodies (immunoglobulins) in which the
variable domain is from the parental antibody generated in an
experimental mammalian animal, such as a rat or mouse, and the
constant domains are obtained from a human antibody, so that the
resulting chimeric antibody will be less likely to elicit an
adverse immune response in a human subject than the parental
mammalian antibody. More preferably, a monoclonal antibody used in
the present invention is a "humanized antibody", in which all or
substantially all of the hypervariable loops (e.g., the
complementarity determining regions or CDRs) in the variable
domains correspond to those of a non-human immunoglobulin, and all
or substantially all of the framework (FR) regions in the variable
domains are those of a human immunoglobulin sequence. A
particularly preferred monoclonal antibody for use in the present
invention is a "fully human antibody", e.g., an antibody that
comprises human immunoglobulin protein sequences only. A fully
human antibody may contain carbohydrate chains from the cell
species in which it is produced, e.g., if produced in a mouse, in a
mouse cell, or in a hybridoma derived from a mouse cell, a fully
human antibody will typically contain murine carbohydrate
chains.
[0081] Monoclonal antibodies used in the present invention may also
include camelized single domain antibodies. See, e.g., Muyldermans
et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999)
J. Immunol. Methods 231:25; WO 94/04678; WO 94/25591; U.S. Pat. No.
6,005,079.
[0082] The antagonistic antibodies used in the present invention
may have modified (or blocked) Fc regions to provide altered
effector functions. See, e.g., U.S. Pat. No. 5,624,821;
WO2003/086310; WO2005/120571; WO2006/0057702. Alterations of the Fc
region include amino acid changes (substitutions, deletions and
insertions), glycosylation or deglycosylation, and adding multiple
Fc. Changes to the Fc can alter the half-life of therapeutic
antibodies, enabling less frequent dosing and thus increased
convenience and decreased use of material. See Presta (2005) J.
Allergy ClM. Immunol. 116:731 at 734-35.
[0083] The antibodies may also be conjugated (e.g., covalently
linked) to molecules that improve stability of the antibody during
storage or increase the half-life of the antibody in vivo. Examples
of molecules that increase the half-life are albumin (e.g., human
serum albumin) and polyethylene glycol (PEG). Albumin-linked and
PEGylated derivatives of antibodies can be prepared using
techniques well known in the art. See, e.g., Chapman, A. P. (2002)
Adv. Drug Deliv. Rev. 54:531-545; Anderson and Tomasi (1988) J.
Immunol. Methods 109:37-42; Suzuki, et al. (1984) Biochim. Biophys.
Acta 788:248-255; and Brekke and Sandlie (2003) Nature Rev.
2:52-62).
[0084] Bispecific antibodies that antagonize both IL-17 and IL-23
activity can be produced by any technique known in the art. For
example, bispecific antibodies can be produced recombinantly using
the co-expression of two immunoglobulin heavy chain/light chain
pairs. See, e.g., Milstein et al. (1983) Nature 305: 537-39.
Alternatively, bispecific antibodies can be prepared using chemical
linkage. See, e.g., Brennan, et al. (1985) Science 229: 81. These
bifunctional antibodies can also be prepared by disulfide exchange,
production of hybrid-hybridomas (quadromas), by transcription and
translation to produce a single polypeptide chain embodying a
bispecific antibody, or transcription and translation to produce
more than one polypeptide chain that can associate covalently to
produce a bispecific antibody. The contemplated bispecific antibody
can also be made entirely by chemical synthesis. The bispecific
antibody may comprise two different variable regions, two different
constant regions, a variable region and a constant region, or other
variations.
[0085] Antibodies used in the present invention will usually bind
with at least a K.sub.D of about 10.sup.-3M, more usually at least
10.sup.-6M, typically at least 10.sup.-7M, more typically at least
10.sup.-8M, preferably at least about 10.sup.-9M, and more
preferably at least 10.sup.-10 M, and most preferably at least
10.sup.-11 M (see, e.g., Presta, et al. (2001) Thromb. Haemost.
85:379-389; Yang, et al. (2001) Crit. Rev. Oncol. Hematol.
38:17-23; Carnahan, et al. (2003) Clin. Cancer Res. (Suppl.)
9:3982s-3990s).
[0086] IL-17 antagonists and IL-23 antagonists are typically
administered to a patient as a pharmaceutical composition in which
the antagonist is admixed with a pharmaceutically acceptable
carrier or excipient, see, e.g., Remington's Pharmaceutical
Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing
Company, Easton, Pa. (1984). The pharmaceutical composition may be
formulated in any manner suitable for the intended route of
administration. Examples of pharmaceutical formulations include
lyophilized powders, slurries, aqueous solutions, suspensions and
sustained release formulations (see, e.g., Hardman, et al. (2001)
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)
(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000)
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0087] The route of administration will depend on the properties of
the antagonist or other therapeutic agent used in the
pharmaceutical composition. A possible administration route is to
administer the pharmaceutical composition topically to the eye in
the form of an ointment, gel or droppable liquids using an ocular
delivery system known to the art such as an applicator or
eyedropper. Alternatively, the pharmaceutical composition may be
administered intraocularly via an polymer implant that is placed
under the under the conjunctiva of the eye or through injection
directly into the eye. Preferably, pharmaceutical compositions
containing IL-17 antagonists and IL-23 antagonists are administered
systemically by oral ingestion, injection or infusion by
intravenous, intraperitoneal, intracerebral, intramuscular,
intraocular, intraarterial, intracerebrospinal, intralesional, or
pulmonary routes, or by sustained release systems such as implants.
Injection of gene transfer vectors into the central nervous system
has also been described (see, e.g., Cua, et al. (2001) J. Immunol.
166:602-608; Sidman et al. (1983) Biopolymers 22:547-556; Langer,
et al. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982)
Chem. Tech. 12:98-105; Epstein, et al. (1985) Proc. Natl. Acad.
Sci. USA 82:3688-3692; Hwang, et al. (1980) Proc. Natl. Acad. Sci.
USA 77:4030-4034; U.S. Pat. Nos. 6,350466 and 6,316,024).
[0088] The pharmaceutical compositions used in the invention may be
administered according to any treatment regimen that ameliorates or
prevents one or more symptoms of the AOID. Selecting the treatment
regimen will depend on several composition-dependent and
patient-dependent factors, including but not limited to the
half-life of the antagonist, the severity of the patient's
symptoms, and the type or length of any adverse effects.
Preferably, an administration regimen maximizes the amount of
therapeutic agent delivered to the patient consistent with an
acceptable level of side effects. Guidance in selecting appropriate
doses of therapeutic antibodies and small molecules is available
(see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific
Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal
Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.;
Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in
Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al.
(2003) New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New
Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New Engl. J.
Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl. J. Med.
342:613-619; Ghosh, et al. (2003) New Engl. J. Med. 348:24-32;
Lipsky, et al. (2000) New Engl. J. Med. 343:1594-1602).
[0089] Biological antagonists such as antibodies may be provided by
continuous infusion, or by doses at intervals of, e.g., once per
day, once per week, or 2 to 7 times per week, once every other
week, or once per month. A total weekly dose for an antibody is
generally at least 0.05 .mu.g/kg body weight, more generally at
least 0.2 .mu.g/kg, most generally at least 0.5 .mu.g/kg, typically
at least 1 mg/kg, more typically at least 10 .mu.g/kg, most
typically at least 100 .mu.g/kg, preferably at least 0.2 mg/kg,
more preferably at least 1.0 mg/kg, most preferably at least 2.0
mg/kg, optimally at least 10 mg/kg, more optimally at least 25
mg/kg, and most optimally at least 50 mg/kg (see, e.g., Yang, et
al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New
Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol.
Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer
Immunol. Immunother. 52:133-144). The desired dose of a small
molecule therapeutic, e.g., a peptide mimetic, natural product, or
organic chemical, is about the same as for an antibody or
polypeptide, on a moles/kg basis. Determination of the appropriate
dose is made by the clinician, e.g., using parameters or factors
known or suspected in the art to affect treatment or predicted to
affect treatment. Generally, the beginning dose is an amount
somewhat less than the optimum dose and the dose is increased by
small increments thereafter until the desired or optimum effect is
achieved relative to any negative side effects.
[0090] Treatment regimens using IL-17 or IL-23 antagonists will
typically be determined by the treating physician and will take
into account the patient's age, medical history, disease symptoms,
and tolerance for different types of medications and dosing
regimens. Generally the treatment regimen is designed to suppress
the overly aggressive immune system, allowing the body to
eventually re-regulate itself, with the result often being that
after the patient has been kept on systemic medications to suppress
the inappropriate immune response for a finite length of time (for
example, one year), medication can then be tapered and stopped
without recurrence of the autoimmune attack. Sometimes resumption
of the attack does occur, in which case the patient must be
re-treated.
[0091] Thus, in some cases, the physician may prescribe the patient
a certain number of doses of the antagonist to be taken over a
prescribed time period, after which therapy with the antagonist is
discontinued. Preferably, after an initial treatment period in
which one or more of the acute symptoms of the disease disappear,
the physician will continue the agonist therapy for some period of
time, in which the amount and/or frequency of antagonist
administered is gradually reduced before treatment is stopped.
[0092] The present invention also contemplates treatment regimens
in which an IL-17 antagonist is used in combination with an IL-23
antagonist. Such regimens may be especially useful in treating the
acute phase of AOID, in which the IL-17 antagonist inhibits the
activity of existing Th.sub.17 cells, while the IL-23 antagonist
prevents the generation of new Th.sub.17 cells. Such combination
therapy may provide effective treatment of AOID using a lower dose
of the IL-17 antagonist and/or administering the IL-17 antagonist
for a shorter period of time. As symptoms ameliorate, therapy with
IL-17 antagonist is preferably discontinued, while administration
of the IL-23 antagonist is continued to prevent generation of new
autoreactive Th.sub.17 cells that could lead to recurrence of the
disease. The two antagonists may be administered at the same time
in a single composition, or in separate compositions. Alternately,
the two antagonists may be administered at separate intervals.
Different doses of the antagonists may also be used. Similarly, a
bispecific antagonist may also be administered during the acute
phase and gradually withdrawn, followed by treatment with an IL-23
antagonist to maintain repression of the disease.
[0093] The treatment regimen may also include use of other
therapeutic agents, to ameliorate one or more symptoms of the AOID
or to prevent or ameliorate adverse effects from the antagonist
therapy. Examples of therapeutic agents that have been used to
treat AOID symptoms are steroids and other anti-inflammatories.
Examples of such therapies include, but are not limited to,
steroids such as dexamethasone, fluorometholone, and prednisolone,
as well as non-steroidal anti-inflammatories such as indomethacin,
aspirin, flubiprofen and diclofenac, antimetabolites (e.g.,
methotrexate, azathioprine), inhibitors of transcription factors
(e.g., cyclosporine, tacrolimus), and DNA cross-linking agents
(e.g., cyclophosphamide, chlorambucil). New agents directed against
cytokines and their receptors, many of which act by inhibiting
important Th1 cytokine rather than signaling pathways, are
beginning to be used for treatment of patients with uveitis. These
include TNF inhibitors such as Infliximab (Remicade.RTM., Centocor,
Malvern, Pa.), Etanercept (Enbrele, Amgen, Thousand Oaks, Calif.),
and Adalimumab (Humira.RTM., Abbott Laboratories, Abbott Park,
Ill.) and specific inhibitors of IL-2 signaling, including
Daclizumab (Zenapax.RTM., Roche Laboratories, Nutley, N.J.) and
Basiliximab (Simulect.RTM., Novartis Pharmaceutical Co., East
Hanover, N.J.).
[0094] In any of the therapies described herein in which two or
more different therapeutic substances are used (e.g., an IL-17
antagonist and an IL-23 antagonist, or an IL-17 antagonist and a
therapeutic agent that does not antagonize IL-17 or IL-23
activity), it will be understood that the different therapeutic
substances are administered in association with each other, that
is, they may be administered concurrently in the same
pharmaceutical composition or as separate compositions or the
substances may be administered at separate times, and in different
orders.
[0095] Diagnosing the presence of an AOID in a patient will
typically involve examining the patient for symptoms known to be
consistent with such diseases. For example, the typical
presentation of anterior uveitis involves pain, photophobia, and
hyperlacrimation. Patients report a deep, dull, aching of the
involved eye and surrounding orbit. Associated sensitivity to
lights may be severe. Excessive tearing occurs secondary to
increased neural stimulation of the lacrimal gland and the patient
does not report a foreign-body sensation. Visual acuity is variable
ranging from mild blur to significant vision loss if synechiae or
cyclitic membranes are present. An examination may reveal mild to
moderate lid swelling resulting in pseudoptosis. A deep, perilimbal
injection of the conjunctiva and episclera is typical, although the
palpebral conjunctiva is characteristically normal. The cornea may
display mild edema.
[0096] The hallmark signs of anterior uveitis include cells and
flare in the anterior chamber. If the anterior chamber reaction is
significant, small gray to brown endothelial deposits known as
keratic precipitates may be present. This can then lead to
endothelial cell dysfunction and corneal edema. Iris findings may
include adhesions to the lens capsule (posterior synechiae) or,
less commonly, to the peripheral cornea (anterior synechiae).
Additionally, granulomatous nodules may appear on the surface of
the iris. Intraocular pressure (TOP) is initially reduced in the
involved eye due to secretory hypotony of the ciliary body.
However, as the reaction persists, inflammatory by-products may
accumulate in the trabeculum. If this debris builds significantly,
and if the ciliary body resumes its normal secretory output, IOP
can rise sharply resulting in a secondary uveitic glaucoma.
[0097] Identifying patients who are susceptible for an AOID will
typically taking a personal and family medical history, and may
include genetic testing. For example, some individuals will have
genetic predisposition to uveitis which is related to autoimmune
disease processes. The most common of these susceptibility genes is
the HLA B27 haplotype, which can predispose to uveitis alone or
also to the Seronegative Spondyloarthropathies and the enteropathic
arthropathies. Examples are ankylosing spondylitis, reactive
arthritis (Reiters syndrome), psoriatic arthritis, irritable bowel
disease and Crohn's disease. A patient may also be diagnosed as
susceptible for an AOID if there was a family history of any of
these autoimmune diseases, or the patient has already been
diagnosed with such a disease.
[0098] The effectiveness of the antagonist therapy for preventing
or treating AOID in a particular patient can be determined using
diagnostic measures such as reduction or occurrence of inflammatory
symptoms of, e.g., the amount of ocular inflammation or level of
inflammatory cytokines in the affected eye(s). The symptoms of
ocular inflammation for the most part depend on the affected area
of the eye. Most common signs and symptoms are: pain redness,
floaters, decreased vision, and light sensitivity. The level of
inflammatory cytokines can be measured, e.g, by contacting a
binding compound for the inflammatory cytokine of interest with a
sample from the patient's eye as well as with a sample from a
control subject or from unaffected tissue or fluid from the
patient, and then comparing the cytokine levels detected by the
binding compound. Expression or activity from a control subject or
control sample can be provided as a predetermined value, e.g.,
acquired from a statistically appropriate group of control
subjects.
EXAMPLES
[0099] The present invention is based upon studies in IL-23p19
knockout (KO) mice and administration of anti-IL-23p19 and
anti-IL-17 antibodies to murine models of autoimmune uveitis. These
experiments were performed according to the Materials and Methods
described in Section II below.
I. Results and Discussion
[0100] In the experiments involving IL-23p19 KO mice, the EAU
susceptibility of IL-23p19 KO (IL-23 deficient) mice were compared
to the EAU susceptibility of IL-12p35 KO (IL-12 deficient) and
IL-12p40 KO (IL-12 and IL-23 deficient) mice. All mice were on the
C57BL/6 background and the EAU induction and scoring was as
described in General Methods below. It was found that IL-12p35 is
not required for generation of IRBP-specific eye tissue
destruction. In contrast, IL-23p19 is essential for development of
EAU (Table 1). Cytokine analysis of lymph node cell cultures
derived from IRBP-immunized mice showed that the EAU susceptible
IL-12 deficient mice (IL-12p35KO) had elevated levels of
IFN-.gamma., IL-6, IL-17 and IL-18, compared to IL-23 deficient
mice (IL-23p19KO and IL-12p40KO). Delayed hypersensitivity (DTH)
responses to IRBP of the 3 KO strains, examined by the ear swelling
assay, showed that DTH response to IRBP was well correlated with
the EAU scores or the respective mice, with significantly lower
responses for p19 and p40 KO and significantly higher responses in
p35 KO compared to wild-type (WT).
TABLE-US-00001 TABLE 1 IL-23, but not IL-12, is essential for EAU
development. DTH Specific EAU swelling +/- Average score +/- SE
IFN-.gamma. IL-6 IL-17 IL-18 SE (.mu.m .times. 10-1) (ng/ml)
(ng/ml) (ng/ml) (ng/ml) Wild type 0.21 .+-. 0.11 44 .+-. 7 39 3.2
2.2 0.25 IL-12p35KO 0.57 .+-. 0.12 57 .+-. 2 16 1.9 4.9 0.29
IL-23p19KO 0 25 .+-. 4 6.5 0.55 1.2 0.10 IL-12p40KO 0 22 .+-. 3
<1 .08 0.85 0.11
[0101] These results were further supported by experiments using an
anti-mouse IL-23p19 antibody in a mouse model of uveitis, in the
highly susceptible B10.RIII strain. It was found that anti-mouse
IL-23p19 antibody treatment significantly blocked immune-mediated
eye inflammation. At the dose of 330 .mu.g per mouse every other
day, the EAU disease index of anti-IL-23p19 treated mice was
dramatically reduced compared to anti-isotype antibody treated and
no antibody controls as determined by histopathology of eyes
collected on day 11 after immunization (Table 2). In addition,
anti-IL-23p19 therapy was as efficacious as Prednisone in blocking
EAU. The expression levels of IL-17, but not IFN-.gamma. mRNA in
the eyes of anti-IL-23p19 treated mice were lower than the control
groups suggesting that targeting IL-23 inhibited EAU by blocking
infiltration of IL-17 producing cells or preventing the expansion
of the pathogenic IL-17 producing cells within the eyes. Neutrophil
elastase and myeloperoxidase mRNA levels of anti-IL-23p19 treated
mice were comparable to naive as well as Prednisone control groups,
whereas the "No antibody" and isotype control treated mice
exhibited 10- to 100-fold increase in expression of these
inflammatory genes. Other proinflammatory factors such as
IL-1.beta., TNF, IL-6, NOS2 and COX2 were somewhat reduced in
anti-IL-23p 19 treated mice. These results demonstrate that
targeting IL-23 inhibits the development of autoimmune uveitis.
TABLE-US-00002 TABLE 2 Anti-IL-23p19 treatment inhibits EAU and
expression of inflammatory cytokines in the eye. Histo- pathology 0
= normal 1 = few monocyte infiltration 4 = severe Eye
Quantitative-PCR gene expression analysis (Shown as expression
relative damage to Ubiquitin). Tissue samples collected on day 11
after IRBP immunization. (individual Neutrophil Myelope eyes)
IFN-.gamma. IL-6 IL-17 TNF IL-1.beta. NOS2 COX2 Elastase r-oxidase
Naive 0 0 0 0 0 0.44 0.16 0 0 3.8 2.7 4.8 0.1 0 mice No 4 4 4 4 4 3
3 6.2 37.6 7.1 37.8 117.8 22.2 24.6 1.23 4.11 mAb 3 2 2 2 1 1 1
control 1 1 1 1 1 Isotype 4 4 4 4 4 4 2 NA 13.6 3.1 28.3 103.0 19.2
14.5 1.35 3.09 mAb 1 1 1 1 1 1 1 control 1 1 1 1 1 0 Anti- 1 1 1 1
1 1 1 6.9 10.3 .013 12.5 64.3 14.7 8.9 0.08 0 IL- 1 1 1 1 1 1 0
23p19 0 0 0 0 Prednisone 4 4 2 1 1 1 1 0.51 1.2 0 17.9 74.6 5.2
14.6 0.55 0 1 1 0 0 0 0 0 0
[0102] Another set of experiments comparing treatment with
anti-IL-23p19 antibodies to treatment with anti-IL-12p40 antibodies
was also performed. In this experiment mice received 500 .mu.g of
the indicated antibodies every other day, starting the day before
immunization, and the eyes and lymphoid organs were collected 17
days after immunization, or 6-7 days after disease onset in
controls. The data indicated that anti-IL-23p19 antibodies were as
effective as anti-p40 antibodies at blocking the onset of uveitis.
The data are shown in Table 3.
[0103] In addition, cytokine protein expression in the lymph nodes
of these mice was assessed by multiplex ELISA. These data show that
treatment with IL-23 antagonists lessens the production of Th1 and
pro-inflammatory cytokines. The data are shown in Table 3.
TABLE-US-00003 TABLE 3 Anti-IL-23p19 treatment inhibits EAU and
systemic cytokine responses to the uveitis antigen. EAU score of
individual IL-2 IL-4 IL-5 IL-6 IL-10 IFN-.gamma. TNF-.alpha. IL-12
IL-17 Sample eyes pg/ml pg/ml pg/ml pg/ml pg/ml pg/ml pg/ml pg/ml
pg/ml Control 3, 3, 3, 3, 3, 247.6 0.4 <3.1 145.7 8.6 1295.3
46.5 2.7 72.9 3, 3, 3, 0, 0.25 Anti IL-23p19 3, 3, 0, 0, 0, 115.0
1.3 19.1 163.7 5.5 1453.3 87.5 2.1 37.0 0, 0, 0, 0, 0 Anti Isotype
3, 3, 3, 3, 3, 205.2 1.4 <3.1 206.7 12.4 2759.6 51.2 3.1 198.0
3, 3, 3, 3, 3 Anti IL-12p40 0.25, 0, 0, 0, 101.9 0.4 <3.1 26.5
4.4 305.5 16.6 <0.8 29.7 0, 0, 0, 0, 0, 0
[0104] A second part of this experiment examined the stage of the
pathogenic process during which IL-23 was required. Mice were
treated with 500 .mu.g of anti-IL-23 p19 antibody every other day
starting 7 days after immunization and the disease was compared to
mice that were treated from day before immunization (as above). EAU
could be prevented by early treatment with either anti-p19 or
anti-p40 antibodies. However, when treatment was started 7 days
after immunization, a time point when uveitogenic effector T cells
have already been primed and can be isolated from the LN and
spleen, EAU development could not be aborted and the disease scores
developed by treated mice were similar to control. This suggests
that the requirement for IL-23 occurs at an early stage of disease
pathogenesis. The data are shown in Table 4.
TABLE-US-00004 TABLE 4 Treatment with anti-p19 antibody prevents,
but does not reverse, EAU. Start of treatment Antibody EAU score
.+-. SE day -1 Anti-isotype 2.9 .+-. 0.1 Anti P19 0.6 .+-. 0.6 Anti
P40 0 .+-. 0 day 7 Anti-isotype 2.05 .+-. 0.5 Anti P19 2.35 .+-.
0.5 Anti P40 2.075 .+-. 0.5
[0105] In the aggregate, these experiments demonstrate that
neutralization of IL-23 prevents, but does not reverse, uveitis in
animal models, and indicate that treatment with IL-23 antagonists
should have a beneficial effect in chronic uveitis in humans by
preventing recruitment of new T cells into the effector pool,
thereby reducing the severity and halting progression of the
disease.
[0106] To test whether IL-17 deficiency can affect EAU development,
IL-17A.sup.-/- mice (see, e.g., Nakae et al. (2002) Immunity
17:375-387) were immunized with a uveitogenic regimen of IRBP.
Inhibition of EAU by genetic IL-17 deficiency was only partial
(Table 5). The relatively modest reduction of EAU scores in
IL-17.sup.-/- mice might be explained by the fact that these mice
are deficient for the IL-17A isoform of the cytokine, and under
conditions of congenital deficiency might compensate with the
usually less abundantly produced IL-17F isoform.
TABLE-US-00005 TABLE 5 Genetic IL-17 deficiency reduces, but does
not abrogate, EAU susceptibility. Expt # WT IL-17A-/- 1 0.5* 0.5
1.5 1.0 0.8 0.9 0.8 0.1 0.4 0.9 1.3 0.6 0.5 2 0.5 0.5 0.9 0.0 1.8
0.3 1.0 0.0 1.5 0.5 Average Score .+-. SE 0.9 .+-. 0.1 0.5 .+-.
0.1
[0107] In contrast, neutralization of IL-17A with IL-17A antibodies
in wild type mice, either through the entire course of disease or
through the effector phase only (starting day 7), was protective.
Importantly, unlike IL-23 neutralization, neutralization of IL-17
could inhibit disease when administered starting day 7 post
immunization, when uveitogenic effectors have already been
generated. Reduction in EAU scores correlated with reduction in the
associated immunological responses, delayed-type hypersensitivity
(DTH) and antigen specific LN cell proliferation. Thus, IL-17 has a
role in the pathogenesis of EAU, and unlike IL-23, appears to
participate in the effector phase of the disease. The data are
shown in Table 6.
TABLE-US-00006 TABLE 6 Treatment with anti-IL-17A antibodies
prevents and reverses EAU Start of EAU score .+-. Proliferation
.+-. SE treatment Antibody SE DTH .+-. SE (.times.10.sup.-3) day -1
Anti-isotype 1.6 .+-. 0.7 16 .+-. 1 19.2 .+-. 1.2 Anti IL-17 0.025
.+-. 0.025 7.6 .+-. 2 6.6 .+-. 6.4 day 7 Anti-isotype 1.6 .+-. 0.6
20.2 .+-. 3 25.4 .+-. 1.4 Anti IL-17 0.5 .+-. 0.5 6.0 .+-. 2 5.9
.+-. 0.3
Section II. Materials and Methods
A. General
[0108] Standard methods in molecular biology are described
(Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;
Sambrook and Russell (2001) Molecular Cloning, 3.sup.rd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel, et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0109] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan, et al. (2001) Current
Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan, et al. (2001)
Current Protcols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0110] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens, et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry,
2.sup.nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical
Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0111] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, P A; Louis, et al. (2002) Basic
Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).
[0112] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al.
(2000) Bioinformatics Applications Note 16:741-742; Wren, et al.
(2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne
(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids
Res. 14:4683-4690).
B. Animals
[0113] IL-23 KO (p19 KO) was described in Cua, et al. (2003) Nature
421:744-748. IL-17.sup.-/- mice were produced as described in
Nakae, et al. (2002) Immunity 17:375-387. IL-12p35 KO (P35 KO),
IL-12p40 KO (P40 KO), IFN-.gamma. KO (GKO) (all on C57BL/6
background) and C57BL/6 and B10RIII, mice were purchased from
Jackson Laboratories. Animals were kept in a specific pathogen-free
facility and given water and standard laboratory chow ad libitum.
Animal care and use were in compliance with institutional
guidelines and with the Association for Research in Vision and
Ophthalmology Statement for the Use of Animals in Ophthalmic and
Vision Research.
C. EAU Induction and Scoring
[0114] CFA was purchased from Sigma. Mycobacterium Tuberculosis
strain H37RA was purchased from Thomas Scientific. Purified
Bordetella PT was purchased from Sigma-Aldrich. IRBP was isolated
from bovine retinas, as described previously, using Con A-Sepharose
affinity chromatography and fast performance liquid chromatography
(see, e.g., Pepperberg et al. (1991) Photochem Photobiol
54:1057-1060). IRBP preparations were aliquoted and stored at
-70.degree. C. Human IRBP-derived peptide 161-180 (Karabezekian, Z.
et al., (2005) Invest Ophthalmol V is Sci. 46(10):3769-76) was
synthesized by Fmoc chemistry (model 432A peptide synthesizer;
Applied Biosystems, Foster City, Calif.).
[0115] Neutralizing anti-mouse IL-23 and anti-mouse IL-17A
antibodies were provided by Schering-Plough Biopharma (Palo Alto,
Calif.). Anti-mouse IL-23 was described previously (see, e.g.,
Langrish et al. (2005) J Exp Med 201:233-240). The C17.8
(anti-IL-12p40, rat IgG2a) hybridoma was provided by the Wistar
Institute, Philadelphia, Pa. Monoclonal antibody was produced in
ascites and purified by ion exchange HPLC by Harlan Bioproducts for
Science (Indianapolis, Ind.). FITC-labeled anti-mouse CD4
(clone-L3T4), PE-labeled anti-mouse IL-17 (clone-TC11-18H10) and
APC-labeled (clone-XMG1.2) and cytokine secretion blocker
(GolgiStop.TM.) were purchased from Becton Dickinson (San Diego,
Calif.). PMA, Ionomycin were purchased from LC Laboratories
(Boston, Mass.).
[0116] EAU was induced by active immunization with 150 .mu.g of
IRBP for C57BL/6 mice and with 7 .mu.g IRBP peptide 161-180 for
B10RIII mice (Jackson Labs, Maine). For C57BL/6 mice, Bordetella
pertussis toxin (0.5 .mu.g/mouse) in PBS containing 2% normal mouse
serum was given by intraperitoneal injection concurrently with
immunization and in some experiments the IRBP was spiked with 500
.mu.g of IRBP peptide 1-20 (Avichezer, D. et al. (2000), Invest
Ophthalmol V is Sci. 41(1):127-31) to enhance the usually modest
disease scores seen in this strain. Antigen solution was emulsified
1:1 v/v in CFA that had been supplemented with Mycobacterium
tuberculosis strain H37RA to 2.5 mg/ml. A total of 200 .mu.l of
emulsion was injected s.c., divided into 3 sites (base of the tail
and both thighs).
[0117] Alternatively, EAU was induced by adoptive transfer of a
uveitogenic T cell line (see below). 1-2 million cells, freshly
stimulated with antigen, were injected intraperitoneally. Clinical
EAU was evaluated by fundoscopy under a binocular microscope after
dilation of the pupil and was graded on a scale of 0-4 using
criteria based on the extent of inflammatory lesions, as described
in detail elsewhere (see,e.g., Agarwal and Caspi, (2004) Methods
Mol Med 102:395-419; and Chan et al. (1990) J Autoimmun 3:247-255).
Eyes harvested 17-21 days after immunization, or 14 days after
adoptive transfer, were prefixed in 4% phosphate-buffered
glutaraldehyde for 1 h (to prevent artifactual detachment of the
retina) and then transferred to 10% phosphate-buffered formaldehyde
until processing. Fixed and dehydrated tissue was embedded in
methacrylate, and 4- to 6-.mu.m sections were stained with standard
H&E. Eye sections cut through pupillary-optic nerve planes were
scored in a masked fashion. Severity of EAU was graded on a scale
of 0-4 in half-point increments using the criteria described
previously, based on the type, number, and size of lesions (see,
Agarwal and Caspi, supra; and Chan et al. supra).
D. Determination of Immunological Responses
[0118] Delayed Type Hypersensitivity (DTH) to IRBP was evaluated by
the ear swelling assay (see, e.g., Tarrant et al. (1998) J Immunol
161:122-127). For Ag-specific lymphocyte proliferation and cytokine
production in primary cultures, the spleen and draining lymph nodes
(inguinal and iliac) (5 per group) were collected at the end of
each experiment as indicated. Lymphoid cells were pooled within the
group, and were incubated with graded doses of Ag in triplicate
0.2-ml cultures, essentially as described (see, e.g., Avichezer et
al. (2000) Invest Ophthalmol V is Sci 41:127-131). Proliferation
was determined by [.sup.3H]thymidine uptake. Cytokines were
quantitated in 48-h Ag-stimulated supernatants using the Pierce
Multiplex SearchLight Arrays technology (see, e.g., Moody et al.
(2001) Biotechniques 31:186-190, 192-184).
E. Neutralization of IL-23, IL-12p40, and IL-17
[0119] B10RIII mice were immunized with IRBP or IRBP uveitogenic
peptide (161-180) as indicated. Mice were injected
intraperitoneally with 0.5 mg per dose of anti-p19, anti-p40, or
anti-IL-17. Treatment was given every other day starting on day--1
through day 15 after immunization, covering both priming and
effector phase (prevention protocol) or starting day 7 through day
15, covering the effector phase only (treatment). Controls were
given the same regimen of isotype (rat IgG1). Eyes and lymphoid
organs were harvested on day 17, 6-7 days after disease onset.
F. Uveitogenic T Cell Line
[0120] The uveitogenic Th1 cell line specific to a peptide of human
IRBP (p16-180) has been described (see, e.g., Silver et al. (1995)
Invest Ophthalmol V is Sci 36:946-954). Briefly, the line was
derived from draining lymph nodes of B10.RIII mice immunized with
human IRBP peptide 161-180, polarized in vitro toward the Th1
phenotype by culture in the presence of antigen, IL-12, and
anti-IL-4. Thereafter the cells were maintained by alternating
cycles of expansion in IL-2 and restimulation with 1 .mu.g/ml of
p161-180 every 2 to 3 weeks in the presence of syngeneic
splenocytes, irradiated with 3000 rads, as APCs. For EAU induction,
cells freshly stimulated with Ag for 48 h were injected i.p. into
naive syngeneic recipients.
G. Detection of Intracellular IL-17 and IFN.gamma.
[0121] Short stimulation: T cell line was stimulated with 1
.mu.g/ml RBP peptide 161-180 in the presence of irradiated APCs for
24 h with the addition of GolgiStop.TM. protein transfer inhibitor
(BD Biosciences, San Jose, Calif.) at the last 4 h. Thereafter,
cells were separated on Ficoll, washed and stained for
extracellular CD4. Than cells were washed, fixed, permeabilized
with Cytofix/Cytoperm.TM. fixation and permeabilization buffer (BD
Biosciences) and stained with PE-conjugated anti Il-17 and
APC-conjugated anti IFN-.gamma. for FACS analysis.
[0122] Long stimulation: T cell line was stimulated for 5 days with
antigen (10 .mu.g/ml IRBP peptide 161-180) or antigen+rIL-23 (10
ng/ml) or antigen+IL-23+anti IFN-.gamma. (10 .mu.g/ml) in the
presence of irradiated APCs. During the last 4 h of incubation
cells were stimulated with PMA and lonomycin with the addition of
GolgiStop.TM. protein transfer inhibitor (BD Biosciences).
Thereafter cells were treated and stained for intracellular IL-17
and IFN-.gamma. as mentioned above.
H. IL-17 and IFN.gamma. Assays
[0123] After 48 h of stimulation with 1 .mu.g/ml IRBP peptide
161-180 in the presence of irradiated APCs the T cell line was
adoptively transferred (2.times.10.sup.6/mouse) i.v. to naive
Thy1.1/0.2 heterozygous mice. Ninety h later spleens were harvested
and splenocytes were stimulated with IRBP peptide 161-180 for 24 h
with the presence of PMA, Ionomycin and GolgiStop.TM. protein
transfer inhibitor (BD Biosciences) at the last 4 h. Thereafter
cells were treated and stained for intracellular IL-17 and
IFN-.gamma. as mentioned above.
I. Statistical Analysis
[0124] Experiments were repeated at least twice, and usually three
or more times. Tables show data compiled from a representative
experiment. Statistical analysis of EAU scores, was by Snedecor and
Cochran's test for linear trend in proportions (nonparametric,
frequency-based) (see, e.g., Snedecor and Cochran (1967)
Statistical Methods Iowa State University Press, Ames, Iowa: p.
248). Each mouse (average of both eyes) was treated as one
statistical event. DTH and proliferation were examined by t-test (2
tailed). Cytokine responses were assayed on pooled samples (usually
5 mice per group).
[0125] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
[0126] All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
document was specifically and individually indicated to be
incorporated by reference. However, citation herein of any
publication or patent document is not intended as an admission that
the cited reference is pertinent prior art, nor does it constitute
any admission as to the contents or effective prior art date of the
reference.
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