U.S. patent application number 10/580164 was filed with the patent office on 2007-11-22 for method for the treatment of multiple sclerosis by inhibiting il-17 activity.
This patent application is currently assigned to Celltech R&D Limited. Invention is credited to Mark Ian Christie, Richard James Mead, Stephen Edward Rapecki, Martyn Kim Robinson.
Application Number | 20070269428 10/580164 |
Document ID | / |
Family ID | 34635441 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070269428 |
Kind Code |
A1 |
Christie; Mark Ian ; et
al. |
November 22, 2007 |
Method for the Treatment of Multiple Sclerosis by Inhibiting Il-17
Activity
Abstract
The present invention provides a method for the treatment and/or
prophylaxis of multiple sclerosis (MS) comprising administering a
therapeutically effective amount of an inhibitor of IL-17
activity.
Inventors: |
Christie; Mark Ian;
(Newmarket, GB) ; Mead; Richard James; (Sheffield,
GB) ; Robinson; Martyn Kim; (High Wycombe, GB)
; Rapecki; Stephen Edward; (Burnham, GB) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR
2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Celltech R&D Limited
208 Bath Road
Slough, Berkshire
GB
SL1 3WE
|
Family ID: |
34635441 |
Appl. No.: |
10/580164 |
Filed: |
November 16, 2004 |
PCT Filed: |
November 16, 2004 |
PCT NO: |
PCT/GB04/04850 |
371 Date: |
May 18, 2006 |
Current U.S.
Class: |
424/133.1 ;
424/130.1 |
Current CPC
Class: |
C07K 2317/55 20130101;
A61K 47/6845 20170801; A61P 25/28 20180101; C07K 2317/76 20130101;
A61P 43/00 20180101; A61P 37/06 20180101; A61K 47/60 20170801; C07K
16/244 20130101; A61K 2039/505 20130101; A61P 37/00 20180101 |
Class at
Publication: |
424/133.1 ;
424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 47/48 20060101 A61K047/48; A61P 37/00 20060101
A61P037/00; C07K 16/18 20060101 C07K016/18; C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 21, 2003 |
GB |
0327181.4 |
Jul 30, 2004 |
GB |
0417115.3 |
Claims
1-9. (canceled)
10. A method for the treatment or prophylaxis of multiple sclerosis
(MS) comprising administering a therapeutically effective amount of
an inhibitor of IL-17 activity to a patient in need thereof.
11. The method according to claim 10, wherein the inhibitor is a
small molecule.
12. The method according to claim 10, wherein the inhibitor is a
nucleic acid.
13. The method according to claim 10, wherein the inhibitor is an
antibody or a functionally active antibody fragment or
derivative.
14. The method according to claim 13, wherein the antibody or
antibody fragment is monoclonal, polyclonal, chimeric, humanized or
bispecific.
15. The method according to claim 13 wherein the antibody fragment
is a Fab, Fab', F(ab').sub.2, scFv or epitope binding fragment.
16. The method according to claim 13 wherein the antibody or
antibody fragment is conjugated to one or more effector
molecule(s).
17. The method according to claim 13 wherein the antibody or
antibody fragment binds to IL-17.
18. The method according to claim 13 wherein the antibody or
antibody fragment binds to IL-17R.
19. The method according to claim 10 wherein the inhibitor of IL-17
activity is administered in combination with one or more other
therapeutically active compounds.
Description
[0001] The present invention relates generally to methods of
treating multiple sclerosis and more specifically to the use of
inhibitors of IL-17 activity for the manufacture of a medicament
for the treatment of multiple sclerosis.
[0002] Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is a
pro-inflammatory cytokine which stimulates the secretion of a wide
range of other cytokines from various non-immune cells. IL-17 is
capable of inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and
G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial
and endothelial cells and is also able to induce ICAM-1 surface
expression, proliferation of T cells, and growth and
differentiation of CD34+human progenitors into neutrophils when
cocultured in presence of irradiated fibroblasts (Fossiez et al.,
1998, Int. Rev. Immunol. 16, 541-551). IL-17 is predominantly
produced by activated memory T cells and acts by binding to a
ubiquitously distributed cell surface receptor (IL-17R) (Yao et
al., 1997, Cytokine, 9, 794-800). A number of homologues of IL-17
have been identified which have both similar and distinct roles in
regulating inflammatory responses. For a review of IL-17
cytokine/receptor families see Dumont, 2003, Expert Opin. Ther.
Patents, 13, 287-303.
[0003] IL-17 may contribute to a number of diseases mediated by
abnormal immune responses, such as rheumatoid arthritis and air-way
inflammation, as well as organ transplant rejection and antitumour
immunity. Inhibitors of IL-17 activity are well known in the art,
for example an IL-17R:Fc fusion protein was used to demonstrate the
role of IL-17 in collagen-induced arthritis (Lubberts et al., J.
Immunol. 2001,167, 1004-1013) and neutralising polyclonal
antibodies have been used to reduce peritoneal adhesion formation
(Chung et al., 2002, J. Exp. Med., 195, 1471-1478). Neutralising
monoclonal antibodies are commercially available (R&D Systems
UK).
[0004] Multiple sclerosis (MS) is a chronic, inflammatory,
demyelinating disease of the central nervous system (CNS), which is
believed to result from a coordinated autoimmune attack against
myelin antigens. There is considerable clinical and pathological
heterogeneity in MS patients and the sequence of events that
initiate the disease remain largely unknown. The clinical
progression of MS may be largely attributed to three disease
processes; inflammation, demyelination and axonal
loss/neurodegeneration.
[0005] Immune mediated inflammatory lesions within the CNS are
thought to result primarily from an infiltration of autoreactive
CD4.sup.+ lymphocytes (Th1) which recognise myelin proteins
presented on MHC class II molecules by antigen presenting cells.
This interaction causes stimulation of Th1 cells which release
proinflammatory cytokines (mainly TNF-.alpha. & IFN-.gamma.)
resulting in proliferation of T-cells, activation of B-cells and
macrophages, upregulation of adhesion molecules and disruption of
the blood-brain barrier. Such events ultimately lead to loss of
oligodendrocytes & axons and the formation of a demyelinated
plaque. This is the hallmark of MS and consists of a demarcated
lesion where myelin sheaths are completely lost and demyelinated
axons are embedded in glial scar tissue. Demyelination may also
occur as a consequence of specific recognition and opsonization of
myelin antigens by autoantibodies. The most important target
antigen is suggested to be myelin oligodendrocyte protein (MOG),
which is present on the surface of the myelin sheath. Destruction
of antibody-opsonized myelin is then accomplished either by
complement or activated macrophages. Axonal loss and
neurodegeneration subsequent to inflammation are thought to be
responsible for the accumulation of irreversible neurological
impairment, characteristic of secondary progressive MS.
[0006] The clinical features of MS vary from headaches and blurred
vision to severe ataxia, blindness and paralysis. MS affects all
ages but first symptoms generally occur between 18 and 50 years and
disease duration has been estimated at >25 years with a
significant proportion of patients dying from causes unrelated to
MS. In the majority of patients (.about.80%) the disease takes a
relapsing-remitting (RR-MS) course with exacerbation of symptoms,
which is rapid in onset (hours to days) followed by a slower
recovery. The frequency and duration of relapses are unpredictable
but average 1.5 per year and can be followed by complete recovery.
With time, recovery from relapses may not be complete and a gradual
worsening of disease occurs. This worsening of disease is
independent of relapse rate and is classified as secondary
progressive MS (SP-MS), accounting for approximately 10% of MS
patients. The remaining 10% of MS patients have a primary
progressive (PP-MS) course where disability worsens at a steadv
rate from onset of the disease.
[0007] Currently licensed therapies are the beta-interferons;
Interferon beta-1b (Betaseron; Berlex), Interferon beta-1a (Avonex;
Biogen, Rebif; Serono) and glatimer acetate (Copaxone; Teva). These
agents have been shown to reduce relapse rate during the
relapsing-remitting phase of the disease in approximately 30% of
patients. There is currently no method available for identifying
the responder population before therapy. Intravenous steroids
(prednisolone is most commonly used) are used to hasten remission
after relapse but do not have long term efficacy. The anti-cancer
agent, mitoxantrone (Novantrone), is approved as an
immunosuppressant in progressive-relapsing and
secondary-progressive patients, but its use and dose is limited by
cardiotoxicity. In Europe azathioprine has also been used as an
immunosuppressant.
[0008] Prescribing decisions seem to be driven by evidence-based
medicine and a recent report by the American Association of
Neurologists (Goodin D S et al; Neurology Jan. 22,
2002;58(2):169-78) is a key document. The consensus amongst many
neurologists is that early, aggressive therapy with
beta-interferons was desirable in increasing the time to first
relapse and limiting the overall disease load, although it was
recognised that there was no evidence that this approach showed
long-term benefit on EDSS score (a measure of disease-related
disability). Beta-interferons were seen as sub-optimal therapy and
glatirimer acetate as having a different mechanism of action, which
may allow it to be used (alone or in combination) in patients that
do not respond to interferons. Individualised therapy based on
mechanistic (MRI, genetic, neurological) markers of disease was
seen as a worthwhile goal, as were therapies with a novel mechanism
of action. There is currently no satisfactory diagnostic marker for
multiple sclerosis.
[0009] There is a clear need for disease modifying therapies.
Agents with different mechanisms of action are needed and may allow
therapy to be tailored to different stages of the disease. An
orally active agent is yet to be licensed in the
relapsing-remitting form of the disease and this would represent a
clear improvement over current therapy if significant efficacy was
associated with the mechanism. Furthermore, there is a clear
requirement for therapies that show efficacy in the primary or
secondary progressive phases of the disease and have a reasonable
side-effect profile.
[0010] Whether IL-17 plays any kind of role in the pathogenesis of
MS is unknown. Microarray analysis of MS lesions obtained at
autopsy have revealed increased transcripts of many different genes
encoding inflammatory cytokines, including, IL-17 (Lock et al.,
2002, Nature Medicine, 8, 500-508). An increased number of IL-17
expressing mononuclear cells have been detected in blood and
cerebrospinal fluid from patients with MS (Matusevicius et al.,
1999, Multiple Sclerosis, 5, 101-104) but as the authors point out
cytokine mRNA expression is not necessarily identical to cytokine
protein production.
[0011] Surprisingly we have been able to demonstrate that
inhibitors of IL-17 activity are active in an animal model of MS.
Specifically we have been able to demonstrate that an anti-IL-17
antibody that inhibits IL-17 activity is active in animal models of
MS. Hence, the present invention provides a method for the
treatment and/or prophylaxis of MS comprising administering a
therapeutically effective amount of an inhibitor of IL-17 activity.
The invention also provides the use of an inhibitor of IL-17
activity for the manufacture of a medicament for the treatment
and/or prophylaxis of multiple sclerosis.
[0012] The term `IL-17 activity` as used herein refers to the
spectrum of activity understood in the art for IL-17 for example,
the induction of secretion of IL-6 or IL-8 from fibroblasts by
IL-17 (Yao et al., 1995, Journal of Immunology, 155,5483-5486).
[0013] An inhibitor of IL-17 activity according to the present
invention is an agent that interferes with the activity of IL-17,
in particular the activity of IL-17 in MS. Particularly preferred
are agents which interfere with the activity of IL-17 in MS in
humans. Inhibitors according to the present invention may partially
or completely inhibit IL-17 activity. Inhibitors of use in the
present invention include without limitation, inhibitors that are
capable of interacting with (e.g. binding to, or recognising) IL-17
or the IL-17 receptor (L17 R) or a nucleic acid molecule encoding
IL-17 or IL-17R, or are capable of inhibiting the expression of
IL-17 or IL-17R or are capable of inhibiting the interaction
between IL-17 and IL-17R. Such inhibitors may be, without
limitation, antibodies, nucleic acids (e.g. DNA, RNA, antisense RNA
and siRNA), carbohydrates, lipids, proteins, polypeptides,
peptides, peptidomimetics, small molecules and other drugs.
[0014] Examples of suitable inhibitors include, but are not limited
to, a synthetic functional fragment of the IL-17 receptor that
binds to IL-17 and interferes with binding to the native IL-17
receptor, an antibody that binds to IL-17 or to the IL-17 receptor
and interferes with IL-17 receptor-ligand interaction, an antisense
nucleic acid molecule that specifically hybridizes to mRNA encoding
IL-17 or the IL-17 receptor or a small molecule or other drug which
inhibits the activity of IL-17 or its receptor.
[0015] Inhibitors of IL-17 activity are well known in the art as
are methods of identifying and producing such inhibitors. Examples
include, IL-17R:Fc fusion proteins (Lubberts et al., J. Immunol.
2001,167, 1004-1013) and neutralising antibodies (Chung et al.,
2002, J. Exp. Med., 195, 1471-1478; Ferretti, 2003, Journal of
Immunology, 170, 2106-2112). Agents that may be suitable inhibitors
can be selected from a wide variety of candidate agents. Examples
of candidate agents include but are not limited to, nucleic acids
(e.g. DNA and RNA), carbohydrates, lipids, proteins, polypeptides,
peptides, peptidomimetics, small molecules and other drugs. Agents
can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is suited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
1997, Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; and
U.S. Pat. No. 5,807,683).
[0016] Examples of suitable methods based on the present
description for the synthesis of molecular libraries can be found
in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad.
Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA
91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et
al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed.
Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233.
[0017] Libraries of compounds may be presented, for example, in
solution (e.g. Houghten, 1992, Bio/Techniques 13:412-421), or on
beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature
364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat.
Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al.,
1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and
Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310).
[0018] In one example, the inhibitor for use in the present
invention may be a nucleic acid. In particular IL-17 or IL-17R
nucleic acid molecules maybe used as anti-sense molecules, to alter
the expression of their respective polypeptides by binding to
complementary nucleic acids. IL-17 or IL-17R nucleic acids may be
obtained using standard cloning techniques from for example genomic
DNA or cDNA or can be synthesised using well known and commercially
available techniques. The IL-17 or IL-17R nucleic acids may contain
one or more nucleotide substitutions, additions or deletions into
the nucleotide sequence of an IL-17 or IL-17R nucleic acid.
Standard techniques known to those of skill in the art can be used
to introduce mutations, including, for example, site-directed
mutagenesis and PCR-mediated mutagenesis. An antisense nucleic acid
according to the present invention includes a IL-17 or IL-17R
nucleic acid capable of hybridising by virtue of some sequence
complementarity to a portion of an RNA (preferably mRNA) encoding
the respective polypeptide. The antisense nucleic acid can be
complementary to a coding and/or non-coding region of an mRNA
encoding such a polypeptide. Most preferably, the antisense nucleic
acids result in inhibition of the expression of the IL-17 or IL-17R
polypeptide. Thus, the present invention provides a method for the
treatment and/or prophylaxis of MS comprising administering a
therapeutically effective amount of an inhibitor of IL-17 activity
wherein the inhibitor comprises at least eight nucleotides that are
antisense to a gene or cDNA encoding a IL-17 or IL-17R polypeptide.
The invention also provides the use of nucleic acids comprising at
least eight nucleotides that are antisense to a gene or cDNA
encoding a IL-17 or IL-17R polypeptide for the manufacture of a
medicament for the treatment and/or prophylaxis of MS.
[0019] Most preferably, an inhibitor for use in the treatment
and/or prophylaxis of MS is an antibody that interacts with (i.e.
binds to or recognises) IL-17 or its receptor and inhibits the
activity of IL-17. Accordingly, there is provided the use of an
antibody that inhibits the activity of IL-17 for the manufacture of
a medicament for the treatment and/or prophylaxis of MS. Also
provided is a method of treatment and/or prophylaxis of MS in a
subject comprising administering to said subject a therapeutically
effective amount of an antibody that inhibits the activity of
IL-17.
[0020] In one example the antibodies selectively interact with
IL-17. Selectively interacting with (e.g. recognising or binding
to) means that the antibodies have a greater affinity for IL-17
polypeptides than for other polypeptides. Examples of suitable
antibodies are those that inhibit the activity of IL-17 by binding
to IL-17 in such a manner as to prevent it being biologically
active, for example by preventing the binding of IL-17 to its
receptor. Accordingly, there is provided by the present invention
the use of an anti-IL-17 antibody for the manufacture of a
medicament for the treatment and/or prophylaxis of MS. Also
provided is a method of treatment and/or prophylaxis of MS in a
subject comprising administering to said subject a therapeutically
effective amount of an anti-IL-17 antibody.
[0021] In another example the antibodies selectively interact with
the IL-17 receptor. Selectively interacting with (e.g. recognising
or binding to) means that the antibodies have a greater affinity
for the IL-17 receptor polypeptide than for other polypeptides.
Examples of suitable antibodies are those that inhibit the activity
of IL-17 by preventing IL-17 mediated signalling from the receptor,
for example by preventing IL-17 from binding to the IL-17 receptor.
Accordingly, there is provided by the present invention the use of
an anti-IL-7R antibody for the manufacture of a medicament for the
treatment and/or prophylaxis of MS. Also provided is a method of
treatment and/or prophylaxis of MS in a subject comprising
administering to said subject a therapeutically effective amount of
an anti-IL-17R antibody.
[0022] IL-17 or IL-17 receptor polypeptides or cells expressing
said polypeptides can be used to produce antibodies which
specifically recognise said polypeptides. The IL-17 and IL-17R
polypeptides may be `mature` polypeptides or biologically active
fragments or derivatives thereof. IL-17 and IL-17R polypeptides may
be prepared by processes well known in the art from genetically
engineered host cells comprising expression systems or they may be
recovered from natural biological sources. In the present
application, the term "polypeptides" includes peptides,
polypeptides and proteins. These are used interchangeably unless
otherwise specified. IL-17 or IL-17R polypeptides may in some
instances be part of a larger protein such as a fusion protein for
example fused to an affinity tag. Antibodies generated against
these polypeptides may be obtained by administering the
polypeptides to an animal, preferably a non-human animal, using
well-known and routine protocols, see for example Handbook of
Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell
Scientific Publishers, Oxford, England, 1986. Many warm-blooded
animals, such as rabbits, mice, rats, sheep, chickens, cows or pigs
may be immunised. However, mice, rabbits, pigs and rats are
generally preferred.
[0023] Anti-IL-17 and anti-IL-17 receptor antibodies for use in the
present invention include whole antibodies and functionally active
fragments or derivatives thereof and may be, but are not limited
to, polyclonal, monoclonal, multi-valent, multi-specific, humanized
or chimeric antibodies, single chain antibodies, Fab fragments,
Fab' and F(ab').sub.2 fragments, fragments produced by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. Antibodies include
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e. molecules that contain an antigen
binding site that specifically binds an antigen. The immunoglobulin
molecules of the invention can be of any class (e.g. IgG, IgE, IgM,
IgD and IgA) or subclass of immunoglobulin molecule.
[0024] Monoclonal antibodies may be prepared by any method known in
the art such as the hybridoma technique (Kohler & Milstein,
1975, Nature, 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72)
and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies
and Cancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).
[0025] Antibodies for use in the invention may also be generated
using single lymphocyte antibody methods by cloning and expressing
immunoglobulin variable region cDNAs generated from single
lymphocytes selected for the production of specific antibodies by
for example the methods described by Babcook, J. et al., 1996,
Proc. Natl. Acad. Sci. USA 93(15):7843-7848 and in WO92/02551.
[0026] Humanized antibodies are antibody molecules from non-human
species having one or more complementarity determining regions
(CDRs) from the non-human species and a framework region from a
human immunoglobulin molecule (see, e.g. U.S. Pat. No.
5,585,089).
[0027] Chimeric antibodies are those antibodies encoded by
immunoglobulin genes that have been genetically engineered so that
the light and heavy chain genes are composed of immunoglobulin gene
segments belonging to different species. These chimeric antibodies
are likely to be less antigenic. Bivalent antibodies may be made by
methods known in the art (Milstein et al., 1983, Nature
305:537-539; WO 93/08829, Traunecker et al., 1991, EMBO J.
10:3655-3659). Multi-valent antibodies may comprise multiple
specificities or may be monospecific (see for example WO
92/22853).
[0028] The antibodies for use in the present invention can also be
generated using various phage display methods known in the art and
include those disclosed by Brinkman et al. (in J. Immunol. Methods,
1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995,
184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994,
24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al.
(Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
Techniques for the production of single chain antibodies, such as
those described in U.S. Pat. No. 4,946,778 can also be adapted to
produce single chain antibodies to IL-17 or IL-17R polypeptides.
Also, transgenic mice, or other organisms, including other mammals,
may be used to express humanized antibodies.
[0029] Antibody fragments and methods of producing them are well
known in the art, see for example Verma et al., 1998, Journal of
Immunological Methods, 216, 165-181.
[0030] Particular examples of antibody fragments for use in the
present invention are Fab' fragments which possess a native or a
modified hinge region. A number of modified hinge regions have
already been described, for example, in U.S. Pat. No. 5,677,425,
WO9915549, and WO9825971 and these are incorporated herein by
reference
[0031] Further examples of particular antibody fragments for use in
the present invention include those described in International
patent applications PCT/GB2004/002810, PCT/GB2004/002870 and
PCT/GB2004/002871 (all filed on 1 Jul. 2004). In particular the
modified antibody Fab fragments described in International patent
application PCT/GB2004/002810 are preferred. These Fab fragments
comprise a heavy and light chain pair, V.sub.H/C.sub.H1 and
V.sub.L/C.sub.L covalently linked through interchain cysteines in
the heavy and light chain constant regions and are characterised in
that the heavy chain constant region terminates at the interchain
cysteine of C.sub.H1. The term `interchain cysteine` refers to a
cysteine in the heavy or light chain constant region that would be
disulphide linked to a cysteine in the corresponding heavy or light
chain constant region encoded in a naturally occurring germline
antibody gene. In particular the interchain cysteines are a
cysteine in the constant region of the light chain (C.sub.L) and a
cysteine in the first constant region of the heavy chain (C.sub.H1)
that are disulphide linked to each other in naturally occurring
antibodies. Examples of such cysteines may typically be found at
position 214 of the light chain and position 233 of the heavy chain
of human IgG1, position 127 of the heavy chain of human IgM, IgE,
IgG2, IgG3, IgG4 and position 128 of the heavy chain of human IgD
and IgA2B, as defined by Kabat et al., 1987, in Sequences of
Proteins of Immunological Interest, US Department of Health and
Human Services, NIH, USA. In murine IgG, interchain cysteines may
be found at position 214 of the light chain and position 235 of the
heavy chain. It will be appreciated that the exact positions of
these cysteines may vary from that of naturally occurring
antibodies if any modifications, such as deletions, insertions
and/or substitutions have been made to the antibody Fab fragment.
These antibody Fab fragments may be prepared by any suitable method
known in the art. For example, the antibody Fab fragment may be
obtained from any whole antibody, especially a whole monoclonal
antibody, using any suitable enzymatic cleavage and/or digestion
techniques, for example by treatment with pepsin or papain and
c-terminal proteases. Preferably these antibody Fab fragments are
prepared by the use of recombinant DNA techniques involving the
manipulation and re-expression of DNA encoding antibody variable
and constant regions. Standard molecular biology techniques may be
used to modify, add or delete further amino acids or domains as
desired. Any alterations to the variable or constant regions are
still encompassed by the terms `variable` and `constant` regions as
used herein. Preferably PCR is used to introduce a stop codon
immediately following the codon encoding the interchain cysteine of
C.sub.H1, such that translation of the C.sub.H1 domain stops at the
interchain cysteine. Methods for designing suitable PCR primers are
well known in the art and the sequences of antibody C.sub.H1
domains are readily available (Kabat et al., supra). Alternatively
stop codons may be introduced using site-directed mutagenesis
techniques such as those described in White (Ed.), PCR Protocols:
Current Methods and Applications (1993). In one example the
constant regions in these fragments are derived from IgG1 and the
interchain cysteine of C.sub.L is at position 214 of the light
chain and the interchain cysteine of C.sub.H1 is at position 233 of
the heavy chain. Examples of human and murine constant region
sequences for use in these fragments are provided in SEQ ID Nos 1-4
and FIG. 13; human heavy chain constant region C.sub.H1 which
terminates at the interchain cysteine (SEQ ID NO:1); human light
chain constant region (SEQ ID NO:2); murine heavy chain constant
region C.sub.H1 which terminates at the interchain cysteine (SEQ ID
NO:3); murine light chain constant region (SEQ ID NO:4).
[0032] If desired an antibody for use in the present invention may
be conjugated to one or more effector molecule(s). The term
effector molecule as used herein includes, for example,
antineoplastic agents, drugs, toxins, biologically active proteins,
for example enzymes, other antibody or antibody fragments,
synthetic or naturally occurring polymers, nucleic acids and
fragments thereof e.g. DNA, RNA and fragments thereof,
radionuclides, particularly radioiodide, radioisotopes, chelated
metals, nanoparticles and reporter groups such as fluorescent
compounds or compounds which may be detected by NMR or ESR
spectroscopy. In one example, anti-IL-17 or anti IL-17R antibodies
can be conjugated to an effector molecule, such as a cytotoxic
agent, a radionuclide or drug moiety to modify a given biological
response. For example, the therapeutic agent may be a drug moiety
which may be a protein or polypeptide possessing a desired
biological activity. Such moieties may include, for example and
without limitation, a toxin such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin, a protein such as tumour necrosis
factor, .alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor or tissue plasminogen activator, a
thrombotic agent or an anti-angiogenic agent, e.g. angiostatin or
endostatin, or, a biological response modifier such as a
lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-6 (IL-6), granulocyte macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
nerve growth factor (NGF) or other growth factor.
[0033] In another example the effector molecules may be cytotoxins
or cytotoxic agents including any agent that is detrimental to
(e.g. kills) cells. Examples include taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Effector molecules also include, but are not
limited to, antimetabolites (e.g. methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g. daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin (AMC), calicheamicins or duocarmycins),
and anti-mitotic agents (e.g. vincristine and vinblastine).
[0034] Other effector molecules may include radionuclides such as
.sup.111In and .sup.90Y, Lu.sup.177, Bismuth.sup.213,
Californium.sup.252, Iridium.sup.192 and
Tungsten.sup.188/Rhenium.sup.188; or drugs such as but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin. Techniques for conjugating such effector molecules to
antibodies are well known in the art (see, Hellstrom et al.,
Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp.
623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik
et al., 1999, Pharmacology and Therapeutics, 83, 67-123). In one
example, the antibody or fragment thereof is fused via a covalent
bond (e.g. a peptide bond), at optionally the N-terminus or the
C-terminus, to an amino acid sequence of another protein (or
portion thereof; preferably at least a 10, 20 or 50 amino acid
portion of the protein). Preferably the antibody, or fragment
thereof, is linked to the other protein at the N-terminus of the
constant domain of the antibody. Recombinant DNA procedures may be
used to create such fusions, for example as described in WO
86/01533 and EP 0392745.
[0035] In another example the effector molecule may increase
half-life in vivo, and/or decrease immunogenicity and/or enhance
the delivery of an antibody across an epithelial barrier to the
immune system. Examples of suitable effector molecules include
polymers and proteins such as albumin and albumin binding proteins.
Examples of suitable polymers include any synthetic or naturally
occurring substantially water-soluble, substantially non-antigenic
polymer including, for example, optionally substituted straight or
branched chain polyalkylene, polyalkenylene, or polyoxyalkylene
polymers or branched or unbranched polysaccharides, e.g. a homo- or
hetero-polysaccharide such as lactose, amylose, dextran or
glycogen. Particular optional substituents which may be present on
the above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups. Particular examples of synthetic polymers
include optionally substituted straight or branched chain
poly(ethyleneglycol), poly(propyleneglycol), poly(vinylalcohol) or
derivatives thereof, especially optionally substituted
poly(ethyleneglycol) such as methoxypoly(ethyleneglycol).
Preferably the polymer is a polyalkylene oxide such as polyethylene
glycol (PEG).
[0036] In one example antibodies for use in the present invention
are attached to poly(ethyleneglycol) (PEG) moieties. In one
particular example the antibody is an antibody fragment and the PEG
molecules may be attached through any available amino acid
side-chain or terminal amino acid functional group located in the
antibody fragment, for example any free amino, imino, thiol,
hydroxyl or carboxyl group. Such amino acids may occur naturally in
the antibody fragment or may be engineered into the fragment using
recombinant DNA methods. See for example U.S. Pat. No. 5,219,996.
Multiple sites can be used to attach two or more PEG molecules.
Preferably PEG molecules are covalently linked through a thiol
group of at least one cysteine residue located in the antibody
fragment. Where a thiol group is used as the point of attachment
appropriately activated effector molecules, for example thiol
selective derivatives such as maleimides and cysteine derivatives
may be used.
[0037] Preferably, the antibody is a modified Fab fragment, such as
a Fab' which is PEGylated, i.e. has PEG (poly(ethyleneglycol))
covalently attached thereto, e.g. according to the method disclosed
in EP 0948544 [see also "Poly(ethyleneglycol) Chemistry,
Biotechnical and Biomedical Applications", 1992, J. Milton Harris
(ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and
Biological Applications", 1997, J. Milton Harris and S. Zalipsky
(eds), American Chemical Society, Washington D.C. and
"Bioconjugation Protein Coupling Techniques for the Biomedical
Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York;
Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545].
The total amount of PEG attached to the fragment may be varied as
desired, but will generally be in an average molecular weight range
from 250 to 100,000Da, preferably from 5,000 to 50,000Da, more
preferably from 10,000 to 40,000Da and still more preferably from
20,000 to 40,000Da The size of PEG may in particular be selected on
the basis of the intended use of the product, for example ability
to localize to certain tissues such as tumors or extend circulating
half-life (for review see Chapman, 2002, Advanced Drug Delivery
Reviews, 54, 531-545).
[0038] In one embodiment PEG is attached to a cysteine in the hinge
region of a Fab'. In one example, a PEG modified Fab' fragment has
a maleimide group covalently linked to a single thiol group in a
modified hinge region. A lysine residue may be covalently linked to
the maleimide group and to each of the amine groups on the lysine
residue may be attached a methoxypoly(ethyleneglycol)polymer having
a molecular weight of approximately 20,000 Da. The total molecular
weight of the PEG attached to the Fab' fragment may therefore be
approximately 40,000 Da.
[0039] In another preferred embodiment an antibody fragment for use
in the present invention is a PEGylated (i.e. has PEG
(poly(ethyleneglycol)) covalently attached thereto) Fab fragment as
described in International Application Number PCT/GB2004/002810
(filed on 1 Jul. 2004). This PEGylated Fab fragment is a Fab
fragment in which the heavy chain terminates at the interchain
cysteine of C.sub.H1 and the PEG attached to the fragment,
preferably PEG-maleimide, is covalently linked to the interchain
cysteine of C.sub.L and the interchain cysteine of C.sub.H1.
Preferably the interchain cysteine of C.sub.L is at position 214 of
the light chain and the interchain cysteine of C.sub.H1 is at
position 233 of the heavy chain. As discussed above the total
amount of PEG attached to the fragment may be varied as desired. In
one example each polymer attached to the Fab preferably has a
molecular weight of approximately 20,000 Da. For example, the
molecular weight may be 15,000-25,000Da, or preferably
18,000-22,000Da, and even more preferably 19,000-21,000Da. The
total molecular weight of the PEG attached to the antibody is
therefore approximately 40,000 Da.
[0040] PEG is attached to these fragments by first reducing the
interchain disulphide bond between the interchain cysteines of
C.sub.L and C.sub.H1 and subsequently attaching the PEG to the free
thiols. Once PEG is attached to the interchain cysteines there is
no interchain disulphide linkage between the heavy and light chain.
Suitable reducing agents for reducing the interchain disulphide
bond are widely known in the art for example those described in
Singh et al., 1995, Methods in Enzymology, 251, 167-73. Particular
examples include thiol based reducing agents such as reduced
glutathione (GSH), .beta.-mercaptoethanol (.beta.-ME),
.beta.-mercaptoethylamine (.beta.-MA) and dithiothreitol (DTT).
Other methods include using electrolytic methods, such as the
method described in Leach et al., 1965, Div. Protein. Chem, 4,
23-27 and using photoreduction method such as the method described
in Ellison et al., 2000, Biotechniques, 28 (2), 324-326. Preferably
however, the reducing agent is a non-thiol based reducing agent,
preferably one of the trialkylphosphine reducing agents (Ruegg U T
and Rudinger, J., 1977, Methods in Enzymology, 47, 111-126; Burns J
et al., 1991, J. Org. Chem, 56, 2648-2650; Getz et al., 1999,
Analytical Biochemistry, 273, 73-80; Han and Han, 1994, Analytical
Biochemistry, 220, 5-10; Seitz et al., 1999, Euro. J. Nuclear
Medicine, 26, 1265-1273), particular examples of which include
tris(2-carboxyethyl)phosphine (TCEP), tris butyl phosphine (TBP),
tris-(2-cyanoethyl) phosphine, tris-(3-hydroxypropyl)phosphine
(THP) and tris-(2-hydroxyethyl)phosphine. Most preferred are the
reducing agents TCEP and THP. It will be clear to a person skilled
in the art that the concentration of reducing agent can be
determined empirically, for example, by varying the concentration
of reducing agent and measuring the number of free thiols produced.
Typically the reducing agent is used in excess over the antibody
fragment for example between 2 and 1000 fold molar excess.
Preferably the reducing agent is in 2, 3, 4, 5, 10, 100 or 1000
fold excess. In one embodiment the reductant is used at between 2
and 5 mM.
[0041] The reduction and PEGylation reactions may generally be
performed in a solvent, for example an aqueous buffer solution such
as acetate or phosphate, at around neutral pH, for example around
pH 4.5 to around pH 8.5, typically pH 4.5 to 8, suitably pH6 to 7.
The reactions may generally be performed at any suitable
temperature, for example between about 5.degree. C. and about
70.degree. C., for example at room temperature. The solvent may
optionally contain a chelating agent such as EDTA, EGTA, CDTA or
DTPA. Preferably the solvent contains EDTA at between 1 and 5 mM,
preferably 2 mM. Alternatively or in addition the solvent may be a
chelating buffer such as citric acid, oxalic acid, folic acid,
bicine, tricine, tris or ADA. The PEG will generally be employed in
excess concentration relative to the concentration of the antibody
fragment. Typically the PEG is in between 2 and 100 fold molar
excess, preferably 5, 10 or 50 fold excess.
[0042] Where necessary, the desired product containing the desired
number of PEG molecules may be separated from any starting
materials or other product generated during the production process
by conventional means, for example by chromatography techniques
such as ion exchange, size exclusion, protein A, G or L affinity
chromatography or hydrophobic interaction chromatography. To
identify inhibitors of IL-17 activity a number of different
approaches may be taken by those skilled in the art. In one example
inhibitors are identified by first identifying agents that interact
with IL-17 or IL-17R and subsequently testing those agents to
identify those that inhibit IL-17 activity. In one such example the
agent is an antibody.
[0043] Agents that interact with IL-17 or IL-17R may be identified
using any suitable method, for example by using a cell-free or
cell-based assay system where the IL-17 or IL-17R polypeptide is
contacted with a candidate agent and the ability of the candidate
agent to interact with the polypeptide is determined. Preferably,
the ability of a candidate agent to interact with a IL-17 or IL-17R
polypeptide is compared to a reference range or control. If
desired, this assay may be used to screen a plurality (e.g. a
library) of candidate agents using a plurality of IL-17 or IL-17R
polypeptide samples. In one example of a cell free assay, a first
and second sample comprising native or recombinant IL-17 or IL-17R
polypeptide are contacted with a candidate agent or a control agent
and the ability of the candidate agent to interact with the
polypeptide is determined by comparing the difference in
interaction between the candidate agent and control agent.
Preferably, the polypeptide is first immobilized, by, for example,
contacting the polypeptide with an immobilized antibody which
specifically recognizes and binds it, or by contacting a purified
preparation of polypeptide with a surface designed to bind
proteins. The polypeptide may be partially or completely purified
(e.g. partially or completely free of other polypeptides) or part
of a cell lysate. Further, the polypeptide may be a fusion protein
comprising the IL-17 or IL-17R polypeptide or a biologically active
portion thereof and a domain such as glutathionine-S-transferase or
the Fc region of IgG1. Alternatively, the polypeptide can be
biotinylated using techniques well known to those of skill in the
art (e.g. biotinylation kit, Pierce Chemicals; Rockford, Ill.). The
ability of the candidate agent to interact with the polypeptide can
be determined by methods known to those of skill in the art for
example, ELISA, BIAcore.TM., Flow cytometry or fluorescent
microvolume assay technology (FMAT). In another example where a
cell-based assay is used, a population of cells expressing IL-17 or
IL-17R is contacted with a candidate agent and the ability of the
candidate agent to interact with the polypeptide is determined.
Preferably, the ability of a candidate agent to interact with IL-17
or IL-17R is compared to a reference range or control. The cell,
for example, can be of eukaryotic origin (e.g. yeast or mammalian)
and can express the IL-17 or IL-17R polypeptide endogenously or be
genetically engineered to express the polypeptide. In some
instances, the IL-17 or IL-17R polypeptide or the candidate agent
is labelled, for example with a radioactive label (such as
.sup.32P, .sup.35S or .sup.125I) or a fluorescent label (such as
fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde or fluorescamine) to enable
detection of an interaction between a polypeptide and a candidate
agent. Alternative methods such as ELISA, flow cytometry and FMAT
may also be used.
[0044] Agents which inhibit IL-17 activity may be identified by any
suitable method, for example by: [0045] (i) comparing the activity
of IL-17 in the presence of a candidate agent with the activity of
said polypeptide in the absence of the candidate agent or in the
presence of a control agent; and [0046] (ii) determining whether
the candidate agent inhibits activity of IL-17.
[0047] Such assays can be used to screen candidate agents, in
clinical monitoring or in drug development.
[0048] As described above, agents may be pre-screened where
appropriate to identify agents (e.g. an antibody) that interact
with IL-17 or IL-17R prior to screening those agents which bind for
their ability to inhibit IL-17 activity.
[0049] In one example a cell-based assay system is used to identify
agents capable of inhibiting the activity of IL-17. In one
particular example the assay used to identify inhibitors of IL-17
activity is the standard IL-6 release assay from fibroblasts (Yao
et al., 1995, Journal of Immunology, 155,5483-5486). Potential
inhibitors are added to the assay and IL-6 release determined by
ELISA. Inhibition is therefore measured as a reduction in IL-6
release compared to controls.
[0050] In another example inhibitors of IL-17 may down-regulate the
expression of the IL-17 or IL-17R polypeptide, for example
antisense inhibitors. Such inhibitors may be identified by any
method known in the art. In one example such inhibitors are
identified in a cell-based assay system. Accordingly, a population
of cells expressing a IL-17 or IL-17R polypeptide or nucleic acid
are contacted with a candidate agent and the ability of the
candidate agent to alter expression of the IL-17 or IL-17R
polypeptide or nucleic acid is determined by comparison to a
reference range or control. In one example, populations of cells
expressing a IL-17 or IL-17R polypeptide are contacted with a
candidate agent or a control agent and the ability of the candidate
agent to alter the expression of the IL-17 or IL-17R polypeptides
or nucleic acids is determined by comparing the difference in the
level of expression of the IL-17 or IL-17R polypeptides or nucleic
acids between the treated and control populations of cells. If
desired, this assay may be used to screen a plurality (e.g. a
library) of candidate agents. The cell, for example, can be of
eukaryotic origin (e.g. yeast or mammalian) and can express an
IL-17 or IL-17R polypeptide endogenously or be genetically
engineered to express a IL-17 or IL-17R polypeptide. The ability of
the candidate agents to alter the expression of a said polypeptides
or nucleic acids can be determined by methods known to those of
skill in the art, for example and without limitation, by flow
cytometry, radiolabelling, a scintillation assay,
immunoprecipitation, Western blot analysis, Northern blot analysis
or RT-PCR.
[0051] Agents that inhibit the activity of IL-17 may be identified
or further tested, for example to determine therapeutically
effective amounts in one or more animal models. Examples of
suitable animals include, but are not limited to, mice, rats,
rabbits, monkeys, guinea pigs, dogs and cats. Preferably, the
animal used represents a model of MS.
[0052] In one example where the agent inhibits the expression of
IL-17 or IL-17R, a first and second group of mammals are
administered with a candidate agent or a control agent and the
ability of the candidate agent to inhibit the expression of IL-17
or IL-17R polypeptide or nucleic acid is determined by comparing
the difference in the level of expression between the first and
second group of mammals. Where desired, the expression levels of
the IL-17 or IL-17R polypeptides or nucleic acid in the first and
second groups of mammals can be compared to the level of IL-17 or
IL-17R polypeptide or nucleic acid in a control group of mammals.
The candidate agent or a control agent can be administered by means
known in the art (e.g. orally, rectally or parenterally such as
intraperitoneally or intravenously). Changes in the expression of a
polypeptide or nucleic acid can be assessed by the methods outlined
above.
[0053] In another example, the inhibition of IL-17 activity can be
determined by monitoring an amelioration or improvement in disease
symptoms, a delayed onset or slow progression of the disease, for
example but without limitation, a reduction in paralysis.
Techniques known to physicians familiar with MS can be used to
determine whether a candidate agent has altered one or more
symptoms associated with the disease.
[0054] A number of different models of MS are known in the art ('t
Hart and Amor 2003, Current Opinion in Neurology 16:375-83). In
particular, experimental autoimmune encephalomyelitis (EAE) in ABH
mice is considered to be a relevant model for MS in humans (Baker
et al., 1990. Journal of Neuroimmunology, 28:261-270). Both acute
and relapsing-remitting models have been developed.
[0055] As discussed herein, inhibitors of IL-17 activity can be
used in the treatment and/or prophylaxis of MS. For such use the
agents will generally be administered in the form of a
pharmaceutical composition.
[0056] Also provided is a pharmaceutical composition comprising an
inhibitor of IL-17 activity and a pharmaceutically acceptable
carrier.
[0057] The term `treatment` includes either therapeutic or
prophylactic therapy. When a reference is made herein to a method
of treating or preventing a disease or condition using a particular
inhibitor or combination of inhibitors, it is to be understood that
such a reference is intended to include the use of that inhibitor
or combination of inhibitors for the manufacture of a medicament
for the treatment and/or prophylaxis of MS.
[0058] The composition will usually be supplied as part of a
sterile, pharmaceutical composition that will normally include a
pharmaceutically acceptable carrier. This composition may be in any
suitable form (depending upon the desired method of administering
it to a patient).
[0059] The inhibitors of use in the invention are preferably
administered to a subject by a variety of other routes such as
orally, transdermally, subcutaneously, intranasally, intravenously,
intramuscularly, intrathecally and intracerebroventricularly. The
most suitable route for administration in any given case will
depend on the particular inhibitor, the subject, and the nature and
severity of the disease and the physical condition of the
subject.
[0060] The inhibitors of use in the invention may be administered
in combination, e.g. simultaneously, sequentially or separately,
with one or more other therapeutically active compounds, which may
be for example other anti-MS therapies or anti-cancer
therapies.
[0061] Pharmaceutical compositions may be conveniently presented in
unit dose forms containing a predetermined amount of an active
agent of the invention per dose. Such a unit may contain for
example but without limitation, 750 mg/kg to 0.1 mg/kg depending on
the condition being treated, the route of administration and the
age, weight and condition of the subject.
[0062] Pharmaceutically acceptable carriers for use in the
invention may take a wide variety of forms depending, e.g. on the
route of administration.
[0063] Compositions for oral administration may be liquid or solid.
Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Oral liquid
preparations may contain suspending agents as known in the art.
[0064] In the case of oral solid preparations such as powders,
capsules and tablets, carriers such as starches, sugars,
microcrystalline cellulose granulating agents, lubricants, binders,
disintegrating agents, and the like may be included. Because of
their ease of administration, tablets and capsules represent the
most advantageous oral dosage unit form in which case solid
pharmaceutical carriers are generally employed. In addition to the
common dosage forms set out above, active agents of the invention
may also be administered by controlled release means and/or
delivery devices. Tablets and capsules may comprise conventional
carriers or excipients such as binding agents for example, syrup,
acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone;
fillers, for example lactose, sugar, maize-starch, calcium
phosphate, sorbitol or glycine; tableting lubricants, for example
magnesium stearate, talc, polyethylene glycol or silica;
disintegrants, for example potato starch; or acceptable wetting
agents such as sodium lauryl sulphate. The tablets may be coated by
standard aqueous or non-aqueous techniques according to methods
well known in normal pharmaceutical practice.
[0065] Pharmaceutical compositions of the present invention
suitable for oral administration may be presented as discrete units
such as capsules, cachets or tablets, each containing a
predetermined amount of the active agent, as a powder or granules,
or as a solution or a suspension in an aqueous liquid, a
non-aqueous liquid, an oil-in-water emulsion or a water-in-oil
liquid emulsion. Such compositions may be prepared by any of the
methods of pharmacy but all methods include the step of bringing
into association the active agent with the carrier, which
constitutes one or more necessary ingredients. In general, the
compositions are prepared by uniformly and intimately admixing the
active agent with liquid carriers or finely divided solid carriers
or both, and then, if necessary, shaping the product into the
desired presentation. For example, a tablet may be prepared by
compression or moulding, optionally with one or more accessory
ingredients.
[0066] Pharmaceutical compositions suitable for parenteral
administration may be prepared as solutions or suspensions of the
active agents of the invention in water suitably mixed with a
surfactant such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0067] The pharmaceutical forms suitable for injectable use include
aqueous or non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the composition isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. Extemporaneous
injection solutions, dispersions and suspensions may be prepared
from sterile powders, granules and tablets.
[0068] Pharmaceutical compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
pharmaceutical composition of the invention can be administered
with a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicaments through the skin; U.S. Pat. No.
4,447,233, which discloses a medication infusion pump for
delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which, discloses a variable flow implantable infusion
apparatus for continuous drug delivery, U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments; and U.S. Pat. No. 4,475,196, which
discloses an osmotic drug delivery system. Many other such
implants, delivery systems, and modules are known to those skilled
in the art.
[0069] Pharmaceutical compositions adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, impregnated dressings,
sprays, aerosols or oils, transdermal devices, dusting powders, and
the like. These compositions may be prepared via conventional
methods containing the active agent. Thus, they may also comprise
compatible conventional carriers and additives, such as
preservatives, solvents to assist drug penetration, emollients in
creams or ointments and ethanol or oleyl alcohol for lotions. Such
carriers maybe present as from about 1% up to about 98% of the
composition. More usually they will form up to about 80% of the
composition. As an illustration only, a cream or ointment is
prepared by mixing sufficient quantities of hydrophilic material
and water, containing from about 5-10% by weight of the compound,
in sufficient quantities to produce a cream or ointment having the
desired consistency.
[0070] Pharmaceutical compositions adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. For example, the active agent may be
delivered from the patch by iontophoresis.
[0071] For applications to external tissues, for example the mouth
and skin, the compositions are preferably applied as a topical
ointment or cream When formulated in an ointment, the active agent
may be employed with either a paraffinic or a water-miscible
ointment base. Alternatively, the active agent may be formulated in
a cream with an oil-in-water cream base or a water-in-oil base.
[0072] Pharmaceutical compositions adapted for topical
administration in the mouth include lozenges, pastilles and mouth
washes.
[0073] Pharmaceutical compositions adapted for topical
administration to the eye include eye drops wherein the active
agent is dissolved or suspended in a suitable carrier, especially
an aqueous solvent. They also include topical ointments or creams
as above.
[0074] Pharmaceutical compositions suitable for rectal
administration wherein the carrier is a solid are most preferably
presented as unit dose suppositories. Suitable carriers include
cocoa butter or other glyceride or materials commonly used in the
art, and the suppositories may be conveniently formed by admixture
of the combination with the softened or melted carrier(s) followed
by chilling and shaping moulds. They may also be administered as
enemas.
[0075] The dosage to be administered of an inhibitor of IL-17
activity will vary according to the particular inhibitor, the type
of MS, the subject, and the nature and severity of the disease and
the physical condition of the subject, and the selected route of
administration; the appropriate dosage can be readily determined by
a person skilled in the art. For the treatment and/or prophylaxis
of MS in humans and animals pharmaceutical compositions comprising
antibodies can be administered to patients (e.g., human subjects)
at therapeutically or prophylactically effective dosages (e.g.
dosages which result in inhibition of MS and/or relief of MS
symptoms) using any suitable route of administration, such as
injection and other routes of administration known in the art for
clinical products, such as antibody-based clinical products.
[0076] The compositions may contain from 0.1% by weight, preferably
from 10-60%, or more, by weight, of the inhibitor of the invention,
depending on the method of administration.
[0077] It will be recognized by one of skill in the art that the
optimal quantity and spacing of individual dosages of an inhibitor
of the invention will be determined by the nature and extent of the
condition being treated, the form, route and site of
administration, and the age and condition of the particular subject
being treated, and that a physician will ultimately determine
appropriate dosages to be used. This dosage may be repeated as
often as appropriate. If side effects develop the amount and/or
frequency of the dosage can be altered or reduced, in accordance
with normal clinical practice.
[0078] In another example, where the inhibitor is a nucleic acid
this may be administered via gene therapy (see for example Hoshida,
T. et al., 2002, Pancreas, 25:111-121; Ikuno, Y. 2002, Invest.
Ophthalmol. Vis. Sci. 2002 43:2406-2411; Bollard, C., 2002, Blood
99:3179-3187; Lee E., 2001, Mol. Med. 7:773-782). Gene therapy
refers to administration to a subject of an expressed or
expressible nucleic acid. In one example this is either the IL-17
or the IL-17R nucleic acid or portions thereof. Any of the methods
for gene therapy available in the art can be used according to the
present invention.
[0079] Delivery of the therapeutic nucleic acid into a patient can
be direct in vivo gene therapy (i.e. the patient is directly
exposed to the nucleic acid or nucleic acid-containing vector) or
indirect ex vivo gene therapy (i.e. cells are first transformed
with the nucleic acid in vitro and then transplanted into the
patient).
[0080] For example for in vivo gene therapy, an expression vector
containing the IL-17 or IL-17R nucleic acid may be administered in
such a manner that it becomes intracellular, i.e. by infection
using a defective or attenuated retroviral or other viral vectors
as described, for example, in U.S. Pat. No. 4,980,286 or by Robbins
et al., 1998, Pharmacol. Ther. 80:35-47.
[0081] The various retroviral vectors that are known in the art are
such as those described in Miller et al. (1993, Meth. Enzymol.
217:581-599) which have been modified to delete those retroviral
sequences which are not required for packaging of the viral genome
and subsequent integration into host cell DNA. Also adenoviral
vectors can be used which are advantageous due to their ability to
infect non-dividing cells and such high-capacity adenoviral vectors
are described in Kochanek (1999, Human Gene Therapy, 10:2451-2459).
Chimeric viral vectors that can be used are those described by
Reynolds et al. (1999, Molecular Medicine Today, 1:25-31). Hybrid
vectors can also be used and are described by Jacoby et al. (1997,
Gene Therapy, 4:1282-1283).
[0082] Direct injection of naked DNA or through the use of
microparticle bombardment (e.g. Gene Gun.RTM.; Biolistic, Dupont)
or by coating it with lipids can also be used in gene therapy.
Cell-surface receptors/transfecting compounds or through
encapsulation in liposomes, microparticles or microcapsules or by
administering the nucleic acid in linkage to a peptide which is
known to enter the nucleus or by administering it in linkage to a
ligand predisposed to receptor-mediated endocytosis (See Wu &
Wu. 1987, J. Biol. Chem., 262:4429-4432) can be used to target cell
types which specifically express the receptors of interest.
[0083] In ex vivo gene therapy, a gene is transferred into cells in
vitro using tissue culture and the cells are delivered to the
patient by various methods such as injecting subcutaneously,
application of the cells into a skin graft and the intravenous
injection of recombinant blood cells such as haematopoietic stem or
progenitor cells.
[0084] Cells into which a IL-17 or IL-17R nucleic acid can be
introduced for the purposes of gene therapy include, for example,
epithelial cells, endothelial cells, keratinocytes, fibroblasts,
muscle cells, hepatocytes and blood cells. The blood cells that can
be used include, for example, T-lymphocytes, B-lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryotcytes,
granulocytes, haematopoietic cells or progenitor cells, and the
like.
[0085] In a one aspect, the pharmaceutical composition of the
present invention comprises an IL-17 or IL-17R nucleic acid, said
nucleic acid being part of an expression vector that expresses an
IL-17 or IL-17R polypeptide or chimeric protein thereof in a
suitable host. In particular, such a nucleic acid has a promoter
operably linked to the polypeptide coding region, said promoter
being inducible or constitutive (and, optionally,
tissue-specific).
[0086] The invention will now be described with reference to the
following examples, which are merely illustrative and should not in
any way be construed as limiting the scope of the present
invention.
FIGURES
[0087] FIG. 1. Effect of Ab#13 mIgG1 on clinical disease when dosed
from day--1 through the acute phase (black arrowheads, days--1, 6,
13, 20). Average clinical score (.+-.sd) plotted against day of
disease induction (a) and average change in weight from day 0
(b).
[0088] FIG. 2. Analysis of the acute phase of disease following
dosing of Ab#13mIgG1 through the acute phase.
[0089] FIG. 3. Analysis of the relapse phase of disease following
dosing of Ab#13mIgG1 through the acute phase.
[0090] FIG. 4. Effect of Ab#13mIgG1 on clinical disease when dosed
through the relapse phase (days 28, 35, 42 and 49, black
arrowheads). Average clinical score (.+-.sd) versus day of disease
induction (a) and average change in weight from day 0 (b).
[0091] FIG. 5. Analysis of the acute phase of disease following
dosing of Ab#13mIgG1 through the reipase phase.
[0092] FIG. 6. Analysis of the relapse phase of disease following
dosing of Ab#13mIgG1 through the relapse phase.
[0093] FIG. 7. Effect of Ab#13 Fab-di-PEG and Ab#13 mIgG1 on
clinical disease when dosed through the relapse phase (black
arrowheads represent dosing days). Average clinical score (.+-.sd)
plotted against day of disease induction (a) and average change in
weight from day 0 (b).
[0094] FIG. 8. Analysis of acute phase prior to dosing with Ab#13
Fab-di-PEG and Ab#13 mIgG1.
[0095] FIG. 9. Analysis of relapse phase following dosing with
Ab#13 Fab-di-PEG and Ab#13 mIgG1 during remission.
[0096] FIG. 10. Effect of Ab#13mIgG1 on clinical disease when dosed
from day-1 (black arrowheads represent dosing days,
prophylactically (a) and therapeutically (b).
[0097] FIG. 11. Analysis of prohylactic dosing regime.
[0098] FIG. 12. Analysis of therapeutic dosing regime.
[0099] FIG. 13. Human and murine constant regions.
EXAMPLES
Example 1
Isolation of an Anti-IL-17 Antibody
[0100] Rabbits were immunised three times with human IL-17 and then
twice with mouse IL-1 7. Using a haemolytic plaque assay with
biotinylated sheep red blood cells coated with murine IL-17 via
streptavidin, 9 antibody genes were isolated using the methods
described by Babcook et al., 1996, Proc. Natl. Acad. Sci,
93,7843-7848 and in WO92/02551. The antibody genes were expressed
in CHO cells and the recombinant antibodies screened for their
ability to neutralise murine IL-17 in a bioassay using mouse
3T3-NIH cells (Yao et al., 1995, Immunity, 3:811-821). All the
antibodies in the panel neutralised murine IL-17 in this assay and
one antibody, m170013 (Ab#13) was selected for in vivo testing. For
testing the efficacy of the antibody in EAE, a chimeric IgG (Ab#13
mIgG1) was produced using the rabbit variable region from antibody
m170013 and mouse constant regions.
Example 2
Effect of Ab#13mIgG1on the Symptoms of EAE
[0101] The MS model, experimental autoimmune encephalomyelitis
(EAE), was used essentially as described by Baker et al., 1990.
Journal of Neuroimmunology, 28:261-270. Female ABH mice 8-10 weeks
of age (Harlan) were immunised with mouse spinal cord homogenate
(SCH, 3.33 mg/ml) in complete freund's adjuvant by subcutaneous
immunisation in either flank (150 .mu.l/site) on days 0 and 7.
[0102] i) Dosing Over the Acute Phase
[0103] Two groups were dosed with antibody at 10 mg/kg, sc on
days--1, 6, 13 and 20. One group (n=14) was dosed with Ab#13 mIgG1
the other (n=13) with 101.4 (isotype control).
[0104] ii) Dosing Over the Relapsing Phase
[0105] A total of 30 mice were followed through the acute phase of
disease and on day 27 analysis of the acute phase of disease was
performed to select two groups with similar disease profiles in the
acute phase (day of onset, peak disease score, cumulative clinical
score and weight loss). Two groups of 12 mice were selected for
dosing with antibody at 10 mg/kg, sc on days 28, 35, 42 and 49. One
group was dosed with Ab#13 mIgG1) the other with 101.4 (isotype
control).
[0106] Weights and clinical scores were recorded daily by an
assessor blinded to treatment and terminal EDTA-Plasma collected.
TABLE-US-00001 Clinical score scale 0 Normal 0.25 Tail dragging 0.5
Partial tail paralysis 1 Complete tail paralysis 2 Incomplete hind
limb paralysis 3 Complete hind paralysis/incontinence 4 Front limb
paralysis/loss of righting reflex
[0107] Statistics
[0108] Pairwise comparisons of clinical scores and day of onset
were performed using Mann-Whitney U test, analysis of incidence was
performed with Fishers exact test, analysis of maximum weight loss
was performed using Students' T test.
[0109] Results
[0110] i) Dosing during acute phase: A significant delay in the
onset of the acute phase and a reduced severity and incidence of
first relapse was observed (FIGS. 1, 2 and 3). FIG. 2 shows that
Ab#13 mIgG1dosed through the acute phase had no effect on the
incidence of disease in the acute phase (Ab#13 mIgG1, 13/14 with
disease vs 13/13 for isotype control, 101.4). The only
statistically significant effect was a delay in the onset of the
acute phase of disease FIG. 2a, p=0.0039, Mann-Whitney U test. No
effects were seen on weight loss (b), maximum clinical score (c) or
cumulative clinical score (d) in the acute phase. FIG. 3 shows that
Ab#13 mIgG1dosed through the acute phase caused a significant
reduction in the incidence of the relapse phase of disease (Ab#13
mIgG1, 6/14 with disease vs 11/11 for isotype control, 101.4,
p=0.0029, Fishers exact test). There was no statistically
significant delay in the onset of the relapse phase of disease for
those animals which entered relapse (a). A significant reduction in
weight loss (b, p=0.0001, Student's T test), maximum clinical score
(c, p=0.0028, Mann-Whitney U test) ) and cumulative clinical score
(d, p=0.0001, Mann-Whitney U test) were observed during the relapse
phase.
[0111] ii) Dosing through the relapsing phase: A reduced incidence,
delayed onset and reduced severity of the relapse phase was
observed (see FIGS. 4, 5 and 6). FIG. 5 shows that the dose groups
selected to have a similar acute phase profile, showed no
significant differences in any of the parameters analysed. FIG. 6
shows that Ab#13 mIgG1dosed through the relapse phase caused a
significant reduction in the incidence of the relapse phase of
disease (Ab#13 mIgG1, 5/12 with disease vs 12/12 for isotype
control, 101.4, p=0.0046, Fishers exact test). There was also a
statistically significant delay in the onset of the relapse phase
of disease for those animals which entered relapse (a, p=0.0061). A
significant reduction in weight loss (b, p<0.000l, Student's T
test), maximum clinical score (c, p=0.001 1, Mann-Whitney U test))
and cumulative clinical score (d, p=0.0023, Mann-Whitney U test)
were also observed during the relapse phase.
[0112] Summary
[0113] Ab#13 mIgG1 antibody was dosed in separate experiments over
the acute phase of disease (prophylactic dosing) and over the
relapse phase (therapeutic dosing). Effects were most pronounced on
relapse with a significant reduction in the incidence and severity
of the relapse phase for both dosing regimes.
Example 3
Effect of Ab#13 Fab-Di-PEG on the Symptoms of EAE
[0114] A Fab fragment, termed Ab#13 Fab-Di-PEG was produced
essentially as described in International Patent Application
PCT/GB2004/002810 (filed on 1 Jul. 2004). The Fab consisted of the
rabbit variable regions of antibody 13 from example 1 and mouse
IgG1 constant regions. In contrast to other Fab fragments, the
heavy chain constant region of this Fab terminates at the
interchain cysteine of C.sub.H1. PCR primers were designed based on
the murine IgG1 CH1 region and PCR mutagenesis used to insert a
stop codon immediately following the interchain cysteine of
C.sub.H1. The mouse constant regions are shown in FIG. 13 and in
SEQ ID Nos 3 (heavy chain) and 4 (light chain). PCR mutagenesis was
also used to replace the cysteine at position 80 of the rabbit
light chain variable region with alanine. TABLE-US-00002 Murine CH1
(SEQ ID NO:3) KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH
TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD C* Murine Kappa
(SEQ ID NO:4) DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV
LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRGEC*
[0115] The Fab fragments were produced in E. coli strain W3110 and
purified using standard methods (Humphreys et al., 2002, Protein
Expression and Purification, 26, 309-320).
[0116] 2.times.20 kDa PEG was attached to the Fab fragment by
attaching a linear 20 kDa PEG to each of the interchain cysteines
(underlined in the sequences above). Reductions and PEGylations
were performed in 50 mM Tris.HCl 5 mM EDTA pH 7.14 with Fab at
20.06 mg/ml. The Fab was reduced at room temperature
(.about.24.degree. C.) for 30 minutes using 10 mM
tris(2-carboxyethyl)phosphine (TCEP) (final). The Fab was desalted
on a PD-10 column (Pharmacia) and then mixed with 4 fold molar
excess of linear 20 kDa PEG-maleimide over Fab. The 20 kDa PEG was
from Nippon Oils and Fats (NOF). PEGylated Fab was separated from
unPEGylated Fab by size exclusion HPLC on analytical Zorbax GF-450
and GF-250 columns in series. These were developed with a 30 min
isocratic gradient of 0.2M phosphate pH 7.0+10% ethanol at 1 ml/min
and Fab detected using absorbance at 214 nm and 280 nm.
[0117] The MS model, experimental autoimmune encephalomyelitis
(EAE), was used essentially as described by Baker et al., 1990.
Journal of Neuroimmunology, 28:261-270.
[0118] Female ABH mice 8-10 weeks of age (Harlan) were immunised
with mouse spinal cord homogenate (SCH, 3.33 mg/ml) in complete
Freund's adjuvant in two sites, sc, on the flanks (150 .mu.l/site)
on days 0 and 7.
[0119] Four groups were dosed prior to onset of first relapse
(during remission). Groups were assigned as follows
[0120] Group 1 (n=11) anti-mouse IL-17 Ab#13 Fab-di-PEG (100 mg/kg,
s.c, weekly)
[0121] Group 2 (n=11) anti-mouse IL-17 Ab#13 Fab-di-PEG (30 mg/kg,
s.c, weekly)
[0122] Group 3 (n=9) anti-mouse IL-17 Ab#13 mIgG1 (10 mg/kg,
sc)
[0123] Group 4 (n=9) PBS control
[0124] Weights and clinical scores were recorded daily and terminal
EDTA-Plasma collected. TABLE-US-00003 Clinical score scale 0 Normal
0.25 Tail dragging 0.5 Partial tail paralysis 1 Complete tail
paralysis 2 Incomplete hind limb paralysis/loss of righting reflex
3 Complete hind paralysis/incontinence 4 Front limb
paralysis/moribund
[0125] Statistics
[0126] Pair wise comparisons of maximum and cumulative clinical
scores and day of onset were performed using Mann-Whitney U test.
Cumulative clinical score is defined as the sum of clinical scores
throughout the disease course for each animal (area under the
curve). Comparisons of disease incidence were performed using
Fishers Exact Test. Maximum weight loss was analysed using one-way
Anova with Bonferroni post test.
[0127] Results
[0128] FIG. 7a shows the effect of anti-IL17 Ab#13 di Fab-PEG and
Ab#13 mIgG1 on clinical disease when dosed from remission (black
arrowheads represent dosing days). When dosed prior to first
relapse all active doses showed a significant reduction in
cumulative and maximum clinical score and incidence.
[0129] FIG. 8 shows that during the acute phase, prior to antibody
treatment, all assigned groups showed no significant differences in
disease onset or clinical severity prior to antibody dosing.
[0130] FIG. 9 shows that anti-mouse IL-17 antibodies dosed prior to
relapse onset during remission showed a significant reduction in
maximum clinical score (Ab#13 di Fab-PEG 100 mg/kg vs. PBS
p<0.05 and IL-17 Ab#13 mIgG1 vs. PBS p<0.001),
[0131] There was also a reduction in cumulative score (Ab#13 di
Fab-PEG (100 mg/kg and 30 mg/kg) and Ab#13 mIgG1 vs. PBS p<0.01,
p<0.05 and p<0.001 respectively). Furthermore there was a
reduction in maximum weight loss (Ab#13 di Fab-PEG 100 mg/kg and
Ab#13 mIgG1 vs. PBS both p<0.05).
[0132] Actual incidence of relapse is summarised in table 1, with
all actively treated groups having significantly lower incidence
than the PBS control group. TABLE-US-00004 TABLE 1 Ab#13 Ab#13
Ab#13 di Fab-PEG di Fab-PEG IgG1 100 mg/kg*** 30 mg/kg* 10 mg/kg***
PBS Animals 0 2 0 7 entering relapse Animals not 11 9 11 2 entering
relapse Total number 11 11 11 9 of animals ***P = 0.005 *p = 0.0216
***P = 0.005
[0133] Summary
[0134] Anti-mouse IL17 antibodies (Ab#13 di Fab-PEG and Ab#13
mIgG1) were dosed in a dose dependent manner during the remission
phase prior to onset of first relapse. Effects were pronounced with
a significant reduction for both antibodies in relapse incidence
and upon maximum and cumulative disease score in comparison to the
PBS control group.
Example 4
Effect of Ab#13 mIgG1 on the Symptoms of Chronic EAE in C57BI/6
Mice
[0135] The chronic EAE model used was essentially as described by
Copray et al., 2004. Journal of Neuroimmunology, 148:41-53.
[0136] Female C57B1/6 mice 6-8 weeks of age (Charles River) were
immunised with Myelin Oligodendrocyte Protein (MOG 35-55, 0.66
mg/ml) in complete Freund's adjuvant (0.4 mg/ml Mycobacterium; 4:1
M.tuberculosis: M butyricum) in two sites, s.c, on the flanks (150
.mu.l/site) on days 0 and 7. Mice were also administered pertussis
toxin (1 .mu.g/ml) on days 0,1,7,8; 200 .mu.l i.p
[0137] Two groups were dosed prophylactically (10 mg/kg; s.c;
commencing on PSD--1 until the end of experiment). One group (n=15)
was dosed with Ab#13 mIgG1, (chimeric rabbit V region, Murine
constant region IgG-1, as described in Example 1), the other (n=15)
with 101.4 (Murine IgG-1 isotype control, 101.4).
[0138] Two groups were dosed therapeutically (upon 50% incidence)
with Ab#13 mIgG1 and control antibody 101.4 (10 mg/kg; s.c
commencing PSD 16 till end of experiment) Weights and clinical
scores were recorded daily and terminal EDTA-Plasma collected.
TABLE-US-00005 Clinical score scale 0 Normal 0.25 Tail dragging 0.5
Partial tail paralysis 1 Complete tail paralysis 2 Incomplete hind
limb paralysis/loss of righting reflex 3 Complete hind
paralysis/incontinence 4 Front limb paralysis/moribund
[0139] Statistics
[0140] Statistical analysis was performed as described in Example
3.
[0141] Results
[0142] FIG. 10 shows that prophylactic dosing (a) of Ab#13 mIgG1
elicited significant delay in the onset of disease, cumulative and
maximum clinical score. Therapeutic dosing (b) significantly
reduced cumulative clinical score. Further analysis showed Ab#13
mIgG1 dosed prophylactically had significant effects in reducing
cumulative score (p=0.0006), delay of onset (p=0.0184) and maximum
clinical score (p=0.0032, all Mann-Whitney U test) (FIG. 11). Ab#13
mIgG1 dosed therapeutically also showed a significant reduction in
cumulative score (b, p=0.0229, Mann-Whitney U test; FIG. 12).
[0143] Summary
[0144] Anti-mouse IL17 antibody Ab#13 mIgG1 was dosed in separate
experiments prophylactically and therapeutically. Effects were most
pronounced prophylactically with a significant reduction in maximum
and cumulative disease score, furthermore incidence of disease was
significantly delayed. Therapeutic treatment showed a reduction in
cumulative disease score.
Sequence CWU 1
1
4 1 103 PRT Homo sapiens 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Lys Val Glu Pro Lys Ser Cys 100 2 108 PRT Homo sapiens 2 Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 1 5 10
15 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
20 25 30 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu 35 40 45 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp 50 55 60 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr 65 70 75 80 Glu Lys His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser 85 90 95 Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 100 105 3 101 PRT Mus musculus 3 Lys Thr
Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala 1 5 10 15
Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe 20
25 30 Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly 35 40 45 Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser 50 55 60 Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro
Ser Glu Thr Val Thr 65 70 75 80 Cys Asn Val Ala His Pro Ala Ser Ser
Thr Lys Val Asp Lys Lys Ile 85 90 95 Val Pro Arg Asp Cys 100 4 105
PRT Mus musculus 4 Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser
Ser Glu Gln Leu 1 5 10 15 Thr Ser Gly Gly Ala Ser Val Val Cys Phe
Leu Asn Asn Phe Tyr Pro 20 25 30 Lys Asp Ile Asn Val Lys Trp Lys
Ile Asp Gly Ser Glu Arg Gln Asn 35 40 45 Gly Val Leu Asn Ser Trp
Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60 Ser Met Ser Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His 65 70 75 80 Asn Ser
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile 85 90 95
Val Lys Ser Phe Asn Arg Gly Glu Cys 100 105
* * * * *