U.S. patent application number 13/774367 was filed with the patent office on 2013-06-20 for methods of treating dementia using a gm-csf antagonist.
This patent application is currently assigned to KaloBios Pharmaceuticals, Inc.. The applicant listed for this patent is KaloBios Pharmaceuticals, Inc.. Invention is credited to Christopher R. Bebbington, Varghese Palath, Geoffrey T. Yarranton.
Application Number | 20130156759 13/774367 |
Document ID | / |
Family ID | 42631152 |
Filed Date | 2013-06-20 |
United States Patent
Application |
20130156759 |
Kind Code |
A1 |
Bebbington; Christopher R. ;
et al. |
June 20, 2013 |
METHODS OF TREATING DEMENTIA USING A GM-CSF ANTAGONIST
Abstract
The invention is based on the discovery that GM-CSF antagonists
can be used for the treatment of a patient that has Alzheimer's
disease or vascular dementia, or is at risk for developing
Alzheimer's disease. Accordingly, the invention provides methods of
administering a GM-CSF antagonist, e.g., a GM-CSF antibody and
pharmaceutical compositions comprising such antagonists.
Inventors: |
Bebbington; Christopher R.;
(South San Francisco, CA) ; Yarranton; Geoffrey T.;
(South San Francisco, CA) ; Palath; Varghese;
(South San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KaloBios Pharmaceuticals, Inc.; |
South San Francisco |
CA |
US |
|
|
Assignee: |
KaloBios Pharmaceuticals,
Inc.
South San Francisco
CA
|
Family ID: |
42631152 |
Appl. No.: |
13/774367 |
Filed: |
February 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12704396 |
Feb 11, 2010 |
8398972 |
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13774367 |
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11944162 |
Nov 21, 2007 |
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12704396 |
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61151750 |
Feb 11, 2009 |
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60902742 |
Feb 21, 2007 |
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60860780 |
Nov 21, 2006 |
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Current U.S.
Class: |
424/133.1 ;
424/178.1 |
Current CPC
Class: |
C07K 2317/55 20130101;
C07K 2317/24 20130101; A61K 45/06 20130101; C07K 2317/92 20130101;
C07K 2317/34 20130101; A61K 31/505 20130101; A61K 31/519 20130101;
A61K 39/3955 20130101; A61P 25/28 20180101; A61K 2039/505 20130101;
C07K 2317/73 20130101; C07K 2317/76 20130101; C07K 16/243 20130101;
A61K 31/505 20130101; A61K 2300/00 20130101; A61K 31/519 20130101;
A61K 2300/00 20130101; A61K 39/3955 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/133.1 ;
424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for treating a patient suffering from vascular dementia
or cerebral amyloid angiopathy, the method comprising administering
a recombinant GM-CSF antagonist to the patient, wherein the
recombinant GM-CSF antagonist is provided in a therapeutically
effective amount.
2. The method of claim 1, wherein the recombinant GM-CSF antagonist
is provided in an amount to prevent cognitive decline.
3. The method of claim 1, wherein the patient has mild cognitive
impairment.
4. The method of claim 1, wherein the patient has cerebral amyloid
angiopathy.
5. The method of claim 1, wherein the patient has elevated levels
of GM-CSF in the cerebrospinal fluid (CSF) or serum, or both CSF
and serum.
6. The method of claim 1, wherein the recombinant GM-CSF antagonist
is administered using intrathecal or perispinal administration.
7. The method of claim 1, wherein the recombinant GM-CSF antagonist
is a neutralizing anti-GM-CSF antibody
8. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody is an antibody fragment that is a Fab, a Fab', a
F(ab').sub.2, a scFv, or a dAB.
9. The method of claim 8, wherein the antibody fragment is
conjugated to polyethylene glycol.
10. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody has a K.sub.D of less than 100 pM.
11. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody is a chimeric antibody.
12. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody is a human antibody.
13. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody comprises a human variable region.
14. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody comprises a human light chain constant region and/or a
human heavy chain constant region.
15. The method of claim 14, wherein the human heavy chain constant
region is a gamma subclass.
16. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody binds to the same epitope as chimeric 19/2.
17. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody comprises a heavy chain variable (V.sub.H) region having
the sequence of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, or SEQ ID NO:13.
18. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody comprises a light chain variable (V.sub.L) region having
the sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID
NO:18.
19. The method of claim 7, wherein the neutralizing anti-GM-CSF
antibody comprises a V.sub.H region having the sequence of SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13;
and a V.sub.L region having the sequence of SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, or SEQ ID NO:18.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/704,396, filed Feb. 11, 2010, which claims benefit of U.S.
provisional application No. 61/151,750, filed Feb. 11, 2009; and is
a continuation-in-part of U.S. patent application Ser. No.
11/944,162, filed Nov. 21, 2007, which claims benefit of U.S.
provisional application No. 60/860,780, filed Nov. 21, 2006; and
U.S. provisional application No. 60/902,742, filed Feb. 21, 2007.
This application is also related to U.S. provisional application
No. 61/048,522, filed Apr. 28, 2008. Each of the noted applications
is herein incorporated by reference.
REFERENCE TO SUBMISSION OF A SEQUENCE LISTING
[0002] The sequence listing written in file-25-4-1.txt, created on
Feb. 21, 2013, 17,200 bytes, machine format IBM-PC, MS-Windows
operating system, is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is
a cytokine that plays a role in the inflammatory response and has
been reported to be involved in mediating aspects of a number of
chronic inflammatory diseases, including rheumatoid arthritis,
psoriasis, ankylosing spondylitis, juvenile idiopathic arthritic,
and systemic lupus erythematosus. Elevated levels of GM-CSF have
been also observed in the cerebrospinal fluid and sera of patients
with Alzheimer's disease and vascular dementia (Tarkowski et al.
Acta Neural. Scand 103:166-174, 2001); however a role of GM-CSF in
the pathology of dementia has not been identified. This invention
is based, in part, on the discovery that a GM-CSF antagonist can be
used for the treatment of dementia, or a patient at risk of
developing dementia.
BRIEF SUMMARY OF THE INVENTION
[0004] The present invention provides methods to treat a patient
suffering from dementia, such as Alzheimer's disease, vascular
dementia, cerebral amyloid angiopathy (CAA); or a patient at risk
of developing dementia such as Alzheimer's disease, vascular
dementia, or CAA, e.g., where the patient is diagnosed with mild
cognitive impairment or a has a family history of familial
Alzheimer's disease. The methods of the invention comprise
administering a GM-CSF antagonist to the patient in a
therapeutically effective amount, which at least partially arrests
symptoms and/or slows the progression or onset of the disease. In
typical embodiments, the GM-CSF antagonist is a recombinant
protein. A patient treated as described herein may be diagnosed
with one of the disease conditions (Alzheimer's, vascular dementia,
or CAA), or may be diagnosed with a combination of these
conditions. Thus, for example, a patient treated in accordance with
the invention may have CAA as well as vascular dementia, or CAA as
well as Alzheimer's, or vascular dementia and Alzheimer's disease,
or all three of the conditions. Treatment with the GM-CSF
antagonist can be performed alone, or in conjunction with other
therapies, such as treatment with an anti-beta-amyloid antibody, a
beta-amyloid vaccine, an acetylcholinesterase inhibitor, an NMDA
receptor antagonist, or IVIG. In some embodiments, the invention
provides a method of administering a GM-CSF antagonist, e.g., an
antibody, to a patient that has dementia, such as Alzheimer's
disease, with the proviso that the GM-CSF antagonist is not human
IVIG.
[0005] The invention additionally provides a GM-CSF antagonist as
described herein for use in treating dementia, or risk or dementia
as described in the preceding paragraph. The methods, uses and
pharmaceutical compositions for treating a patient in accordance
with the invention can employ any GM-CSF antagonist described
herein including any of the following antagonists:
[0006] In some embodiments of the invention, the GM-CSF antagonist
is recombinantly produced, e.g., a recombinant monoclonal antibody.
In other embodiments, the GM-CSF antagonist, e.g., purified
anti-GM-CSF from human plasma, is purified from a natural source.
In some embodiments, the GM-CSF antagonist is a recombinant
anti-GM-CSF antibody, an anti-GM-CSF receptor antibody; a GM-CSF
analog, e.g., such as a peptide analog, a soluble GM-CSF receptor;
a cytochrome b562 antibody mimetic; an adnectin, a lipocalin
scaffold antibody mimetic; a calixarene antibody mimetic, or an
antibody-like binding peptidomimetic.
[0007] In many embodiments, the GM-CSF antagonist is an antibody to
GM-CSF, i.e., an anti-GM-CSF antibody. In typical embodiments, the
anti-GM-CSF antibody is a recombinant antibody. In various
embodiments, the antibody can be a polyclonal antibody, a
monoclonal antibody, or an antibody such as a nanobody or a camelid
antibody. In some embodiments, the antibody is an antibody
fragment, such as a Fab, a Fab', a F(ab').sub.2, a scFv, or a
domain antibody (dAB). The antibody can also be modified, e.g., to
enhance stability. For example, in some embodiments, the antibody
is conjugated to polyethylene glycol.
[0008] In some embodiments, the antibody has an affinity of about
100 pM to about 10 nM, e.g., from about 100 pM, about 200 pM, about
300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM,
about 800 pM, about 900 pM, or about 1 nM to about 10 nM. In
further embodiments, the antibody has an affinity of about 1 pM to
about 100 pM, e.g., an affinity of about 1 pM, about 5 pM, about 10
pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40
pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, or about 90
pM to about 100 pM. In some embodiments, the antibody has an
affinity of from about 10 to about 30 pM. In some embodiments, the
antibody has from 10-1000 fM affinity.
[0009] In some embodiments, the antibody is a neutralizing
antibody. In further embodiments, the antibody is a recombinant or
chimeric antibody. In some embodiments, the antibody is a human
antibody. In some embodiments, the antibody comprises a human
variable region. In some embodiments, the antibody comprises a
human light chain constant region. In some embodiments, the
antibody comprises a human heavy chain constant region, such as a
gamma chain.
[0010] In further embodiments, the antibody competes for binding to
the same epitope as a chimeric 19/2 antibody, which is a chimeric
antibody obtained from the mouse monoclonal antibody LMM102. The
antibody can, e.g., comprise the V.sub.H and V.sub.L regions of
chimeric 19/2. The antibody can also comprise a human heavy chain
constant region such as a gamma region. In some embodiments, the
antibody comprises the CDR1, CDR2, and CDR3 of the V.sub.H region
of chimeric 19/2. In further embodiments, the antibody comprises
the CDR1, CDR2, and CDR3 of the V.sub.L region of chimeric 19/2. In
additional embodiments, the antibody comprises the CDR1, CDR2, and
CDR3 of the V.sub.H and V.sub.L regions of a chimeric 19/2
antibody. In some embodiments, the antibody comprises the V.sub.H
region CDR3 and V.sub.L region CDR3 of chimeric 19/2.
[0011] In some embodiments, an anti-GM-CSF antibody for use in the
invention comprises a V.sub.H region that has a CDR3 binding
specificity determinant RQRFPY (SEQ ID NO:1) or RDRFPY (SEQ ID
NO:2), a J segment, and a V-segment, wherein the J-segment
comprises at least 95% identity to human JH4 (YFDYWGQGTLVTVSS; SEQ
ID NO:3) and the V-segment comprises at least 90% identity to a
human germ line VH1 1-02 or VH1 1-03 sequence. In some embodiments,
the antibody comprise a V.sub.H region that comprises a CDR3
binding specificity determinant comprising RQRFPY (SEQ ID NO:1). In
some embodiments, the J segment comprises YFDYWGQGTLVTVSS (SEQ ID
NO:3). In some embodiments, the anti-GM-CSF antibody has a CDR3
that comprises RQRFPYYFDY (SEQ ID NO:4) or RDRFPYYFDY (SEQ ID
NO:5). In some embodiments the V.sub.H region CDR1 is a human
germline VH1 CDR1; the V.sub.H region CDR2 is a human germline VH1
CDR2; or both V.sub.H region the CDR1 and CDR2 are human germline
VH1. In some embodiments, the antibody comprises a V.sub.H CDR1, or
a V.sub.H CDR2, or both a V.sub.H CDR1 and a V.sub.H CDR2 as shown
in a V.sub.H region set forth in FIG. 1. In some embodiments, the
V-segment sequence has a V.sub.H V segment sequence shown in FIG.
1. In further embodiments, the V.sub.H has the sequence of VH#1,
VH#2, VH#3, VH#4, or VH#5 set forth in FIG. 1.
[0012] In some embodiments, an anti-GM-CSF antibody of the
invention, e.g., an anti-GM-CSF antibody having V.sub.H region as
described herein, comprises a V.sub.L region that comprises a CDR3
binding specificity determinant FNK or FNR. In some embodiments,
the antibody comprises a human germline JK4 region. In some
embodiments, the V.sub.L region CDR3 comprises QQFN(K/R)SPLT (SEQ
ID NO:6). In some embodiments, the V.sub.L region comprises a CDR1,
or a CDR2, or both a CDR1 and CDR2 of a sequence V.sub.L region
shown in FIG. 1. In some embodiments, the V.sub.L region comprises
a V segment that has at least 95% identity to the VKIIIA27
V-segment sequence as shown in FIG. 1. In some embodiments, the
V.sub.L region has the sequence of VK#1, VK#2, VK#3, or VK#4 set
forth in FIG. 1.
[0013] In some embodiments, an anti-GM-CSF antibody for use in the
invention has a half-life of about 7 to about 25 days.
[0014] In some embodiments of the methods of the invention, the
GM-CSF antagonist, e.g., an anti-GMCSF antibody, is administered by
injection or by infusion. For example, the GM-CSF antagonist can be
administered intravenously over a period between about 15 minutes
and about 2 hours.
[0015] In other embodiments, the GM-CSF antagonist is administered
subcutaneously by bolus injection.
[0016] In further embodiments, the GM-CSF antagonist is
administered by intranasal administration, perispinal
administration, intrathecal administration, or subcutaneous
administration.
[0017] A GM-CSF antibody can, for example, be administered at a
dose between about 1 mg/kg of body weight and about 10 mg/kg of
body weight.
[0018] In some embodiments, treatment with the GM-CSF antagonist
comprises a second administration of the GM-CSF antagonist.
[0019] The invention also provides a method of treating a patient
having Alzheimer's disease, vascular dementia, or CAA, or who is at
risk for developing Alzheimer's disease or vascular dementia, the
method comprising administering an anti-GM-CSF antibody as
described herein to the patient in a therapeutically effective
amount.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 provides exemplary V.sub.H (SEQ ID NOS:8 and 10-13)
and V.sub.L (SEQ ID NOS:15-18) sequences of anti-GM-CSF antibodies.
VH1 1-02=SEQ ID NO:7; VH1 1-03=SEQ ID NO:9, VKIII A27=SEQ ID
NO:14.
[0021] FIG. 2. Uptake of 22E9 into the hippocampus region of the
brain of a hAPP751-SL transgenic mouse. The mouse was administered
22E9 antibody (rat anti-mouse anti-GM-CSF neutralizing antibody) 48
hours prior to sacrifice and sectioning of the brain. 22E9 is
detected using anti-rat IgG specific antibody. The figure shows
22E9 antibody uptake associated with amyloid plaques in the
hippocampus region of the brain after A) intravenous (i.v.)
administration or B) intranasal (i.n.) administration
[0022] FIG. 3. Performance of anti-GM-CSF antibody 22E9 treated and
control mice in the Morris Water Maze beginning 44 days after
treatment initiation. Mean swimming path length is shown (+standard
error). * Difference between rat IgG2a treated and 22E9 treated
mice is statistically significant.
[0023] FIG. 4. Performance of anti-GM-CSF antibody 22E9 treated and
control mice in the Contextual fear conditioning analysis beginning
51 days after treatment initiation. Context conditioned freezing
response is shown for animals transferred to the training chamber
24 hours after initial training. Results are means+standard
errors.
[0024] FIG. 5. Mean size of amyloid plaques in the brains of mice
treated with anti-GM-CSF antibody 22E9 or control-treated mice.
Mean plaque size (+standard error) from 6 mice in each group was
determined by staining with 6E10 antibody to human amyloid. Data
were analyzed by one-way Analysis of variance (ANOVA).
DETAILED DESCRIPTION OF THE INVENTION
[0025] As used herein, "Alzheimer's disease" refers to senile
dementia as diagnosed using commonly accepted criteria in the art,
such as the criteria set forth by The National Institute of
Neurological and Communicative Disorders and Stroke and the
Alzheimer's disease and Related Disorders Association and/or the
criteria as listed in the Diagnostic and Statistical Manual of
Mental Disorders (DSM-IV-TR) published by the American Psychiatric
Association. The Diagnostic and Statistical Manual of Mental
Disorders (Fourth Edition, revised in 2000), also known as the
DSM-IV-TR, outlines a detailed set of criteria for the diagnosis of
Alzheimer's disease. For purposes of this application, the terms
"Alzheimer's" and "Alzheimer's disease" and "AD" are used
interchangeably.
[0026] "Vascular dementia" is a common form of dementia. The term
"vascular dementia" refers to a group of syndromes relating to
different vascular mechanisms. Various subtypes of vascular
dementia have been described to date. The spectrum of disease
includes (1) mild vascular cognitive impairment, (2) multi-infarct
dementia, (3) vascular dementia due to a strategic single infarct,
(4) vascular dementia due to lacunar lesions, (5) vascular dementia
due to hemorrhagic lesions, (6) Binswanger disease, (7) subcortical
vascular dementia, and (8) mixed dementia (combination of AD and
vascular dementia). Vascular dementia is sometimes further
classified as cortical or subcortical dementia. Vascular dementia
can be diagnosed by clinical criteria, often in combination with
brain imaging.
[0027] "Cerebral amyloid angiopathy" refers to a disorder
characterized by deposition of amyloid within the walls of the
cerebral arteries. Severe CAA is associated with vasculopathic
changes, vessel rupture, and cerebral hemorrhage. CAA is a
component of any disorder in which amyloid is deposited in the
brain, e.g., Alzheimer's disease, and it is not associated with
systemic amyloidosis.
[0028] In the current invention, a patient with "mild to moderate"
dementia, or early-stage Alzheimer's disease can be identified
using neurological testing and other clinical endpoints. For
example, a subject with mild to moderate dementia, e.g.,
Alzheimer's disease, can be identified using the Mini-Mental State
Examination (MMSE), Typically, a score of 16 to 26 (both inclusive)
is indicative of mild to moderate Alzheimer's disease. Patients
with advanced Alzheimer's disease can also be identified based on
clinical parameters. Subjects with this form of Alzheimer's disease
may no longer respond to therapy with acetylcholinesterase
inhibitors, and may have a markedly reduced acetylcholine
level.
[0029] As used herein, "Granulocyte Macrophage-Colony Stimulating
Factor" (GM-CSF) refers to a small, naturally occurring
glycoprotein with internal disulfide bonds having a molecular
weight of approximately 23 kDa. In humans, it is encoded by a gene
located within the cytokine cluster on human chromosome 5. The
sequence of the human gene and protein are known. The protein has
an N-terminal signal sequence, and a C-terminal receptor binding
domain (Rasko and Gough In: The Cytokine Handbook, A. Thomson, et
al, Academic Press, New York (1994) pages 349-369). Its
three-dimensional structure is similar to that of the interleukins,
although the amino acid sequences are not similar. GM-CSF is
produced in response to a number of inflammatory mediators by
mesenchymal cells present in the hemopoietic environment and at
peripheral sites of inflammation. GM-CSF is able to stimulate the
production of neutrophilic granulocytes, macrophages, and mixed
granulocyte-macrophage colonies from bone marrow cells and can
stimulate the formation of eosinophil colonies from fetal liver
progenitor cells. GM-CSF can also stimulate some functional
activities in mature granulocytes and macrophages.
[0030] The term "granulocyte macrophage-colony stimulating factor
receptor" (GM-CSFR)" refers to a membrane bound receptor expressed
on cells that transduces a signal when bound to granulocyte
macrophage colony-stimulating factor (GM-CSF). GM-CSFR consists of
a ligand-specific low-affinity binding chain (GM-CSFR alpha) and a
second chain that is required for high-affinity binding and signal
transduction. This second chain is shared by the ligand-specific
alpha-chains for the interleukin 3 (IL-3) and IL-5 receptors and is
therefore called beta common (beta c). The cytoplasmic region of
GM-CSFR alpha consists of a membrane-proximal conserved region
shared by the alpha 1 and alpha 2 isoforms and a C-terminal
variable region that is divergent between alpha 1 and alpha 2. The
cytoplasmic region of beta-c contains membrane proximal serine and
acidic domains that are important for the proliferative response
induced by GM-CSF.
[0031] The term "soluble granulocyte macrophage-colony stimulating
factor receptor" (sGM-CSFR) refers to a non-membrane bound receptor
that binds GM-CSF, but does not transduce a signal when bound to
the ligand.
[0032] As used herein, a "peptide GM-CSF antagonist" refers to a
peptide that interacts with GM-CSF, or its receptor, to reduce or
block (either partially or completely) signal transduction that
would otherwise result from the binding of GM-CSF to its cognate
receptor expressed on cells. GM-CSF antagonists may act by reducing
the amount of GM-CSF ligand available to bind the receptor (e.g.,
antibodies that once bound to GM-CSF increase the clearance rate of
GM-CSF) or prevent the ligand from binding to its receptor either
by binding to GM-CSF or the receptor (e.g., neutralizing
antibodies). GM-CSF antagonists may also include other peptide
inhibitors, which may include polypeptides, that bind GM-CSF or its
receptor to partially or completely inhibit signaling. A peptide
GM-CSF antagonist can be, e.g., an antibody; a natural or synthetic
GM-CSF receptor ligand that antagonizes GM-CSF, or other
polypeptides. An exemplary assay to detect GM-CSF antagonist
activity is provided in Example 1. Typically, a peptide GM-CSF
antagonist, such as a neutralizing antibody, has an EC.sub.50 of 10
nM or less.
[0033] A "purified" GM-CSF antagonist as used herein refers to a
GM-CSF antagonist that is substantially or essentially free from
components that normally accompany it as found in its native state.
For example, a GM-CSF antagonist such as an anti-GM-CSF antibody,
that is purified from blood or plasma is substantially free of
other blood or plasma components such as other immunoglobulin
molecules. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified. Typically, "purified" means that the
protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure relative to the components
with which the protein naturally occurs.
[0034] As used herein, an "antibody" refers to a protein
functionally defined as a binding protein and structurally defined
as comprising an amino acid sequence that is recognized by one of
skill as being derived from the framework region of an
immunoglobulin-encoding gene of an animal that produces antibodies.
An antibody can consist of one or more polypeptides substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0035] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains, respectively.
[0036] The term antibody includes antibody fragments that retain
binding specificity. For example, there are a number of well
characterized antibody fragments. Thus, for example, pepsin digests
an antibody C-terminal to the disulfide linkages in the hinge
region to produce F(ab)'.sub.z, a dimer of Fab which itself is a
light chain joined to VH-CH1 by a disulfide bond. The F(ab)'.sub.2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region thereby converting the (Fab').sub.2 dimer into
an Fab' monomer. The Fab' monomer is essentially a Fab with part of
the hinge region (see, Fundamental Immunology, W.E. Paul, ed.,
Raven Press, N.Y. (1993), for a more detailed description of other
antibody fragments). While various antibody fragments are defined
in terms of the digestion of an intact antibody, one of skill will
appreciate that fragments can be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody, as used herein also includes antibody fragments
either produced by the modification of whole antibodies or
synthesized using recombinant DNA methodologies.
[0037] Antibodies include dimers such as V.sub.H-V.sub.L dimers,
V.sub.H dimers, or V.sub.L dimers, including single chain
antibodies (antibodies that exist as a single polypeptide chain),
such as single chain Fv antibodies (sFv or scFv) in which a
variable heavy and a variable light region are joined together
(directly or through a peptide linker) to form a continuous
polypeptide. The single chain Fv antibody is a covalently linked
V.sub.H-V.sub.L heterodimer which may be expressed from a nucleic
acid including V.sub.H- and V.sub.L-encoding sequences either
joined directly or joined by a peptide-encoding linker (e.g.,
Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988).
While the V.sub.H and V.sub.L are connected to each as a single
polypeptide chain, the V.sub.H and V.sub.L domains associate
non-covalently. Alternatively, the antibody can be another
fragment, such as a disulfide-stabilized Fv (dsFv). Other fragments
can also be generated, including using recombinant techniques. The
scFv antibodies and a number of other structures converting the
naturally aggregated, but chemically separated light and heavy
polypeptide chains from an antibody V region into a molecule that
folds into a three dimensional structure substantially similar to
the structure of an antigen-binding site are known to those of
skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405,
and 4,956,778). In some embodiments, antibodies include those that
have been displayed on phage or generated by recombinant technology
using vectors where the chains are secreted as soluble proteins,
e.g., scFv, Fv, Fab, (Fab').sub.2 or generated by recombinant
technology using vectors where the chains are secreted as soluble
proteins. Antibodies for use in the invention can also include
diantibodies and miniantibodies.
[0038] Antibodies of the invention also include heavy chain dimers,
such as antibodies from camelids. Since the V.sub.H region of a
heavy chain dimer IgG in a camelid does not have to make
hydrophobic interactions with a light chain, the region in the
heavy chain that normally contacts a light chain is changed to
hydrophilic amino acid residues in a camelid. V.sub.H domains of
heavy-chain dimer IgGs are called VHH domains. Antibodies for use
in the current invention include single domain antibodies (dAbs)
and nanobodies (see, e.g., Cortez-Retamozo, et al., Cancer Res.
64:2853-2857, 2004).
[0039] As used herein, "V-region" refers to an antibody variable
region domain comprising the segments of Framework 1, CDR1,
Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4,
which segments are added to the V-segment as a consequence of
rearrangement of the heavy chain and light chain V-region genes
during B-cell differentiation. A "V-segment" as used herein refers
to the region of the V-region (heavy or light chain) that is
encoded by a V gene.
[0040] As used herein, the term "J-segment" refers to a subsequence
of the encoded variable region comprising a C-terminal portion of a
CDR3 and the FR4. An endogenous J-segment is encoded by an
immunoglobulin J-gene.
[0041] As used herein, "complementarity-determining region (CDR)"
refers to the three hypervariable regions in each chain that
interrupt the four "framework" regions established by the light and
heavy chain variable regions. The CDRs are primarily responsible
for binding to an epitope of an antigen. The CDRs of each chain are
typically referred to as CDR1, CDR2, and CDR3, numbered
sequentially starting from the N-terminus, and are also typically
identified by the chain in which the particular CDR is located.
Thus, for example, a V.sub.H CDR3 is located in the variable domain
of the heavy chain of the antibody in which it is found, whereas a
V.sub.L CDR1 is the CDR1 from the variable domain of the light
chain of the antibody in which it is found.
[0042] The sequences of the framework regions of different light or
heavy chains are relatively conserved within a species. The
framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three dimensional space.
[0043] The amino acid sequences of the CDRs and framework regions
can be determined using various well known definitions in the art,
e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT),
and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk,
1987, Canonical structures for the hypervariable regions of
immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al.,
1989, Conformations of immunoglobulin hypervariable regions. Nature
342, 877-883; Chothia C. et al., 1992, structural repertoire of the
human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al.,
J. Mol. Biol. 1997, 273(4)). Definitions of antigen combining sites
are also described in the following: Ruiz et al., IMGT, the
international ImMunoGeneTics database. Nucleic Acids Res., 28,
219-221 (2000); and Lefranc, M.-P. IMGT, the international
ImMunoGeneTics database. Nucleic Acids Res. Jan 1; 29(1):207-9
(2001); MacCallum et al, Antibody-antigen interactions: Contact
analysis and binding site topography, J. Mol. Biol., 262 (5),
732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86,
9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153,
(1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et
al, In Sternberg M. J. E. (ed.), Protein Structure Prediction.
Oxford University Press, Oxford, 141-172 1996).
[0044] "Epitope" or "antigenic determinant" refers to a site on an
antigen to which an antibody binds. Epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
[0045] As used herein, "neutralizing antibody" refers to an
antibody that binds to GM-CSF and prevents signaling by the GM-CSF
receptor, or inhibits binding of GM-CSF to its receptor.
[0046] As used herein, "chimeric antibody" refers to an
immunoglobulin molecule in which (a) the constant region, or a
portion thereof, is altered, replaced or exchanged so that the
antigen binding site (variable region) is linked to a constant
region of a different or altered class, effector function and/or
species, or an entirely different molecule that confers new
properties to the chimeric antibody, e.g., an enzyme, toxin,
hormone, growth factor, drug, etc.; or (b) the variable region, or
a portion thereof, is altered, replaced or exchanged with a
variable region, or portion thereof, having a different or altered
antigen specificity; or with corresponding sequences from another
species or from another antibody class or subclass.
[0047] As used herein, "humanized antibody" refers to an
immunoglobulin molecule in CDRs from a donor antibody are grafted
onto human framework sequences. Humanized antibodies may also
comprise residues of donor origin in the framework sequences. The
humanized antibody can also comprise at least a portion of a human
immunoglobulin constant region. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. Humanization can be
performed using methods known in the art (e.g., Jones et al.,
Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327,
1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr.
Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No. 4,816,567),
including techniques such as "superhumanizing" antibodies (Tan et
al., J. Immunol. 169: 1119, 2002) and "resurfacing" (e.g., Staelens
et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.
Natl. Acad. Sci. USA 91: 969, 1994).
[0048] A "HUMANEERED.TM." antibody in the context of this invention
refers to an engineered human antibody having a binding specificity
of a reference antibody. A engineered human antibody for use in
this invention has an immunoglobulin molecule that contains minimal
sequence derived from a donor immunoglobulin. In some embodiments,
the engineered human antibody may retain only the minimal essential
binding specificity determinant from the CDR3 regions of a
reference antibody. Typically, an engineered human antibody is
engineered by joining a DNA sequence encoding a binding specificity
determinant (BSD) from the CDR3 region of the heavy chain of the
reference antibody to human V.sub.H segment sequence and a light
chain CDR3BSD from the reference antibody to a human V.sub.L
segment sequence. A "BSD" refers to a CDR3-FR4 region, or a portion
of this region that mediates binding specificity. A binding
specificity determinant therefore can be a CDR3-FR4, a CDR3, a
minimal essential binding specificity determinant of a CDR3 (which
refers to any region smaller than the CDR3 that confers binding
specificity when present in the V region of an antibody), the D
segment (with regard to a heavy chain region), or other regions of
CDR3-FR4 that confer the binding specificity of a reference
antibody. Methods for engineering human antibodies are provided in
US patent application publication no. 20050255552 and US patent
application publication no. 20060134098.
[0049] The term "human antibody" as used herein refers to an
antibody that is substantially human, i.e., has FR regions, and
often CDR regions, from a human immune system. Accordingly, the
term includes humanized and humaneered antibodies as well as
antibodies isolated from mice reconstituted with a human immune
system and antibodies isolated from display libraries.
[0050] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not normally found in the same
relationship to each other in nature. For instance, the nucleic
acid is typically recombinantly produced, having two or more
sequences, e.g., from unrelated genes arranged to make a new
functional nucleic acid. Similarly, a heterologous protein will
often refer to two or more subsequences that are not found in the
same relationship to each other in nature.
[0051] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, e.g., recombinant cells
express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all. By the term "recombinant nucleic acid" herein is meant nucleic
acid, originally formed in vitro, in general, by the manipulation
of nucleic acid, e.g., using polymerases and endonucleases, in a
form not normally found in nature. In this manner, operably linkage
of different sequences is achieved. Thus an isolated nucleic acid,
in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that are not normally joined, are both
considered recombinant for the purposes of this invention. It is
understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of
the host cell rather than in vitro manipulations; however, such
nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. Similarly, a "recombinant protein"
is a protein made using recombinant techniques, i.e., through the
expression of a recombinant nucleic acid.
[0052] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,
refers to a binding reaction where the antibody binds to the
antigen of interest. In the context of this invention, the antibody
typically binds to the antigen, e.g., GM-CSF, with an affinity of
500 nM or less, and has an affinity of 5000 nM or greater, for
other antigens.
[0053] The terms "identical" or percent "identity," in the context
of two or more polypeptide (or nucleic acid) sequences, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues (or nucleotides) that
are the same (i.e., about 60% identity, preferably 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site). Such
sequences are then said to be "substantially identical."
"Substantially identical" sequences also includes sequences that
have deletions and/or additions, as well as those that have
substitutions, as well as naturally occurring, e.g., polymorphic or
allelic variants, and man-made variants. As described below, the
preferred algorithms can account for gaps and the like. Preferably,
protein sequence identity exists over a region that is at least
about 25 amino acids in length, or more preferably over a region
that is 50-100 amino acids=in length, or over the length of a
protein.
[0054] A "comparison window", as used herein, includes reference to
a segment of one of the number of contiguous positions selected
from the group consisting typically of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0055] Preferred examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity
include the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul
et al., J. Mol. Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are
used, with the parameters described herein, to determine percent
sequence identity for the nucleic acids and proteins of the
invention. The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,
N=-4 and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as defaults a wordlength of 3, and
expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0056] An indication that two polypeptides are substantially
identical is that the first polypeptide is immunologically cross
reactive with the antibodies raised against the second polypeptide.
Thus, a polypeptide is typically substantially identical to a
second polypeptide, e.g., where the two peptides differ only by
conservative substitutions.
[0057] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high performance liquid chromatography. A protein that is the
predominant species present in a preparation is substantially
purified. The term "purified" in some embodiments denotes that a
protein gives rise to essentially one band in an electrophoretic
gel. Preferably, it means that the protein is at least 85% pure,
more preferably at least 95% pure, and most preferably at least 99%
pure.
[0058] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers, those containing modified
residues, and non-naturally occurring amino acid polymer.
[0059] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, e.g., an
.alpha. carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs may
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0060] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0061] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g.,
naturally contiguous, sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode most proteins. For instance, the codons GCA, GCC, GCG
and GCU all encode the amino acid alanine. Thus, at every position
where an alanine is specified by a codon, the codon can be altered
to another of the corresponding codons described without altering
the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, often silent variations of a nucleic acid
which encodes a polypeptide is implicit in a described sequence
with respect to the expression product, but not with respect to
actual probe sequences.
[0062] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables and
substitution matrices such as BLOSUM providing functionally similar
amino acids are well known in the art. Such conservatively modified
variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
Typical conservative substitutions for one another include: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)).
I. Introduction
[0063] The invention relates to methods of administering a GM-CSF
antagonist to a patient for the treatment of dementia, such as
Alzheimer's disease, vascular dementia or CAA. Patient to be
treated in accordance with the invention include patients having
Alzheimer's disease, vascular dementia, or CAA; or a patient at
risk for developing Alzheimer's disease, or vascular dementia.
GM-CSF antagonists may include anti-GM-CSF antibodies, anti-GM-CSF
receptor antibodies, or other inhibitors that prevent signaling
that normally results from the binding of GM-CSF to its cognate
receptor.
[0064] Antibodies, e.g., anti-GM-CSF or anti-GM-CSF receptor
antibodies, suitable for use with the present invention may be
monoclonal, polyclonal, chimeric, humanized, engineered human
antibodies that contain only minimal sequence from a reference
antibody, or human. Other GM-CSF antagonists suitable for use with
the present invention may include naturally occurring or synthetic
ligands (or fragments thereof) that compete with GM-CSF for binding
to the receptor, but do not result in signaling when bound to the
receptor. Additional non-limiting GM-CSF antagonists may include
polypeptides, nucleic acids, small molecules and the like that
either partially or completely block signaling that would naturally
result from the binding of GM-CSF to its receptor in the absence of
the GM-CSF antagonist.
[0065] In the context of the present specification, "beta-amyloid",
".beta.-amyloid", "amyloid-beta", "amyloid-.beta." and "a.beta."
are used interchangeably to refer to beta amyloid peptides and
include reference to various peptides, including a.beta..sub.1-40,
a.beta..sub.1-42, a.beta..sub.1-43, a.beta..sub.1-44, and the
like.
II. Patients
Alzheimer's Disease
[0066] Typical patients to be treated with the GM-CSF antagonist
are those diagnosed with Alzheimer's disease or who have a familial
disposition to Alzheimer's disease. For the purposes of this
invention, an Alzheimer's patient is diagnosed according to
accepted clinical criteria. There are two alternative diagnoses
standards that are commonly employed to clinically diagnose
Alzheimer's disease. The National Institute of Neurological and
Communicative Disorders and Stroke and the Alzheimer's disease and
Related Disorders Association (also referred to as NINCDS-ADRDA)
established one set of commonly used diagnostic criteria for
Alzheimer's disease, although other criteria can also be used
(e.g., Dubois et al, Lancet Neurol 6 (8): 734-46, 2007). For the
NINCDS-ADRDA criteria, the presence of cognitive impairment, and a
suspected dementia syndrome is confirmed by neuropsychological
testing for a clinical diagnosis of possible or probable AD. A
definitive diagnosis is obtained by histopathological confirmation.
As understood in the art, for the purposes of this invention, the
terms "diagnosed with Alzheimer's disease" or a patient "having
Alzheimer's disease" includes patients that have probable or
possible Alzheimer's, as a definitive diagnosis under NINCDS-ADRDA
diagnostic criteria is based on histopathologic evidence that is
often obtained post-mortem. NINCDS-ADRDA diagnostic criteria
are:
[0067] Definite Alzheimer's disease: The patient meets the criteria
for probable Alzheimer's disease and has histopathologic evidence
of AD via autopsy or biopsy.
[0068] Probable Alzheimer's disease: Dementia has been established
by clinical and neuropsychological examination. Cognitive
impairments is progressive and is present in two or more areas of
cognition. The onset of the deficits has been between the ages of
40 and 90 years and there is an absence of other diseases capable
of producing a dementia syndrome.
[0069] Possible Alzheimer's disease: There is a dementia syndrome
with an atypical onset, presentation or progression; and without a
known etiology; but no co-morbid diseases capable of producing
dementia are believed to be in the origin of it.
[0070] Unlikely Alzheimer's disease: The patient presents a
dementia syndrome with a sudden onset, focal neurologic signs, or
seizures or gait disturbance early in the course of the
illness.
[0071] Alzheimer's disease can also be diagnosed based on the
criteria in the Diagnostic and Statistical Manual of Mental
Disorders (DSM-IV-TR, 2000) published by the American Psychiatric
Association. Briefly, the criteria for diagnosis of Alzheimer's
disease under DSM-IV-TR include the development of multiple
cognitive deficits manifested by both (1) memory impairment
(impaired ability to learn new information or to recall previously
learned information) and (2) one (or more) of the following
cognitive disturbances: (a) aphasia (language disturbance), (b)
apraxia (impaired ability to carry out motor activities despite
intact motor function), (c) agnosia (failure to recognize or
identify objects despite intact sensory function), (d) disturbance
in executive functioning (i.e., planning, organizing, sequencing,
abstracting). The cognitive deficits cause significant impairment
in social or occupational functioning and represent a significant
decline from a previous level of functioning. The course is
characterized by gradual onset and continuing cognitive decline,
and the all other specific causes of dementia are excluded by
history, physical examination, and/or laboratory tests.
[0072] Other diagnostic procedures can also be used to diagnose
Alzheimer's disease (see, e.g., Dubois et al., Lancet Neurol, Vol.
6:734-746, 2007). For example, such procedures include tests of
episodic memory, e.g. delayed recall and double memory tests to
differentiate between memory storage or encoding problems, which
are indicative of Alzheimer's disease, and problems involving
memory retrieval. Biochemical tests can also be used for diagnosis.
For example, low amyloid .beta..sub.(1-42) concentrations,
increased total tau concentrations, or increased phospho-tau
concentrations or combinations of these three in a CSF sample, or
other appropriate sample, from a patient is indicative of
Alzheimer's disease. Neopterin levels in the serum of a patient,
may also be evaluated to determine whether an elevated level, which
is associated with Alzheimer's disease, is present (Leblhuber et
al., Clin. Chem. Lab. Med. 37:429-431, 1999). Structural and
metabolic evaluation can also be performed on the brain, e.g., PET
scanning to identify diminished glucose metabolism in the bilateral
temporoparietal regions and posterior cingulate. The presence of
atrophy in the medial temporal lobe regions of the brain, including
volume loss of hippocampi, entorhinal cortex, and amygdala, may
also be determined using computed tomographic scanning (CT), and
magnetic resonance imaging (MRI) (Leedom and Miller, "CT, MRI, and
NMR Spectroscopy in Alzheimer's disease," Alzheimer's disease,
Current Research in Early Diagnosis, Becker and Giacobini (eds.),
pp. 297-313, 1990).
[0073] Genetic risk factors may also be evaluated to either aid in
the diagnosis of Alzheimer's or to evaluate increased risk for
developing Alzheimer's. In some embodiments, subjects that may have
an increased risk for developing Alzheimer's disease can also be
treated with a GM-CSF antagonist, e.g., an antibody as described
herein.
[0074] In assessing genetic risk factors, subjects can be screened
based on a number of biochemical and genetic markers. For example,
genetic abnormality in a few families has been traced to chromosome
21 (St. George-Hyslop et al., Science 235:885-890, 1987). One
genetic marker is, for example, the presence of mutations in the
APP gene, particularly mutations at position 717 and positions 670
and 671, which are referred to as the Hardy and Swedish mutations,
respectively. Other markers of risk are mutations in the presenilin
genes, PS1 and PS2, the ApoE4 profile of a subject, family history
of Alzheimer's disease, and the presence of risk factors such as
hypercholesterolemia or atherosclerosis. Subjects with APP, PS1 or
PS2 mutations are highly likely to develop Alzheimer's disease.
Subjects having the E4 isoform of ApoE (ApoE4 isoform) have an
increased risk of developing Alzheimer's disease.
[0075] Alzheimer's patients that can be treated in accordance with
the methods of the invention include those with mild or moderate
impairment as well as patients with more advanced impairment.
[0076] In some embodiments, a patient who has mild cognitive
impairment (MCI) may be treated with a GM-CSF antagonist. MCI
patients are at risk for development of Alzheimer's disease. MCI
can be diagnosed and evaluated using any of the many objective
tests or criteria well-known and accepted in the fields of
psychology or psychiatry. For example, one criterion for the
diagnosis of MCI is that the patient receives a clinical dementia
rating of 0.5 as described, e.g., in Hughes et al., Brit. J.
Psychiat. 140:566-572, 1982 and Morris, Neurology 43:2412-2414,
1993. In determining the clinical dementia rating, a patient is
typically assessed and rated in each of six cognitive and
behavioural categories: memory, orientation, judgment and problem
solving, community affairs, home and hobbies, and personal care.
The patient is assessed and rated in each of these areas and the
overall rating, (0, 0.5, 1.0, 2.0 or 3.0) determined A rating of 0
is considered normal. A rating of 1.0 is considered to correspond
to mild dementia. A patient with cognitive impairment demonstrates
impaired performance on a memory task test. Memory may be measured
by such tests known in the art as the Wechsler Memory Scale or a
pair-associated memory task. A patient is considered to exhibit
impaired performance on such a test if the score is below the
education and age-adjusted cutoff for that test.
[0077] In the current invention a patient diagnosed with
Alzheimer's disease or at risk of developing Alzheimer's disease,
e.g., has mild cognitive impairment, a family history of familial
Alzheimer's, or a risk factor for developing Alzheimer's such as
the Alzheimer's disease-associated ApoE subtype (ApoE4 subtype),
may be treated with a GM-CSF antagonist as described herein.
Vascular Dementia
[0078] A GM-CSF antagonist, e.g., an anti-GM-CSF antibody, may also
be used to treat a patient with vascular dementia. Vascular
dementia is also known as ischemic vascular dementia or
multi-infarct dementia. These terms refer to a group of syndromes
caused by different mechanisms all resulting in vascular lesions in
the brain. The main subtypes of vascular dementia described to date
are vascular mild cognitive impairment, multi-infarct dementia,
vascular dementia due to a strategic single infarct, vascular
dementia due to hemorrhagic lesions, small vessel disease (which
includes vascular dementia due to lacunar lesions and Binswanger
disease), and Alzheimer's disease mixed with vascular dementia.
Vascular lesions can be the result of diffuse cerebrovascular
disease or focal lesions (or a combination of both, which is what
is observed in the majority of cases). Vascular dementia is
diagnosed based on clinical criteria, often in conjunction with
neurological imaging to detect ischemic lesions. Mixed dementia is
diagnosed when patients have evidence of Alzheimer's disease and
cerebrovascular disease, either clinically or based on neuroimaging
evidence of ischemic lesions.
[0079] In some embodiments, a patient who is at risk for vascular
dementia, e.g., has transient ischemic episodes or has evidence of
amyloid deposits in blood vessels of the brain, may be treated with
a GM-CSF antagonist.
Cerebral Amyloid Angiopathy
[0080] In some embodiments, a patient treated with a GM-CSF
antagonist, e.g., an anti-GM-CSF antibody, may have cerebral
amyloid angiopathy (CAA), in which amyloid is deposited in the
walls of the cerebral arteries. Although CAA has been recognized as
one of the morphologic hallmarks of Alzheimer's disease, it is also
often found in the brains of elderly patients who are otherwise
neurologically healthy. While often asymptomatic, CAA may lead to
dementia, intracranial hemorrhage, or transient neurologic events.
Incranial hemorrhage (ICH) is the most recognized result of CAA.
More than 40% of patients with ICH-related hemorrhage have some
degree of dementia. In some cases, the cognitive changes can
precede the ICH. Thus patients having cerebral amyloid angiopathy
may have Alzheimer's disease, vascular dementia, or both, or may be
at risk of developing Alzheimer's disease and/or vascular
dementia.
[0081] A commonly used guideline for the diagnosis of CAA is the
Boston Cerebral Amyloid Angiopathy Group guidelines. Often, CAA is
accompanied by hemorrhage. The Boston Criteria for the diagnosis of
CAA-related hemorrhage are based on a combination of clinical,
radiologic, and pathologic data to differentiate lobar
intracerebral hemorrhage into categories of possible, probable, or
definite based on the likelihood of underlying cerebral amyloid
angiopathy. As used herein in the context of this invention, a
patient diagnosed with CAA may be diagnosed with possible or
probable CAA and need not be definitively diagnosed, as definite
CAA is typically determined postmortem.
Definite CAA: Full postmortem examination reveals lobar, cortical,
or corticosubcortical hemorrhage and evidence of severe CAA.
Probable CAA with supporting pathological evidence: The clinical
data and pathological tissue (evacuated hematoma or cortical biopsy
specimen) demonstrate a hemorrhage with certain characteristics and
some degree of vascular amyloid deposition. Probable CAA: Clinical
data and MRI findings (in the absence of a pathological specimen)
demonstrate multiple hematomas (as described above) in a patient
older than 60 years. Possible CAA: This is considered if the
patient is older than 60 years, and clinical and MRI data reveal a
single lobar, cortical, or corticosubcortical hemorrhage without
another cause, multiple hemorrhages with a possible but not a
definite cause, or some hemorrhage in an atypical location.
[0082] In some embodiments, a patient that has Alzheimer's disease,
vascular dementia, CAA, or is a candidate for developing
Alzheimer's disease, vascular dementia, or both and is treated with
a GM-CSF antagonist exhibits elevated GM-CSF levels, in comparison
to normal healthy controls, in the cerebrospinal fluid or other
sample, e.g., serum. Elevated levels of GM-CSF can be detected
using many techniques commonly known in the art, e.g., an
immunoassay.
III. GM-CSF Antagonists
[0083] As noted above, the invention provides methods for treating
Alzheimer's disease, vascular dementia, or CAA by administering a
GM-CSF antagonist to a patient suffering from the disease, or at
risk of developing the disease. GM-CSF antagonists suitable for use
in the invention selectively interfere with the induction of
signaling by the GM-CSF receptor by causing a reduction in the
binding of GM-CSF to the receptor. Such antagonists may include
antibodies that bind the GM-CSF receptor, antibodies that bind to
GM-CSF, GM-CSF analogs such as E21R, and other proteins or small
molecules that compete for binding of GM-CSF to its receptor or
inhibit signaling that normally results from the binding of the
ligand to the receptor.
[0084] In many embodiments, the GM-CSF antagonist used in the
invention is a polypeptide e.g., an anti-GM-CSF antibody, an
anti-GM-CSF receptor antibody, a soluble GM-CSF receptor, or a
modified GM-CSF polypeptide that competes for binding with GM-CSF
to a receptor, but is inactive. Such proteins are often produced
using recombinant expression technology. Such methods are widely
are widely known in the art. General molecular biology methods,
including expression methods, can be found, e.g., in instruction
manuals, such as, Sambrook and Russell (2001) Molecular Cloning: A
laboratory manual 3rd ed. Cold Spring Harbor Laboratory Press;
Current Protocols in Molecular Biology (2006) John Wiley and Sons
ISBN: 0-471-50338-X.
[0085] A variety of prokaryotic and/or eukaryotic based protein
expression systems may be employed to produce a GM-CSF antagonist
protein. Many such systems are widely available from commercial
suppliers. These include both prokaryotic and eukaryotic expression
systems.
GM-CSF Antibodies
[0086] In some embodiments, the GM-CSF antagonist is an antibody
that binds to GM-CSF or an antibody that binds to the GM-CSF
receptor .alpha. or .beta. subunit. The antibodies can be raised
against GM-CSF (or GM-CSF receptor) proteins, or fragments, or
produced recombinantly. Antibodies to GM-CSF for use in the
invention can be neutralizing or can be non-neutralizing antibodies
that bind GM-CSF and increase the rate of in vivo clearance of
GM-CSF such that the GM-CSF level in the circulation is reduced.
Often, the GM-CSF antibody is a neutralizing antibody.
[0087] Methods of preparing polyclonal antibodies are known to the
skilled artisan (e.g., Harlow & Lane, Antibodies, A Laboratory
manual (1988); Methods in Immunology). Polyclonal antibodies can be
raised in a mammal by one or more injections of an immunizing agent
and, if desired, an adjuvant. The immunizing agent includes a
GM-CSF or GM-CSF receptor protein, e.g., a human GM-CSF or GM-CSF
receptor protein, or fragment thereof.
[0088] In some embodiment, a GM-CSF antibody for use in the
invention is purified from human plasma. In such embodiments, the
GM-CSF antibody is typically a polyclonal antibody that is isolated
from other antibodies present in human plasma. Such an isolation
procedure can be performed, e.g., using known techniques, such as
affinity chromatography.
[0089] In some embodiments, the GM-CSF antagonist is a monoclonal
antibody. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler & Milstein, Nature
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent, such as human GM-CSF, to elicit lymphocytes that produce or
are capable of producing antibodies that will specifically bind to
the immunizing agent. Alternatively, the lymphocytes may be
immunized in vitro. The immunizing agent preferably includes human
GM-CSF protein, fragments thereof, or fusion protein thereof.
[0090] Human monoclonal antibodies can be produced using various
techniques known in the art, including phage display libraries
(Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); Marks et
al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol.
147(1):86-95 (1991)). Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody
production is observed, which closely resembles that seen in humans
in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, e.g., in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0091] In some embodiments the anti-GM-CSF antibodies are chimeric
or humanized monoclonal antibodies. As noted supra, humanized forms
of antibodies are chimeric immunoglobulins in which residues from a
complementary determining region (CDR) of human antibody are
replaced by residues from a CDR of a non-human species such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity.
[0092] An antibody that is employed in the invention can be in any
format. For example, in some embodiments, the antibody can be a
complete antibody including a constant region, e.g., a human
constant region, or can be a fragment or derivative of a complete
antibody, e.g., an Fd, a Fab, Fab', F(ab').sub.2, a scFv, an Fv
fragment, or a single domain antibody, such as a nanobody or a
camelid antibody. Such antibodies may additionally be recombinantly
engineered by methods well known to persons of skill in the art. As
noted above, such antibodies can be produced using known
techniques.
[0093] In some embodiments of the invention, the antibody is
additionally engineered to reduced immunogenicity, e.g., so that
the antibody is suitable for repeat administration. Methods for
generating antibodies with reduced immunogenicity include
humanization/humaneering procedures and modification techniques
such as de-immunization, in which an antibody is further
engineered, e.g., in one or more framework regions, to remove T
cell epitopes.
[0094] In some embodiments, the antibody is a humaneered antibody.
A humaneered antibody is an engineered human antibody having a
binding specificity of a reference antibody, obtained by joining a
DNA sequence encoding a binding specificity determinant (BSD) from
the CDR3 region of the heavy chain of the reference antibody to
human VH segment sequence and a light chain CDR3BSD from the
reference antibody to a human VL segment sequence. Methods for
humaneering are provided in US patent application publication no.
20050255552 and US patent application publication no. 20060134098.
Methods for signal-less secretion of antibody fragments from E.
coli are described in US patent application 20070020685.
[0095] An antibody can further be de-immunized to remove one or
more predicted T-cell epitopes from the V-region of an antibody.
Such procedures are described, for example, in WO 00/34317.
[0096] In some embodiments, the variable region is comprised of
human V-gene sequences. For example, a variable region sequence can
have at least 80% identity, or at least 85% identity, at least 90%
identity, at least 95% identity, at least 96% identity, at least
97% identity, at least 98% identity, or at least 99% identity, or
greater, with a human germ-line V-gene sequence.
[0097] An antibody used in the invention can include a human
constant region. The constant region of the light chain may be a
human kappa or lambda constant region. The heavy chain constant
region is often a gamma chain constant region, for example, a
gamma-1, gamma-2, gamma-3, or gamma-4 constant region.
[0098] In some embodiments, e.g., where the antibody is a fragment,
the antibody can be conjugated to another molecule, e.g., to
provide an extended half-life in vivo such as a polyethylene glycol
(pegylation) or serum albumin. Examples of PEGylation of antibody
fragments are provided in Knight et al (2004) Platelets 15: 409
(for abciximab); Pedley et al (1994) Br. J. Cancer 70: 1126 (for an
anti-CEA antibody) Chapman et al (1999) Nature Biotech. 17:
780.
Antibody Specificity
[0099] An antibody for use in the invention binds to GM-CSF or
GM-CSF receptor. Any number of techniques can be used to determine
antibody binding specificity. See, e.g., Harlow & Lane,
Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity of an antibody.
[0100] An exemplary antibody suitable for use with the present
invention is c19/2 (a human mouse chimeric anti-GM-CSF antibody).
In some embodiments, a monoclonal antibody that competes for
binding to the same epitope as c19/2, or that binds the same
epitope as c19/2, is used. The ability of a particular antibody to
recognize the same epitope as another antibody is typically
determined by the ability of the first antibody to competitively
inhibit binding of the second antibody to the antigen. Any of a
number of competitive binding assays can be used to measure
competition between two antibodies to the same antigen. For
example, a sandwich ELISA assay can be used for this purpose. This
is carried out by using a capture antibody to coat the surface of a
well. A subsaturating concentration of tagged-antigen is then added
to the capture surface. This protein will be bound to the antibody
through a specific antibody:epitope interaction. After washing a
second antibody, which has been covalently linked to a detectable
moiety (e.g., HRP, with the labeled antibody being defined as the
detection antibody) is added to the ELISA. If this antibody
recognizes the same epitope as the capture antibody it will be
unable to bind to the target protein as that particular epitope
will no longer be available for binding. If however this second
antibody recognizes a different epitope on the target protein it
will be able to bind and this binding can be detected by
quantifying the level of activity (and hence antibody bound) using
a relevant substrate. The background is defined by using a single
antibody as both capture and detection antibody, whereas the
maximal signal can be established by capturing with an antigen
specific antibody and detecting with an antibody to the tag on the
antigen. By using the background and maximal signals as references,
antibodies can be assessed in a pair-wise manner to determine
epitope specificity.
[0101] A first antibody is considered to competitively inhibit
binding of a second antibody, if binding of the second antibody to
the antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the first antibody using any of the assays described above.
Epitope Mapping
[0102] In some embodiments of the invention, an antibody is
employed that competes with binding, or bind, to the same epitope
as a known antibody, e.g., c19/2. Method of mapping epitopes are
well known in the art. For example, one approach to the
localization of functionally active regions of human
granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to
map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal
antibodies. For example, the epitope to which c19/2 (which has the
same variable regions as the neutralizing antibody LMM102) binds
has been defined using proteolytic fragments obtained by enzymic
digestion of bacterially synthesized hGM-CSF (Dempsey, et al.,
Hybridoma 9:545-558, 1990). RP-HPLC fractionation of a tryptic
digest resulted in the identification of an immunoreactive "tryptic
core" peptide containing 66 amino acids (52% of the protein).
Further digestion of this "tryptic core" with S. aureus V8 protease
produced a unique immunoreactive hGM-CSF product comprising two
peptides, residues 86-93 and 112-127, linked by a disulfide bond
between residues 88 and 121. The individual peptides, were not
recognized by the antibody.
Determining Binding Affinity
[0103] In some embodiments, the antibodies suitable for use with
the present invention have a high affinity binding for human GM-CSF
or GM-CSF receptor. High affinity binding between an antibody and
an antigen exists if the dissociation constant (K.sub.D) of the
antibody is <about 10 nM, typically<1 nM, and preferably
<100 pM. In some embodiments, the antibody has a dissociation
rate of about 10.sup.-4 per second or better.
[0104] A variety of methods can be used to determine the binding
affinity of an antibody for its target antigen such as surface
plasmon resonance assays, saturation assays, or immunoassays such
as ELISA or RIA, as are well known to persons of skill in the art.
An exemplary method for determining binding affinity is by surface
plasmon resonance analysis on a BJAcore.TM. 2000 instrument
(Biacore AB, Freiburg, Germany) using CM5 sensor chips, as
described by Krinner et al., (2007) Mol. Immunol. February;
44(5):916-25. (Epub 2006 May 11)).
Cell Proliferation Assay for Identifying Neutralizing
Antibodies
[0105] In some embodiments, the GM-CSF antagonists are neutralizing
antibodies to GM-CSF, or its receptor, which bind in a manner that
interferes with the binding of GM-CSF. In some embodiments, an
anti-GM-CSF antibody for use in the invention inhibits binding to
the alpha subunit of the GM-CSF receptor. Such an antibody can, for
example, bind to GM-CSF at the region where GM-CSF binds to the
receptor and thereby inhibit binding.
[0106] Neutralizing antibodies and other GM-CSF antagonists may be
identified using any number of assays that assess GM-CSF function.
For example, cell-based assays for GM-CSF receptor signaling, such
as assays which determine the rate of proliferation of a
GM-CSF-dependent cell line in response to a limiting amount of
GM-CSF, are conveniently used. The human TF-1 cell line is suitable
for use in such an assay. See, Krinner et al., (2007) Mol. Immunol.
In some embodiments, the neutralizing antibodies of the invention
inhibit GM-CSF-stimulated TF-1 cell proliferation by at least 50%
when a GM-CSF concentration is used which stimulates 90% maximal
TF-1 cell proliferation. In other embodiments, the neutralizing
antibodies inhibit GM-CSF stimulated proliferation by at least 90%.
Thus, typically, a neutralizing antibody, or other GM-CSF
antagonist for use in the invention, has an EC.sub.50 of less than
10 nM (e.g., Table 1). Additional assays suitable for use in
identifying neutralizing antibodies suitable for use with the
present invention will be well known to persons of skill in the
art.
Exemplary Antibodies
[0107] Antibodies for use in the invention are known in the art and
can be produced using routine techniques. Exemplary antibodies are
described. It is understood that the exemplary antibodies can be
engineered in accordance with the procedures known in the art and
summarized herein to produce antibody fragments, chimeras, and the
like by either chemical or recombinant technology.
[0108] An exemplary chimeric antibody suitable for use as a GM-CSF
antagonist is c19/2. The c19/2 antibody binds GM-CSF with a
monovalent binding affinity of about 10 pM as determined by surface
plasmon resonance analysis. The heavy and light chain variable
region sequences of c19/2 are known (e.g., WO03/068920). The CDRs,
as defined according to Kabat, are:
TABLE-US-00001 CDRH1 DYNIH (SEQ ID NO: 19) CDRH2 YIAPYSGGTGYNQEFKN
(SEQ ID NO: 20) CDRH3 RDRFPYYFDY (SEQ ID NO: 5) CDRL1 KASQNVGSNVA
(SEQ ID NO: 21) CDRL2 SASYRSG (SEQ ID NO: 22) CDRL3 QQFNRSPLT. (SEQ
ID NO: 23)
The CDRs can also be determined using other well known definitions
in the art, e.g., Chothia, international ImMunoGeneTics database
(IMGT), and AbM.
[0109] In some embodiments, an antibody used in the invention
competes for binding to, or binds to, the same epitope as c19/2.
The GM-CSF epitope recognized by c19/2 has been identified as a
product that has two peptides, residues 86-93 and residues 112-127,
linked by a disulfide bond between residues 88 and 121. The c19/2
antibody inhibits the GM-CSF-dependent proliferation of a human
TF-1 leukemia cell line with an EC.sub.50 of 30 pM when the cells
are stimulated with 0.5 ng/ml GM-CSF. In some embodiments, the
antibody used in the invention binds to the same epitope as
c19/2.
[0110] An antibody for administration, such as c19/2, can be
additionally humaneered. For example, the c19/2 antibody can be
further engineered to contain human V gene segments.
[0111] In some embodiments, a GM-CSF-binding antibody of the
invention is generated where, an antibody that has a CDR from one
of the V.sub.H-regions of the invention shown in FIG. 1, is
combined with one an antibody having a CDR of one of the
V.sub.L-regions shown in FIG. 1, and expressed in any of a number
of formats in a suitable expression system. Thus the antibody may
be expressed as a scFv, Fab, Fab' (containing an immunoglobulin
hinge sequence), F(ab').sub.2, (formed by di-sulfide bond formation
between the hinge sequences of two Fab' molecules), whole
immunoglobulin or truncated immunoglobulin or as a fusion protein
in a prokaryotic or eukaryotic host cell, either inside the host
cell or by secretion. A methionine residue may optionally be
present at the N-terminus, for example, in polypeptides produced in
signal-less expression systems. Each of the V.sub.H-regions
described herein may be paired with each of the V.sub.L regions to
generate an anti-GM-CSF antibody. Exemplary combinations of heavy
and light chains are shown in the table in FIG. 1.
[0112] In some embodiment, the antibody VL region, e.g., VK#1,
VK#2, VK#3, or VK#4 of FIG. 1, is combined with a human kappa
constant region to form the complete light-chain. Further, in some
embodiments, the VH region is combined a human gamma-1 constant
regions. Any suitable gamma-1 allotype can be chose, such as the
f-allotype. Thus, in some embodiments, the antibody is an IgG,
e.g., having an f-allotype, that has a V.sub.H selected from VH#1,
VH#2, VH#3, VH#4, or VH#5 (FIG. 1), and a V.sub.L selected from
VK#1, VK#2, VK#3, or VK#4 (FIG. 1.)
[0113] In some embodiments, e.g., where the antibody is a fragment,
the antibody can be conjugated to another molecule, e.g.,
polyethylene glycol (PEGylation) or serum albumin, to provide an
extended half-life in vivo. Examples of PEGylation of antibody
fragments are provided in Knight et al. Platelets 15:409, 2004 (for
abciximab); Pedley et al., Br. J. Cancer 70:1126, 1994 (for an
anti-CEA antibody); Chapman et al., Nature Biotech. 17:780, 1999;
and Humphreys, et al., Protein Eng. Des. 20: 227, 2007).
[0114] In some embodiments, the antibodies of the invention are in
the form of a Fab' fragment. A full-length light chain is generated
by fusion of a V.sub.L-region to human kappa or lambda constant
region. Either constant region may be used for any light chain;
however, in typical embodiments, a kappa constant region is used in
combination with a Vkappa variable region and a lambda constant
region is used with a Vlambda variable region.
[0115] The heavy chain of the Fab' is a Fd' fragment generated by
fusion of a V.sub.H-region of the invention to human heavy chain
constant region sequences, the first constant (CH1) domain and
hinge region. The heavy chain constant region sequences can be from
any of the immunoglobulin classes, but is often from an IgG, and
may be from an IgG1, IgG2, IgG3 or IgG4. The Fab' antibodies of the
invention may also be hybrid sequences, e.g., a hinge sequence may
be from one immunoglobulin sub-class and the CH1 domain may be from
a different sub-class.
[0116] Two other examples of neutralizing anti-GM-CSF antibody are
the human E10 antibody and human G9 antibody described in Li et
al., (2006) PNAS 103(10):3557-3562. E10 and G9 are IgG class
antibodies. E10 has an 870 pM binding affinity for GM-CSF and G9
has a 14 pM affinity for GM-CSF. Both antibodies are specific for
binding to human GM-CSF and show strong neutralizing activity as
assessed with a TF1 cell proliferation assay.
[0117] An additional exemplary neutralizing anti-GM-CSF antibody is
the MT203 antibody described by Krinner et al., (Mol. Immunol.
44:916-25, 2007; Epub 2006 May 112006). MT203 is an IgG1 class
antibody that binds GM-CSF with picomolar affinity. The antibody
shows potent inhibitory activity as assessed by TF-1 cell
proliferation assay and its ability to block IL-8 production in
U937 cells.
[0118] Additional antibodies suitable for use with the present
invention will be known to persons of skill in the art.
[0119] GM-CSF antagonists that are anti-GM-CSF receptor antibodies
can also be employed in the invention. Such GM-CSF antagonists
include antibodies to the GM-CSF receptor alpha chain or beta
chain. An anti-GM-CSF receptor antibody employed in the invention
can be in any antibody format as explained above, e.g., intact,
chimeric, monoclonal, polyclonal, antibody fragment, humanized,
humaneered, and the like. Examples of anti-GM-CSF receptor
antibodies, e.g., neutralizing, high-affinity antibodies, suitable
for use in the invention are known (see, e.g., U.S. Pat. No.
5,747,032 and Nicola et al., Blood 82: 1724, 1993).
Non Antibody GM-CSF Antagonists
[0120] Other proteins that may interfere with the productive
interaction of GM-CSF with its receptor include mutant GM-CSF
proteins and secreted proteins comprising at least part of the
extracellular portion of one or both of the GM-CSF receptor chains
that bind to GM-CSF and compete with binding to cell-surface
receptor. For example, a soluble GM-CSF receptor antagonist can be
prepared by fusing the coding region of the sGM-CSFRalpha with the
CH2-CH3 regions of murine IgG2a. An exemplary soluble GM-CSF
receptor is described by Raines et al. (1991) Proc. Natl. Acad.
Sci. USA 88: 8203. An example of a GM-CSFRalpha-Fc fusion protein
is provided, e.g., in Brown et al (1995) Blood 85: 1488. In some
embodiments, the Fc component of such a fusion can be engineered to
modulate binding, e.g., to increase binding, to the Fc
receptor.
[0121] Other GM-CSF antagonists include GM-CSF mutants. For
example, GM-CSF having a mutation of amino acid residue 21 of
GM-CSF to Arginine or Lysine (E21R or E21K) described by Hercus et
al. Proc. Natl. Acad. Sci. USA 91:5838, 1994 has been shown to have
in vivo activity in preventing dissemination of GM-CSF-dependent
leukemia cells in mouse xenograft models (Iversen et al. Blood
90:4910, 1997). As appreciated by one of skill in the art, such
antagonists can include conservatively modified variants of GM-CSF
that have substitutions, such as the substitution noted at amino
acid residue 21, or GM-CSF variants that have, e.g., amino acid
analogs to prolong half-life.
[0122] In some embodiments, the GM-CSF antagonist may be a peptide.
For example, A GM-CSF peptide antagonist may be a peptide designed
to structurally mimic the positions of specific residues on the B
and C helices of human GM-CSF that are implicated in receptor
binding and bioactivity (e.g., Monfardini et al., J. Biol. Chem.
271:2966-2971, 1996).
[0123] In other embodiments, the GM-CSF antagonist is an "antibody
mimetic" that targets and binds to the antigen in a manner similar
to antibodies. Certain of these "antibody mimics" use
non-immunoglobulin protein scaffolds as alternative protein
frameworks for the variable regions of antibodies. For example, Ku
et al. (Proc. Natl. Acad. Sci. U.S.A. 92(14):6552-6556 (1995))
discloses an alternative to antibodies based on cytochrome b562 in
which two of the loops of cytochrome b562 were randomized and
selected for binding against bovine serum albumin. The individual
mutants were found to bind selectively with BSA similarly with
anti-BSA antibodies.
[0124] U.S. Pat. Nos. 6,818,418 and 7,115,396 disclose an antibody
mimic featuring a fibronectin or fibronectin-like protein scaffold
and at least one variable loop. Known as Adnectins, these
fibronectin-based antibody mimics exhibit many of the same
characteristics of natural or engineered antibodies, including high
affinity and specificity for any targeted ligand. The structure of
these fibronectin-based antibody mimics is similar to the structure
of the variable region of the IgG heavy chain. Therefore, these
mimics display antigen binding properties similar in nature and
affinity to those of native antibodies. Further, these
fibronectin-based antibody mimics exhibit certain benefits over
antibodies and antibody fragments. For example, these antibody
mimics do not rely on disulfide bonds for native fold stability,
and are, therefore, stable under conditions which would normally
break down antibodies. In addition, since the structure of these
fibronectin-based antibody mimics is similar to that of the IgG
heavy chain, the process for loop randomization and shuffling may
be employed in vitro that is similar to the process of affinity
maturation of antibodies in vivo.
[0125] Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903
(1999)) disclose an antibody mimic based on a lipocalin scaffold
(Anticalin.RTM.). Lipocalins are composed of a .beta.-barrel with
four hypervariable loops at the terminus of the protein. The loops
were subjected to random mutagenesis and selected for binding with,
for example, fluorescein. Three variants exhibited specific binding
with fluorescein, with one variant showing binding similar to that
of an anti-fluorescein antibody. Further analysis revealed that all
of the randomized positions are variable, indicating that
Anticalin.RTM. would be suitable to be used as an alternative to
antibodies. Thus, Anticalins.RTM. are small, single chain peptides,
typically between 160 and 180 residues, which provides several
advantages over antibodies, including decreased cost of production,
increased stability in storage and decreased immunological
reaction.
[0126] U.S. Pat. No. 5,770,380 discloses a synthetic antibody
mimetic using the rigid, non-peptide organic scaffold of
calixarene, attached with multiple variable peptide loops used as
binding sites. The peptide loops all project from the same side
geometrically from the calixarene, with respect to each other.
Because of this geometric confirmation, all of the loops are
available for binding, increasing the binding affinity to a ligand.
However, in comparison to other antibody mimics, the
calixarene-based antibody mimic does not consist exclusively of a
peptide, and therefore it is less vulnerable to attack by protease
enzymes. Neither does the scaffold consist purely of a peptide, DNA
or RNA, meaning this antibody mimic is relatively stable in extreme
environmental conditions and has a long life span. Further, since
the calixarene-based antibody mimic is relatively small, it is less
likely to produce an immunogenic response.
[0127] Murali et al. (Cell Mol Biol 49(2):209-216 (2003)) describe
a methodology for reducing antibodies into smaller peptidomimetics,
they term "antibody-like binding peptidomimetics" (ABiP) which may
also be useful as an alternative to antibodies.
[0128] In addition to non-immunoglobulin protein frameworks,
antibody properties have also been mimicked in compounds comprising
RNA molecules and unnatural oligomers (e.g., protease inhibitors,
benzodiazepines, purine derivatives and beta-turn mimics).
Accordingly, non-antibody GM-CSF antagonists can also include such
compounds.
III. Therapeutic Administration
[0129] The methods of the invention typically comprise
administering a GM-CSF antagonist, (e.g., an anti-GM-CSF antibody)
as a pharmaceutical composition to a patient suffering from one or
more of Alzheimer's disease, vascular dementia, or CAA; or to a
patient at risk of developing Alzheimer's and/or vascular dementia,
in a therapeutically effective amount using a dosing regimen
suitable for treatment of the disease.
[0130] In the present invention, a therapeutically effective amount
is an amount that at least partially arrests symptoms and/or slows
the progression or onset of Alzheimer's disease, vascular dementia,
or CAA. For example, a therapeutically effective amount may slow
deposition of amyloid, or reduce the size or number of amyloid
plaques, in the brain and/or blood vessels. For example, the
methods of the invention successfully treat a patient having
Alzheimer's disease or vascular dementia by improving performance
of memory task tests and/or slowing or preventing the rate of, or
extent of, cognitive decline. Effectiveness may also be measured by
assessing other parameters, such as biochemical markers or
evaluating brain structure using CT scanning, MRI or PET
scanning.
[0131] The composition can be formulated for use in a variety of
drug delivery systems. One or more physiologically acceptable
excipients or carriers can also be included in the compositions for
proper formulation. Suitable formulations for use in the present
invention are found in Remington: The Science and Practice of
Pharmacy, 21st Edition, Philadelphia, Pa. Lippincott Williams &
Wilkins, 2005. For a brief review of methods for drug delivery,
see, Langer, Science 249: 1527-1533 (1990).
[0132] The GM-CSF antagonist for use in the methods of the
invention is provided in a solution suitable for injection into the
patient such as a sterile isotonic aqueous solution for injection.
The GM-CSF antagonist is dissolved or suspended at a suitable
concentration in an acceptable carrier. In some embodiments the
carrier is aqueous, e.g., water, saline, phosphate buffered saline,
and the like. The compositions may contain auxiliary pharmaceutical
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, and the like.
[0133] Amounts that are administered that are effective will depend
upon the severity of the disease and the general state of the
patient's health, including other factors such as age, weight,
gender, administration route, etc. Single or multiple
administrations of the antagonist may be administered depending on
the dosage and frequency as required and tolerated by the patient.
In any event, the methods provide a sufficient quantity of GM-CSF
antagonist in an amount to effectively treat the patient.
[0134] In some embodiments, the quantity of GM-CSF antagonist,
e.g., anti-GM-CSF administered to the patient is an amount that
results in improvement in performance on cognition tests used to
measure cognitive function in dementia (see, e.g. Qaseem et al.,
Annals of Internal Medicine 148:370-378, 2008). The primary scales
used to measure the domain of cognition deficits include the
Alzheimer's Disease Assessment Scale (ADAS) cognitive subscale
(ADAS-cog), noncognitive subscale (ADAS-noncog) and total score
(ADAS-tot); Mini-Mental State Examination (MMSE) or standardized
MMSE; and the Severe Impairment Battery (SIB). For the domain of
global assessment, the primary scale used include clinician-based
impression of change (CIBIC) (with caregiver input [CIBIC-plus] and
other modified versions). A patient receiving a therapeutically
effective amount of a GM-CSF antagonist, e.g., an anti-GM-CSF
antibody, may demonstrate improvement in performance on any one of
these tests, or alternative measures of cognitive function known in
the art, or on multiple tests. Improvement is generally determined
relative to a baseline value for cognitive function in the patient
prior to treatment with a GM-CSF antagonist. In some embodiments,
administration of an GM-CSF antagonist, e.g., an anti-GM-CSF
antibody, to a patient results in maintained cognitive function
where a patient does not exhibit a decline in cognitive function as
measure by any a standard test.
[0135] In another embodiment of the invention, the GM-CSF
antagonist used to treat a patient suffering from at least one of
Alzheimer's disease, vascular dementia, or CAA; or who is at risk
for developing Alzheimer's disease and/or vascular dementia, is
provided in combination with one or more additional therapeutic
agents for the treatment of the disease. Patients can receive the
one or more additional therapeutic agents as concomitant therapy.
Alternatively, patients may be treated sequentially, in any order,
with the additional therapeutic agent(s) and GM-CSF antagonist.
Examples of additional therapeutic agents include an
anti-beta-amyloid antibody, e.g., bapineuzumab; an amyloid-beta
(a.beta.) vaccine; cholesterol-lowering agents such as statins or
inhibitors of acyl-coenzyme A:cholesterol acyltransferase (ACAT);
nonsteroidal anti-inflammatory drugs; an acetyl cholinesterase
inhibitor, such as ARICEPT.RTM. (donepezil), EXELON.RTM.
(rivastigmine), or RAZADYNE.RTM. (galantamine); an NMDA receptor
antagonist, such as NAMENDA.RTM. (memantine); an antagonist to the
receptor for advanced glycation endproducts (RAGE); a
beta-secretase inhibitor; a gamma secretase inhibitor; IVIG, or a
neuroprotective agent such as DIMEBON.RTM. (dimebolin).
[0136] A. Administration
[0137] In some embodiments, the GM-CSF antagonist is administered
by injection or infusion through any suitable route including but
not limited to intravenous, perispinal, subcutaneous,
intramuscular, intranasal, perispinal, intrathecal, intraspinal or
intraperitoneal routes. In an exemplary embodiment, the GM-CSF
antagonist is diluted in a physiological saline solution for
injection prior to administration to the patient. Such an
antagonist is administered, for example, by intravenous infusion
over a period of between 15 minutes and 2 hours. In still other
embodiments, the administration procedure is via sub-cutaneous or
intramuscular injection.
[0138] In some embodiments, the GM-CSF antagonist, e.g., an
anti-GM-CSF antibody, is administered by a perispinal route.
Perispinal administration involves anatomically localized delivery
performed so as to place the therapeutic molecule directly in the
vicinity of the spine at the time of initial administration.
Perispinal administration is described, e.g., in U.S. Pat. No.
7,214,658 and in Tobinick & Gross, J. Neuroinflammation 5:2,
2008.
[0139] B. Dosing
[0140] The dose of GM-CSF antagonist is chosen in order to provide
effective therapy for a patient that has been diagnosed with one or
more of Alzheimer's disease, vascular dementia, or CAA, or is at
risk for developing Alzheimer's disease or vascular dementia. The
dose is typically in the range of about 0.1 mg/kg body weight to
about 25 mg/kg body weight or in the range about 1 mg to about 2 g
per patient. The dose is often in the range of about 1 to about 10
mg/kg or approximately about 50 mg to about 1000 mg/patient. The
dose may be repeated at an appropriate frequency which may be in
the range once per day to once every three months, depending on the
pharmacokinetics of the antagonists (e.g. half-life of the antibody
in the circulation) and the pharmacodynamic response (e.g. the
duration of the therapeutic effect of the antibody). In some
embodiments where the antagonist is an antibody or modified
antibody fragment, the in vivo half-life of between about 7 and
about 25 days and antibody dosing is repeated between once per week
and once every 3 months. In other embodiments, the antibody is
administered approximately once per month.
EXAMPLES
Example 1
Exemplary Humaneered Antibodies to GM-CSF
[0141] A panel of engineered Fab' molecules with the specificity of
c19/2 were generated from epitope-focused human V-segment libraries
as described in US patent application publication nos. 20060134098
and 20050255552. Full-length engineered V-regions from a
Vh1-restricted library were selected that supported binding to
recombinant human GM-CSF.
[0142] The "full-length" V-kappa library was used as a base for
construction of "cassette" libraries as described in US patent
application publication no. 20060134098, in which only part of the
murine c19/2 V-segment was initially replaced by a library of human
sequences. Two types of cassettes were constructed. Cassettes for
the V-kappa chains were made by bridge PCR with overlapping common
sequences within the framework 2 region. In this way "front-end"
and "middle" human cassette libraries were constructed for the
human V-kappa III isotype. Human V-kappa III cassettes which
supported binding to GM-CSF were identified by colony-lift binding
assay and ranked according to affinity in ELISA. The V-kappa human
"front-end" and "middle" cassettes were fused together by bridge
PCR to reconstruct a fully human V-kappa region that supported
GM-CSF binding activity. The engineered Fabs thus consist of
engineered V-heavy and V-kappa regions that support binding to
human GM-CSF.
[0143] Binding activity was determined by surface plasmon resonance
(spr) analysis. Biotinylated GM-CSF was captured on a
streptavidin-coated CMS biosensor chip. Humaneered Fab fragments
expressed from E. coli were diluted to a starting concentration of
30 nM in 10 mM HEPES, 150 mM NaCl, 0.1 mg/ml BSA and 0.005% P20 at
pH 7.4. Each Fab was diluted 4 times using a 3-fold dilution series
and each concentration was tested twice at 37 degrees C. to
determine the binding kinetics with the different density antigen
surfaces. The data from all three surfaces were fit globally to
extract the dissociation constants.
[0144] Binding kinetics were analyzed by Biacore 3000 surface
plasmon resonance (SPR). Recombinant human GM-CSF antigen was
biotinylated and immobilized on a streptavidin CMS sensor chip. Fab
samples were diluted to a starting concentration of 3 nM and run in
a 3 fold dilution series. Assays were run in 10 mM HEPES, 150 mM
NaCl, 0.1 mg/mL BSA and 0.005% p20 at pH 7.4 and 37.degree. C. Each
concentration was tested twice. Fab' binding assays were run on two
antigen density surfaces providing duplicate data sets. The mean
affinity (K.sub.D) for each of 6 various humaneered anti-GM-CSF Fab
clones, calculated using a 1:1 Langmuir binding model, is shown in
Table 1.
[0145] Fabs were tested for GM-CSF neutralization using a TF-1 cell
proliferation assay. GM-CSF-dependent proliferation of human TF-1
cells was measured after incubation for 4 days with 0.5 ng/ml
GM-CSF using a MTS assay (Cell titer 96, Promega) to determine
viable cells. All Fabs inhibited cell proliferation in this assay
indicating that these are neutralizing antibodies. There is a good
correlation between relative affinities of the anti-GM-CSF Fabs and
EC.sub.50 in the cell-based assay. Anti-GM-CSF antibodies with
monovalent affinities in the range 18 pM-104 pM demonstrate
effective neutralization of GM-CSF in the cell-based assay.
[0146] Exemplary engineered anti-GM-CSF V region sequences are
shown in FIG. 1.
TABLE-US-00002 TABLE 1 Affinity of anti-GM-CSF Fabs determined by
surface plasmon resonance analysis in comparison with activity
(EC.sub.50) in a GM-CSF dependent TF-1 cell proliferation assay
Monovalent EC.sub.50 (pM) in binding affinity TF-1 cell determined
by proliferation Fab SPR (pM) assay 94 18 165 104 19 239 77 29 404
92 58 539 42 104 3200 44 81 7000
Example 2
Administration of a GM-CSF Antibody in a Mouse Model of Alzheimer's
Disease
[0147] Transgenic mice over-expressing a mutant human amyloid
precursor protein (hAPP) were used to evaluate the role of
anti-mouse GM-CSF antibody 22E9 in an animal model of amyloid
deposit-associated dementia. Mice over-expressing human APP(751)
protein with three point mutations (K670M, N671L and V717I) under
the control of the murine Thy-1 promoter were used (APP751-SL mice)
on a C57BL/6 XDBA genetic background. This line of transgenic mice
shows a consistent age-dependent increase in accumulation of
amyloid beta peptides 40 and 42 (A.beta.40, A.beta.42) in the
brain. The mice develop plaques in the brain consisting of amyloid
deposits, starting at approximately 4 to 6 months and show
progressive deficits in learning and memory. By 8 months of age,
the mice show a strongly developed amyloid pathology accompanied by
inflammatory processes including prominent astrocytosis and
microgliosis around mature neuritic amyloid plaques (Wang et al.,
Vaccine 25:3041, 2007; Hutter-Paier et al., Neuron 44:227,
2004).
[0148] For investigation of uptake of anti-GM-CSF antibody into the
brain, 22E9 rat monoclonal anti-mouse GM-CSF antibody (R&D
Systems) was administered by three different routes to female
hAPP751-SL mice aged at least 10 months. Intracerebroventricular
injection (i.c.); intranasal administration (i.n.); and intravenous
injection (i.v.) were evaluated using a single dose of antibody
(0.25 mg i.n., 0.25 mg iv. and 25 pg i.c). Mice were sacrificed 48
hours post inoculation and brain histology was carried out.
[0149] Male APP751-SL mice at an age of 8 months (.+-.2 weeks) were
randomly allocated to treatment groups (13 mice per group) and
treated with rat anti-mouse GM-CSF antibody 22E9 (R&D Systems),
or with rat IgG2a isotype control antibody, or with the vehicle
phosphate buffered saline solution (PBS). Antibody or vehicle were
administered intravenously twice weekly for 2 months.
Behavioral Tests
Morris Water Maze (MWM)
[0150] Forty four days after the initiation of treatment, mice were
trained in the MWM spatial navigation task in which mice swim to
locate a hidden platform using visual cues. The task is based on
the principle that mice are motivated to escape from a water
environment by the most direct route. The Morris Water Maze task
was conducted in a black circular pool of diameter 100 cm filled
with water at a temperature of 21.+-.2.degree. C., divided
virtually into four sectors. A transparent platform was placed in
the southwest quadrant 0.5 cm beneath the water surface. One day
before the training session, animals were evaluated in a "pre-test"
consisting of two 60 s trials, to ensure that the vision of each
animal was normal. Only animals that completed this task
successfully were included in the MWM testing. n the MWM task, mice
were evaluated in three trials per day (each lasting 1 minute) for
four consecutive days. Escape latency (time to find the hidden
platform) and length of swimming path were measured. Differences
between animals in treatment and control groups were analyzed by
Student's t-tests to determine statistical significance.
Contextual Fear Conditioning Task (CFC)
[0151] The fear conditioning task was conducted in an automated box
provided by TSE-Systems, Germany. Mice were trained and tested on 2
consecutive days starting at Day 51 of the study. Training
consisted of placing a subject into the test chamber and allowing
exploration for 2 min. Thereafter, an auditory cue [2 Hz;
conditional stimulus (CS)] was presented for 15 sec. An electric
shock [1.5 mAmp; unconditioned stimulus (US)] was given for the
final 2 sec of the CS. This procedure was repeated, and mice were
removed from the chamber. Twenty hours after training, mice were
returned to the training chambers (context conditioned response),
and freezing behavior was recorded automatically. At the end of the
5 min contextual testing, mice were returned to their home cage.
Approximately 1 h later, freezing was recorded in a novel
environment and in response to the cue. In the CFC task, duration
of freezing behavior of each subject, expressed as a percentage of
each part of the test, was recorded.
Histology
[0152] Animals were sacrificed 48 hours after a single
administration of antibody or on day 54 of the therapeutic study.
Brains were rapidly removed and right hemispheres were fixed in 4%
Paraformaldehyde/PBS (pH 7.4) for one hour at room temperature,
transferred to a 15% sucrose PBS solution for 24 hours to ensure
cryoprotection, and stored at -80.degree. C. Brain hemispheres from
6 animals in each treatment group were used to provide sagittal
sections (10 .mu.m thick) using a cryotome for the determination of
plaque load visualized by a ThioflavinS staining or with an amyloid
specific antibody (anti-hAPP clone 6E10 (Signet 1: 5000)) and
fluorescent Cy3-labeled secondary antibody (Jackson
Immunoresearch). The estimation of plaque size, area and number
were determined using computer-aided quantification. Uptake of 22E9
antibody into the brain was visualized using an anti-rat IgG
antibody labeled with HistoGreen (Linaris.RTM.).
Results
[0153] Uptake of 22E9 into the brain was first evaluated by three
different routes of administration in hAPP751-SL mice:
intracerebroventricular injection (i.c.); intranasal administration
(i.n.); and intravenous injection (i.v.). A single dose of antibody
was administered (0.25 mg i.n., 0.25 mg iv. and 25 pg i.c) to mice
aged at least 10 months and brain histology was carried out 48
hours post-administration. Staining with anti-rat IgG-specific
antibody demonstrated that i.c. treatment led to clear uptake of
22E9 with particularly intense labeling of regions associated with
hippocampal and thalamic plaques. Both i.v. and i.n. treatment also
led to significant antibody uptake into the brain where
accumulation was detected around microglia, particularly associated
with hippocampal and thalamic amyloid plaques (see FIG. 2). This
provides alternative routes of administration of anti-GM-CSF
antibodies allowing uptake into the brain in the context of
amyloid-related disease without the need for direct stereotactic
injection into the cerebroventricular space.
[0154] Eight-month old male transgenic APP751-SL mice were then
treated by twice weekly i.v. administration with 22E9 anti-GM-CSF
antibody at 10 mg/kg or with isotype-matched (rat IgG2a) control
antibody at the same dose level or vehicle alone (PBS). From Day 44
after the initiation of treatment (after 13 doses of antibody),
mice were evaluated in the Morris Water Maze. Mice treated with
anti-GM-CSF antibody 22E9 showed improved learning ability in this
task over the 4 days of evaluation compared with vehicle treated or
isotype control-treated mice (see FIG. 3). The difference in
swimming path length between anti-GM-CSF treated and isotype
control treated mice achieved statistical significance by Day 4 of
the test. The escape latency also showed a trend to improvement by
Day 4 of the test in the anti-GM-CSF treated mice compared with the
group receiving the control antibody but the difference did not
achieve statistical significance.
[0155] At Day 51 of the study (after 15 doses), mice were evaluated
in a contextual fear study in which the freezing behavior in
response to a training chamber and auditory cue was evaluated.
Anti-GM-CSF treated mice showed a tendency to freeze for a longer
time when placed in the training chamber compared with vehicle or
isotype-control treated mice (see FIG. 4). No differences in
response to auditory cues were observed in this experiment.
[0156] These data indicate that anti-GM-CSF treatment led to
reduction in the learning deficit in mice transgenic for mutant
human APP using two tests for visuo-spatial learning ability (the
Morris Water Maze and Contextual fear conditioning).
[0157] Histological analysis was carried out on regions of the
brain of mice at the end of the therapeutic study (Day 54)
Immunohistochemistry using antibody 6E10 specific for human amyloid
indicated the presence of amyloid plaques in all evaluated mice but
the mean plaque size was significantly reduced in the hippocampus
of mice treated with 22E9 antibody compared with isotype-control
treated mice (see FIG. 5). Plaque size also appeared to be reduced
in the cortex of anti-GM-CSF treated mice compared with control
animals. Plaque number was also quantified but showed no
significant differences between the treated and control animals in
this study at the time point that was analyzed. Staining for
ThioS-positive did not reveal significant differences between the
mature, condensed neuritic plaques of treated and control mice at
Day 54 in this experiment.
[0158] The effects of administration of an anti-GM-CSF antibody in
another transgenic AD mouse model were also investigated, the
Tg2576 model. This Alzheimer's disease mouse model was generated
using mutant human APP gene 695 amino acid isoform with a double
mutation (K670N and M671L). Administration of anti-GM-CSF directly
into the brains of Tg2576 mice decreased soluble a.beta..sub.1-42
production and suppressed microglial activity in the mice (Maczak
et al., Hum. Molec. Genet. 18:3876-3893, 2009).
[0159] The above examples are provided by way of illustration only
and not by way of limitation. Those of skill in the art will
readily recognize a variety of noncritical parameters that could be
changed or modified to yield essentially similar results.
[0160] All publications, patent applications, accession numbers,
and other references cited in this specification are herein
incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference.
Sequence CWU 1
1
2416PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody V-H region CDR3
binding specificity determinant 1Arg Gln Arg Phe Pro Tyr1 5
26PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody V-H region CDR3
binding specificity determinant 2Arg Asp Arg Phe Pro Tyr1 5
315PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody J segment JH4 3Tyr
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 10 15
410PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody V-H region CDR3
4Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr1 5 10 510PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (anti-GM-CSF) antibody V-H region CDR3, chimeric antibody
c19/2 CDRH3 5Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr1 5 10
69PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody V-L region CDR3
6Gln Gln Phe Asn Xaa Ser Pro Leu Thr1 5 798PRTHomo sapienshuman
germ line VH1 1-02 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80 Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg8119PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
antibody V-H region VH#1 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Arg Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 998PRTHomo
sapienshuman germ line VH1 1-03 9Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp
Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg10119PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
antibody V-H region VH#2 10Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly
Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 11119PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (anti-GM-CSF) antibody V-H region VH#3 11Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40
45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
50 55 60 Gln Gly Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser Thr
Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gln Arg Phe Pro Tyr Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
12119PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody V-H region VH#4
12Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn
Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys
Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Arg Asp
Thr Ser Ala Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Gln Arg
Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 13119PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
antibody V-H region VH#5 13Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Arg Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 1496PRTHomo
sapienshuman germ line VKIII A27 V-segment 14Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Gly Ser Ser Pro 85 90 95 15107PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
antibody V-L region VK#1 15Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40 45 Tyr Ser Thr Ser
Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Arg Ser Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
16107PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) antibody V-H region VK#2
16Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Thr
Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Val Leu Ile 35 40 45 Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr
Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Phe Asn Lys Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 17107PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (anti-GM-CSF) antibody V-H region VK#3 17Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ser Asn 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40
45 Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn
Arg Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 18107PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
antibody V-H region VK#4 18Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Ile Gly Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40 45 Tyr Ser Thr Ser
Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Lys Ser Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
195PRTArtificial Sequencesynthetic anti-granulocyte-macrophage
colony-stimulating factor (anti-GM-CSF) chimeric antibody c19/2
CDRH1 19Asp Tyr Asn Ile His1 5 2017PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
chimeric antibody c19/2 CDRH2 20Tyr Ile Ala Pro Tyr Ser Gly Gly Thr
Gly Tyr Asn Gln Glu Phe Lys1 5 10 15 Asn2111PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (anti-GM-CSF) chimeric antibody c19/2 CDRL1 21Lys Ala Ser
Gln Asn Val Gly Ser Asn Val Ala1 5 10 227PRTArtificial
Sequencesynthetic anti-granulocyte-macrophage colony-stimulating
factor (anti-GM-CSF) chimeric antibody c19/2 CDRL2 22Ser Ala Ser
Tyr Arg Ser Gly1 5 239PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
chimeric antibody c19/2 CDRL3 23Gln Gln Phe Asn Arg Ser Pro Leu
Thr1 5 249PRTArtificial Sequencesynthetic
anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF)
antibody V-L region CDR3 24Gln Gln Phe Asn Lys Ser Pro Leu Thr1
5
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