U.S. patent application number 15/150958 was filed with the patent office on 2016-12-15 for compounds for use in the treatment of alzheimer's disease.
This patent application is currently assigned to UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG. The applicant listed for this patent is UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG. Invention is credited to Bianca Da Costa Dias, Danielle Gonsavles, Katarina Jovanovic, Stefan Knackmuss, Melvyn Little, Uwe Reusch, Marc Saul Weinberg, Stefan Franz Thomas Weiss.
Application Number | 20160361413 15/150958 |
Document ID | / |
Family ID | 47116142 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160361413 |
Kind Code |
A1 |
Weiss; Stefan Franz Thomas ;
et al. |
December 15, 2016 |
COMPOUNDS FOR USE IN THE TREATMENT OF ALZHEIMER'S DISEASE
Abstract
The invention relates to a method of modulating concentration of
Alzheimer's Disease (AD) relevant proteins amyloid precursor
protein (APP), beta (.beta.) and gamma (.gamma.) secretases and
amyloid beta peptide (A.beta.), and also relates to a method of
reducing A.beta. shedding. Furthermore, this invention extends to a
compound for use in the treatment of AD, and also to a method of
treating AD.
Inventors: |
Weiss; Stefan Franz Thomas;
(Johannesburg, ZA) ; Jovanovic; Katarina;
(Edenvale, ZA) ; Gonsavles; Danielle;
(Johannesburg, ZA) ; Da Costa Dias; Bianca;
(Johannesburg, ZA) ; Knackmuss; Stefan;
(Plankstadt, DE) ; Reusch; Uwe; (Maikammer,
DE) ; Little; Melvyn; (St. Peter-Ording, DE) ;
Weinberg; Marc Saul; (Midrand, ZA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG |
Johannesburg |
|
ZA |
|
|
Assignee: |
UNIVERSITY OF THE WITWATERSRAND,
JOHANNESBURG
JOHANNESBURG
ZA
|
Family ID: |
47116142 |
Appl. No.: |
15/150958 |
Filed: |
May 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14345770 |
Mar 19, 2014 |
9365647 |
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PCT/IB2012/054968 |
Sep 19, 2012 |
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15150958 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 39/3955 20130101; C07K 2317/76 20130101; C07K 16/26 20130101;
C07K 16/28 20130101; A61K 31/7105 20130101; C12N 15/1138
20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 19, 2011 |
ZA |
2011/06804 |
Claims
1-62. (canceled)
63. A method for reducing the concentration of amyloid beta peptide
(A.beta.) in a human or animal suffering from Alzheimer's Disease
(AD), the method comprising: contacting a cell surface protein with
a cell surface protein specific antibody, or any fragment thereof,
such that binding occurs between a surface epitope of the cell
surface protein and the cell surface protein specific antibody
therein hindering the proteolytic cleavage of APP by beta (.beta.)
and gamma (.gamma.) secretases, which in turn causes a decrease in
the concentration of amyloid beta peptide (A.beta.).
64. The method according to claim 63, wherein the cell surface
protein is a laminin receptor protein.
65. The method according to claim 64, wherein the laminin receptor
protein is 37 kDa/67 kDa laminin receptor (LRP/LR) or a protein
having at least 80% or greater homology to LRP/LR.
66. The method according to claim 63, wherein the antibody or
fragment thereof is raised against the cell surface protein.
67. The method according to claim 63, wherein the antibody or
fragment thereof is an anti-laminin receptor specific antibody or
fragment thereof.
68. The method according to claim 67, wherein the anti-laminin
receptor specific antibody or fragment thereof is an anti-LRP/LR
specific antibody or fragment thereof or an antibody or fragment
thereof having at least 80% or greater homology to anti-LRP/LR
specific antibody or fragment thereof.
69. The method according to claim 68, wherein the anti-LRP/LR
specific antibody or fragment thereof is IgG1-iS18 or a fragment
thereof.
70. The method according to claim 63, wherein the cell surface
protein is 37 kDa/67 kDa laminin receptor (LRP/LR) and the cell
surface protein specific antibody is IgG1-iS18 or a fragment
thereof.
Description
FIELD OF INVENTION
[0001] This invention relates to a compound for the modulation of
37 kDa/67 kDa laminin receptor in humans and/or animals. The
invention extends to a method of modulating concentration of
Alzheimer's Disease (AD) relevant proteins amyloid precursor
protein (APP), beta (.beta.) and gamma (.gamma.) secretases and
amyloid beta peptide (A.beta.). Furthermore, this invention extends
to a compound for use in the treatment of AD, and also to a method
of treating AD.
BACKGROUND
[0002] Alzheimer's disease (AD) is notably the most prevalent form
of dementia afflicting the elderly and is associated with a
multitude of genetic, environmental, epigenetic, dietary and
lifestyle risk factors.sup.1. AD is said to affect in excess of 37
million people globally.sup.4.
[0003] The neuropathological hallmarks of AD include intracellular
neurofibrillary tangle formation and extracellular amyloid beta
peptide (A.beta.) plaque deposition.sup.5. The sequential cleavage
of the amyloid precursor protein (APP) by beta (.beta.) and gamma
(.gamma.) secretases.sup.2 results in the shedding of the 4 kDa
A.beta. which aggregates to form amyloid plaques. A.beta., as a
soluble oligomer, as well as plaque-incorporated aggregate, is the
predominant focus of investigative efforts to treat AD.
[0004] A.beta. and more specifically the 42 amino acid isoform
(A.beta..sub.42), is largely considered the primary disease causing
agent in AD (as A.beta. accumulation is a pre-requisite for tau
hyperphosporylation, another AD-associated protein).sup.6.
Specifically, A.beta. is generated through the proteolytic cleavage
of the type I transmembrane protein APP by .beta.- and
.gamma.-secretase. The mechanisms underlying A.beta. induction of
neuronal loss (one of the key pathophysiological features of AD)
are yet to be firmly established. It is proposed that the
neurotoxicity of A.beta. is partially mediated through its
interactions with cellular receptors.sup.3. These interactions may
include binding of A.beta. to a surface receptor on a neuron
thereby changing its biochemical structure, which negatively
affects neuronal communication. It is proposed that A.beta. may
affect neuronal communication by eliciting alterations in signal
transduction pathways through direct binding to cell surface
receptors, (such as N-methyl-d-aspartate (NMDA) receptors, insulin
receptors, .alpha.-7 nicotinic receptors).sup.3,7. Alternatively,
A.beta. may alter signal transduction pathways indirectly via
incorporation into lipid membranes of the plasma membrane and, to a
lesser extent, cellular organelles.sup.8. This is thought to induce
structural and functional alterations in lipid bound receptors and
consequently results in aberrant signal transduction
pathways.sup.8.
[0005] There is a need for compounds which in use modulate the
production and concentrations of APP, (.beta.) and (.gamma.)
secretases and A.beta. in a human or animal in order to treat AD.
There is a further need for compounds that modulate intracellular
neurofibrillary tangle formation and extracellular A.beta. plaque
deposition in order to treat AD.
SUMMARY
[0006] According to a first aspect of this invention there is
provided a method for reducing concentration of at least one
Alzheimer's Disease (AD) relevant protein selected from the group
including, but not limited to, amyloid precursor protein (APP),
beta (.beta.) and gamma (.gamma.) secretases and amyloid beta
peptide (A.beta.), the method comprising contacting a cell surface
protein, preferably an extracellular matrix glycoprotein, with a
cell surface protein specific antibody, preferably a monoclonal
antibody, or any fragment of the aforementioned, such that binding
occurs between a surface epitope of the cell surface protein and
the cell surface protein specific antibody causing a decrease in
the concentration of the at least one AD relevant proteins.
[0007] The reduction in A.beta. concentration may be a reduction
relative to A.beta. concentration in a normal healthy human or
animal, or it may be a reduction relative to A.beta. concentration
in a human or animal suffering from AD.
[0008] It is to be understood that the binding between the surface
epitope of the cell surface protein and the cell surface protein
specific antibody at least hinders, preferably prevents, binding of
the at least one AD relevant proteins to the cell surface
protein.
[0009] The cell surface protein may be a laminin receptor protein.
In a preferred embodiment of the invention the laminin receptor is
37 kDa/67 kDa laminin receptor (LRP/LR) of a human and/or animal.
LRP/LR is also known as LAMR, RPSA and p40. The cell surface
protein may also be a protein showing at least 80% or greater
homology to the laminin receptor protein, preferably showing at
least 80% or greater homology to LRP/LR.
[0010] In a preferred embodiment of the invention the AD relevant
protein whose concentration is reduced via the method of this
invention is A.beta.. The reduced amount of A.beta. causes reduced
intracellular neurofibrillary tangle formation and/or reduced
extracellular A.beta. plaque deposition in human and/or animal
cells, preferably neuronal cells, therein treating and/or
preventing AD.
[0011] The cell surface protein specific antibody may be any
antibody, or fragment thereof, raised against the cell surface
protein. In a preferred embodiment the antibody is raised against
LRP/LR or against a protein having 80% or greater homology with
LRP/LR. The antibody, or fragment thereof, may be a F(ab')2
fragment, a Fab fragment scFv, a bi-specific scFv, a tri-specific
scFv, a single chain or tandem diabody, a single domain antibody
(dAb), a minibody or a molecular recognition unit (MRU).
Furthermore, the antibody, or fragment thereof, may be monovalent,
bivalent or multivalent. The antibody, or fragment thereof, may
additionally comprise at least one further antigen-interaction site
and/or at least one further effector domain.
[0012] In a preferred embodiment of the invention, the antibody or
fragment thereof may be an anti-laminin receptor specific antibody,
preferably an anti-LRP/LR specific antibody, further preferably
IgG1-iS18.
[0013] In a preferred embodiment of the invention the cell surface
protein and/or the cell surface protein specific antibody is a
human or animal cell surface protein and/or cell surface protein
specific antibody. The cell surface protein may be located on
murine neuronal cells (N2a), human neuronal cells (SH-SY5Y), baby
hamster kidney cells (BHK) and human embryonic kidney cells (HEK293
and/or HEK293 FT).
[0014] In a preferred embodiment of the invention, the method for
reducing concentration of at least one AD relevant protein selected
from the group including, but not limited to, APP, beta (.beta.)
and gamma (.gamma.) secretases and A.beta., the method comprises
contacting LRP/LR with IgG1-iS18, or any fragment thereof, such
that binding occurs between LRP/LR and IgG1-iS18, or any fragment
thereof, causing a decrease in the concentration of A.beta..
[0015] According to a second aspect of this invention there is
provided a method for reducing amyloid beta peptide (A.beta.)
shedding caused by the proteolytic cleavage of amyloid precursor
protein (APP) by beta (.beta.) and gamma (.gamma.) secretases, the
method comprising contacting a cell surface protein, preferably an
extracellular matrix glycoprotein, with a cell surface protein
specific antibody, preferably a monoclonal antibody, or any
fragment of the aforementioned, such that binding occurs between a
surface epitope of the cell surface protein and the cell surface
protein specific antibody therein hindering the proteolytic
cleavage of APP by beta (.beta.) and gamma (.gamma.)
secretases.
[0016] The reduction in A.beta. shedding may be a reduction
relative to A.beta. shedding in a normal healthy human or animal,
or it may be a reduction relative to A.beta. shedding in a human or
animal suffering from AD.
[0017] It is to be understood that the binding between the surface
epitope of the cell surface protein and the cell surface protein
specific antibody at least hinders, preferably prevents, binding of
at least one of the AD relevant proteins APP, beta (.beta.) and
gamma (.gamma.) secretases and A.beta. to the cell surface protein.
It is to be understood that this binding causes a reduction in
A.beta. shedding.
[0018] The cell surface protein may be a laminin receptor protein.
In a preferred embodiment of the invention the laminin receptor is
37 kDa/67 kDa laminin receptor (LRP/LR) of a human and/or animal.
LRP/LR is also known as LAMR, RPSA and p40. The cell surface
protein may also be a protein showing at least 80% or greater
homology to the laminin receptor protein, preferably showing at
least 80% or greater homology to LRP/LR.
[0019] The cell surface protein specific antibody may be any
antibody, or fragment thereof, raised against the cell surface
protein. In a preferred embodiment the antibody is raised against
LRP/LR or against a protein having 80% or greater homology with
LRP/LR. The antibody, or fragment thereof, may be a F(ab')2
fragment, a Fab fragment scFv, a bi-specific scFv, a tri-specific
scFv, a single chain or tandem diabody, a single domain antibody
(dAb), a minibody or a molecular recognition unit (MRU).
Furthermore, the antibody, or fragment thereof, may be monovalent,
bivalent or multivalent. The antibody, or fragment thereof, may
additionally comprise at least one further antigen-interaction site
and/or at least one further effector domain.
[0020] In a preferred embodiment of the invention, the antibody or
fragment thereof may be an anti-laminin receptor specific antibody,
preferably an anti-LRP/LR specific antibody, further preferably
IgG1-iS18.
[0021] In a preferred embodiment of the invention the cell surface
protein and/or the cell surface protein specific antibody is a
human or animal cell surface protein and/or cell surface protein
specific antibody. The cell surface protein may be located on
murine neuronal cells (N2a), human neuronal cells (SH-SY5Y), baby
hamster kidney cells (BHK) and human embryonic kidney cells (HEK293
and/or HEK 293 FT).
[0022] In a preferred embodiment of the invention, the method for
reducing A.beta. shedding caused by the proteolytic cleavage of APP
by beta (.beta.) and gamma (.gamma.) secretases, the method
comprises contacting LRP/LR with IgG1-iS18, or any fragment
thereof, such that binding occurs between LRP/LR and IgG1-iS18, or
any fragment thereof, causing a reduction in A.beta. shedding.
[0023] According to a third aspect of this invention there is
provided a method for reducing concentration of at least one
Alzheimer's Disease (AD) relevant protein selected from the group
including, but not limited to, amyloid precursor protein (APP),
beta (.beta.) and gamma (.gamma.) secretases and amyloid beta
peptide (A.beta.), the method comprising contacting a cell surface
protein, preferably an extracellular matrix glycoprotein, with a
nucleotide sequence, preferably an RNA sequence, further preferably
a short hairpin RNA (shRNA) sequence or a short interfering RNA
(siRNA) sequence or a micro RNA (miRNA) sequence, such that binding
occurs between mRNA of the cell surface protein and the nucleotide
sequence causing a downregulation of the cell surface protein which
in turn causes a decrease in the concentration of the at least one
AD relevant proteins.
[0024] It is to be understood that the binding between the mRNA of
the cell surface protein and the nucleotide sequence downregulates
the cell surface protein such that there are fewer cell surface
proteins present on the cell when compared to regular physiological
functioning. Therefore, there are fewer binding sites available for
the at least one AD relevant proteins to bind to. A reduction in
binding sites leads to reduced concentrations of the at least one
AD relevant proteins APP, beta (.beta.) and gamma (.gamma.)
secretases and A.beta..
[0025] The cell surface protein may be a laminin receptor protein.
In a preferred embodiment of the invention the laminin receptor is
37 kDa/67 kDa laminin receptor (LRP/LR) of a human and/or animal.
LRP/LR is also known as LAMR, RPSA and p40. The cell surface
protein may also be a protein showing at least 80% or greater
homology to the laminin receptor protein, preferably showing at
least 80% or greater homology to LRP/LR.
[0026] Preferably, when binding between the nucleotide sequence and
the mRNA occurs, such binding is between the nucleotide sequence
and LRP mRNA.
[0027] In a preferred embodiment of the invention the AD relevant
protein whose concentration is reduced via the method of this
invention is A.beta.. The reduced amount of A.beta. causes reduced
intracellular neurofibrillary tangle formation and/or reduced
extracellular A.beta. plaque deposition in human and/or animal
cells, preferably neuronal cells, therein treating and/or
preventing AD. The reduction in A.beta. concentration may be a
reduction relative to A.beta. concentration in a normal healthy
human or animal, or it may be a reduction relative to A.beta.
concentration in a human or animal suffering from AD.
[0028] The nucleotide sequence is preferably shRNA, further
preferably at least one of shRNA1 and shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively.
[0029] In a preferred embodiment of the invention the cell surface
protein and/or the nucleotide sequence is a human or animal cell
surface protein and/or nucleotide sequence. The cell surface
protein may be located on murine neuronal cells (N2a), human
neuronal cells (SH-SY5Y), baby hamster kidney cells (BHK) and human
embryonic kidney cells (HEK293 and/or HEK 293 FT).
[0030] In a preferred embodiment of the invention, the method for
reducing concentration of at least one AD relevant protein selected
from the group including, but not limited to, APP, beta (.beta.)
and gamma (.gamma.) secretases and A.beta., the method comprises
contacting LRP mRNA with shRNA1 and/or shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively, such that
binding occurs between the LRP mRNA and shRNA1 and/or shRNA7
causing a decrease in the concentration of A.beta..
[0031] According to a fourth aspect of this invention there is
provided a method for reducing A.beta. shedding caused by the
proteolytic cleavage of amyloid precursor protein (APP) by beta
(.beta.) and gamma (.gamma.) secretases, the method comprising
contacting a cell surface protein, preferably an extracellular
matrix glycoprotein, with a nucleotide sequence, preferably an RNA
sequence, further preferably a short hairpin RNA (shRNA) sequence
or a short interfering RNA (siRNA) sequence or a mirco RNA (miRNA)
sequence, such that binding occurs between mRNA of the cell surface
protein and the nucleotide sequence causing a downregulation of the
cell surface protein which in turn causes a decrease in the
proteolytic cleavage of APP by beta (.beta.) and gamma (.gamma.)
secretases.
[0032] The reduction in A.beta. shedding may be a reduction
relative to A.beta. shedding in a normal healthy human or animal,
or it may be a reduction relative to A.beta. shedding in a human or
animal suffering from AD.
[0033] It is to be understood that the binding between the mRNA of
the cell surface protein and the nucleotide sequence downregulates
the cell surface protein such that there are fewer cell surface
proteins present on the cell when compared to regular physiological
functioning. Therefore, there are fewer binding sites available for
the at least one AD relevant proteins to bind to. A reduction in
binding sites leads to a reduction in A.beta. shedding.
[0034] The cell surface protein may be a laminin receptor protein.
In a preferred embodiment of the invention the laminin receptor is
37 kDa/67 kDa laminin receptor (LRP/LR) of a human and/or animal.
LRP/LR is also known as LAMR, RPSA and p40. The cell surface
protein may also be a protein showing at least 80% or greater
homology to the laminin receptor protein, preferably showing at
least 80% or greater homology to LRP/LR.
[0035] Preferably, when binding between the nucleotide sequence and
the mRNA occurs, such binding is between the nucleotide sequence
and LRP mRNA.
[0036] The nucleotide sequence is preferably a shRNA, further
preferably at least one of shRNA1 and shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively.
[0037] In a preferred embodiment of the invention the cell surface
protein and/or the nucleotide sequence is a human or animal cell
surface protein and/or nucleotide sequence. The cell surface
protein may be located on murine neuronal cells (N2a), human
neuronal cells (SH-SY5Y), baby hamster kidney cells (BHK) and human
embryonic kidney cells (HEK293 and/or HEK 293 FT).
[0038] In a preferred embodiment of the invention, the method for
reducing A.beta. shedding caused by the proteolytic cleavage of APP
by beta (.beta.) and gamma (.gamma.) secretases, the method
comprises contacting LRP mRNA with shRNA1 and/or shRNA7 having
sequence listing as set forth in SEQ ID NO: 1 and 2, respectively,
such that binding occurs between LRP mRNA and shRNA1 and/or shRNA7
reducing LRP/LR cell surface levels causing a reduction in A.beta.
shedding.
[0039] According to a fifth aspect of this invention there is
provided a method for reducing concentration of at least one
Alzheimer's Disease (AD) relevant protein selected from the group
including, but not limited to, amyloid precursor protein (APP),
beta (.beta.) and gamma (.gamma.) secretases and amyloid beta
peptide (A.beta.), the method comprising contacting a cell surface
protein of the first aspect of the invention with the antibody of
the first aspect of the invention and the nucleotide sequence of
the third aspect of the invention.
[0040] According to a sixth aspect of this invention there is
provided a method for reducing A.beta. shedding caused by the
proteolytic cleavage of amyloid precursor protein (APP) by beta
(.beta.) and gamma (.gamma.) secretases, the method comprising the
method comprising contacting a cell surface protein of the second
aspect of the invention with the antibody the second aspect of the
invention and the nucleotide sequence of the fourth aspect of the
invention.
[0041] According to a seventh aspect of this invention there is
provided for use of an anti-laminin receptor specific antibody in
the manufacture of a pharmaceutical composition to treat
Alzheimer's Disease (AD).
[0042] In a preferred embodiment of the invention the anti-laminin
receptor specific antibody may be anti-37 kDa/67 kDa laminin
receptor (LRP/LR) specific antibody, further preferably the
anti-LRP/LR specific antibody may be IgG1-iS18.
[0043] According to an eighth aspect of this invention there is
provided an anti-laminin receptor specific antibody for use in
treating Alzheimer's Disease (AD). In a preferred embodiment of the
invention the anti-laminin receptor specific antibody may be
anti-37 kDa/67 kDa laminin receptor (LRP/LR) specific antibody,
further preferably the anti-LRP/LR specific antibody may be
IgG1-iS18.
[0044] According to a ninth aspect of this invention there is
provided for use of a nucleotide sequence in the manufacture of a
pharmaceutical composition to treat Alzheimer's Disease (AD).
[0045] In a preferred embodiment of the invention the nucleotide
sequence may be a shRNA, further preferably at least one of shRNA1
and shRNA7 having sequence listing as set forth in SEQ ID NO: 1 and
2, respectively. The use may further include an anti-laminin
specific receptor antibody, preferably an anti-LRP/LR specific
antibody, further preferably IgG1-iS18.
[0046] According to a tenth aspect of this invention there is
provided a nucleotide sequence for use in treating Alzheimer's
Disease (AD).
[0047] In a preferred embodiment of the invention the nucleotide
sequence may be a shRNA, further preferably at least one of shRNA1
and shRNA7 having sequence listing as set forth in SEQ ID NO: 1 and
2, respectively. There is provided for the nucleotide sequence to
be used together with an anti-laminin specific receptor antibody,
preferably an anti-LRP/LR specific antibody, further preferably
IgG1-iS18.
[0048] According to an eleventh aspect of this invention there is
provided a method of treating Alzheimer's Disease (AD) comprising
administering an anti-laminin specific receptor antibody,
preferably an anti-37 kDa/67 kDa laminin receptor (LRP/LR) specific
antibody, further preferably the anti-LRP/LR specific antibody may
be IgG1-iS18 to a human or animal in need thereof.
[0049] According to a twelfth aspect of this invention there is
provided a method of treating Alzheimer's Disease (AD) comprising
administering a nucleotide sequence, preferably shRNA, further
preferably at least one of shRNA1 and shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively, to a
human or animal in need thereof. The method may further include
administering an anti-laminin specific receptor antibody,
preferably an anti-LRP/LR specific antibody, further preferably
IgG1-iS18, to the human or animal in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The invention is now described, by way of example only, with
reference to the accompanying diagrammatic drawings, in which
[0051] FIG. 1a shows co-localisation of 37 kDa/67 kDa laminin
receptor (LRP/LR) with the Alzheimer's Disease (AD) relevant
proteins amyloid precursor protein (APP), beta (.beta.) and gamma
(.gamma.) secretases and amyloid beta peptide (A.beta.), on the
surface of human embryonic kidney cells (HEK293) cells via
immunofluorescence microscopy;
[0052] FIG. 1aa shows black and white line drawings of FIG. 1a;
[0053] FIG. 1b shows co-localisation of LRP/LR with the AD relevant
proteins APP, beta (.beta.) and gamma (.gamma.) secretases and
A.beta., on the surface of murine neuroblastoma (N2a) cells via
immunofluorescence microscopy;
[0054] FIG. 1bb shows black and white line drawings of FIG. 1b;
[0055] FIG. 2a shows A.beta. concentrations in HEK293 and human
neuronal cells (SH-SY5Y) cells after treatment with antibodies
IgG1-i518 and IgG1-HD37 as detected by an A.beta. ELISA (Human
Amyloid .beta.(1-x) Assay Kit (IBL)) after 18 hours of antibody
incubation;
[0056] FIG. 2b shows A.beta. concentrations after SH-SY5Y cells
were treated with varying doses of IgG1-i518 for 18 hours, as
determined by an A.beta. ELISA;
[0057] FIG. 2c shows flow cytometric analysis of APP,
.beta.-secretase and .gamma.-secretase levels on the surface of
human embryonic kidney cells (HEK293FT) and SH-SY5Y cells post
treatment with IgG1-iS18;
[0058] FIG. 2d shows a Western blot analysis depicting sAPP.beta.
(SAPP.beta. is a shedded cleavage product of APP generated by the
action of .beta.-secretase) levels from cell culture medium after
SH-SY5Y cells were treated with varying concentrations (0-100
.mu.g/ml) of IgG1-iS18 for 18 hours;
[0059] FIG. 3 shows LRP/LR target sequences and structure of short
hairpin RNA1 (shRNA1) and short hairpin RNA7 (shRNA7);
[0060] FIG. 4a shows a Western Blot analysis of HEK293 cells
transfected with LRP-specific shRNA1 and shRNA7 (as well as a
scrambled control, shRNAscr);
[0061] FIG. 4b shows the effects of shRNA on downregulation of
LRP/LR wherein the A.beta. concentration of the cell culture medium
of shRNA-transfected HEK293 cells was analysed using an A.beta.
ELISA;
[0062] FIG. 4c shows flow cytometric analysis of APP,
.beta.-secretase and .gamma.-secretase levels on the surface of
shRNA-transfected HEK293 cells;
[0063] FIG. 4d shows sAPP.beta. levels in shRNA-transfected HEK293
cells were analysed by Western blotting;
[0064] FIG. 4e shows A.beta. concentration of the cell culture
medium of shRNAscr-transfected and mock-transfected HEK293
cells;
[0065] FIG. 5a shows flow cytometry histogram overlay plots for
.beta.-secretase, .gamma.-secretase and APP after antibody
treatment to HEK293 cells;
[0066] FIG. 5b shows flow cytometry histogram overlay plots for
.beta.-secretase, .gamma.-secretase and APP after antibody
treatment to SH-SY5Y cells;
[0067] FIG. 5c shows flow cytometry histogram overlay plots for
.beta.-secretase, .gamma.-secretase and APP after shRNA treatment
to HEK293 cells were transfected with either shRNA1, shRNA7 or
shRNAscr;
[0068] FIG. 6a(i) shows a pull down assay of cell lysates
containing recombinantly expressed LRP/LR::FLAG co-incubated with
exogenous A.beta.;
[0069] FIG. 6a(ii) shows an immunoblot employed to validate the
position of LRP::FLAG (.about.38 kDa);
[0070] FIG. 6b shows cellular viability of HEK293 cells, as
determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (1 mg/ml) assay, post exogenous treatment with
synthetic A.beta..sub.42 and upon co-incubation with anti-LRP/LR
IgG1-iS18 or IgG1-HD37 (negative control);
[0071] FIG. 6c shows cellular viability of SH-SY5Y cells;
[0072] FIG. 6d shows cellular viability of N2a cells;
[0073] FIG. 6e shows cellular proliferation of N2a cells as
determined by colorimetric 5-bromo-2'-deoxyuridine (BrdU)
non-isotopic immunoassay (Calbiochem.RTM.), allowing 4 h for BrdU
incorporation into cultured cells; and
[0074] FIG. 7 shows post pull down assay protein detection of
control samples.
DETAILED DESCRIPTION OF THE DRAWINGS
[0075] According to a first aspect of this invention there is
provided a method for reducing concentration of at least one
Alzheimer's Disease (AD) relevant protein selected from the group
including, but not limited to, amyloid precursor protein (APP),
beta (.beta.) and gamma (.gamma.) secretases and amyloid beta
peptide (A.beta.), the method comprising contacting a cell surface
protein, preferably an extracellular matrix glycoprotein, with a
cell surface protein specific antibody, preferably a monoclonal
antibody, or any fragment of the aforementioned, such that binding
occurs between a surface epitope of the cell surface protein and
the cell surface protein specific antibody causing a decrease in
the concentration of the at least one AD relevant proteins.
[0076] The reduction in concentration of the at least one AD
relevant protein is a reduction relative to the amount of AD
relevant proteins in a normal healthy human or animal, or relative
to a human or animal suffering from AD.
[0077] It is to be understood that the binding between the surface
epitope of the cell surface protein and the cell surface protein
specific antibody at least hinders, preferably prevents, binding of
at least one of the AD relevant proteins to the cell surface
protein.
[0078] The cell surface protein may be a laminin receptor protein.
However, in a preferred embodiment of the invention the laminin
receptor is 37 kDa/67 kDa laminin receptor (LRP/LR) of a human
and/or animal. LRP/LR is also known as LAMR, RPSA and p40. The cell
surface protein may also be a protein showing at least 80% or
greater homology to the laminin receptor protein, preferably
showing at least 80% or greater homology to LRP/LR.
[0079] In the examples illustrated and/or exemplified below the AD
relevant protein whose concentration is reduced via the method of
this invention is A.beta.. The reduced amount of A.beta. causes
reduced intracellular neurofibrillary tangle formation and/or
reduced extracellular A.beta. plaque deposition in human and/or
animal cells, preferably neuronal cells, therein treating and/or
preventing AD. When exercising this method of reducing A.beta.
concentration in an effort to treat and/or prevent AD, the
contacting typically takes place in vivo.
[0080] The cell surface protein specific antibody may be any
antibody, or fragment thereof, raised against the cell surface
protein. Typically, the antibody is raised against LRP/LR or
against a protein having 80% or greater homology with LRP/LR. The
antibody, or fragment thereof, may be a F(ab')2 fragment, a Fab
fragment scFv, a bi-specific scFv, a tri-specific scFv, a single
chain or tandem diabody, a single domain antibody (dAb), a minibody
or a molecular recognition unit (MRU). Furthermore, the antibody,
or fragment thereof, may be monovalent, bivalent or multivalent.
The antibody, or fragment thereof, may additionally comprise at
least one further antigen-interaction site and/or at least one
further effector domain. Preferably, the antibody or fragment
thereof an anti-laminin receptor specific antibody, further
preferably anti-LRP/LR specific antibody, still further preferably
IgG1-iS18.
[0081] The cell surface protein and/or the cell surface protein
specific antibody is typically a human or animal cell surface
protein and/or cell surface protein specific antibody. The cell
surface protein may be located on murine neuronal cells (N2a),
human neuronal cells (SH-SY5Y), baby hamster kidney cells (BHK) and
human embryonic kidney cells (HEK293 and/or HEK 293 FT).
[0082] In a preferred embodiment of the invention, the method for
reducing concentration of at least one AD relevant protein
comprises contacting LRP/LR with IgG1-iS18, or any fragment
thereof, such that binding occurs between LRP/LR and IgG1-iS18, or
any fragment thereof, causing a decrease in the concentration of
A.beta.. From a practical point of view, when exercising this
method of reducing A.beta. concentration in an effort to treat
and/or prevent AD, the antibody (typically IgG1-iS18, or any
fragment thereof) is formulated into a pharmaceutical composition
and further formulated into a pharmaceutical dosage form to be
administered to a human or animal in need of treatment. The
pharmaceutical composition may include excipients. The dosage form
may be formulated to deliver the pharmaceutical composition via
oral and/or parenteral means.
[0083] According to a second aspect of this invention there is
provided a method for reducing A.beta. shedding caused by the
proteolytic cleavage of APP by beta (.beta.) and/or gamma (.gamma.)
secretases, the method comprising contacting the cell surface
protein described above, with the cell surface protein specific
antibody described above, such that binding occurs between a
surface epitope of the cell surface protein and the cell surface
protein specific antibody therein hindering the proteolytic
cleavage of APP by beta (.beta.) and/or gamma (.gamma.)
secretases.
[0084] The reduction in A.beta. shedding may be a reduction
relative to A.beta. shedding in a normal healthy human or animal,
or it may be a reduction relative to A.beta. shedding in a human or
animal suffering from AD.
[0085] It is to be understood that the binding between the surface
epitope of the cell surface protein and the cell surface protein
specific antibody at least hinders, preferably prevents, binding of
at least one of the AD relevant proteins APP, beta (.beta.) and
gamma (.gamma.) secretases and A.beta. to the cell surface protein.
It is further to be understood that it is this binding that causes
a reduction in A.beta. shedding.
[0086] As mentioned in the first aspect of the invention, in a
preferred embodiment of the invention the cell surface protein is a
laminin receptor, preferably the laminin receptor is LRP/LR of a
human and/or animal. The cell surface protein may also be a protein
showing at least 80% or greater homology to the laminin receptor
protein, preferably showing at least 80% or greater homology to
LRP/LR.
[0087] The cell surface protein specific antibody may be any
antibody, or fragment thereof, raised against the cell surface
protein. In a preferred embodiment the antibody is raised against
LRP/LR or against a protein having 80% or greater homology with
LRP/LR. The antibody, or fragment thereof, may be a F(ab')2
fragment, a Fab fragment scFv, a bi-specific scFv, a tri-specific
scFv, a single chain or tandem diabody, a single domain antibody
(dAb), a minibody or a molecular recognition unit (MRU).
Furthermore, the antibody, or fragment thereof, may be monovalent,
bivalent or multivalent. The antibody, or fragment thereof, may
additionally comprise at least one further antigen-interaction site
and/or at least one further effector domain.
[0088] In a preferred embodiment of the invention, the antibody or
fragment thereof may be an anti-laminin receptor specific antibody,
preferably an anti-LRP/LR specific antibody, further preferably
IgG1-iS18.
[0089] In a preferred embodiment of the invention the cell surface
protein and/or the cell surface protein specific antibody is a
human or animal cell surface protein and/or cell surface protein
specific antibody. The cell surface protein may be located on
murine neuronal cells (N2a), human neuronal cells (SH-SY5Y), baby
hamster kidney cells (BHK) and human embryonic kidney cells (HEK293
and/or HEK 293 FT).
[0090] In a preferred embodiment of the invention, the method for
reducing A.beta. shedding caused by the proteolytic cleavage of APP
by beta (.beta.) and gamma (.gamma.) secretases, the method
comprises contacting LRP/LR with IgG1-iS18, or any fragment
thereof, such that binding occurs between LRP/LR and IgG1-iS18, or
any fragment thereof, causing a reduction in A.beta. shedding. As
described in the first aspect of the invention above, the IgG1-iS18
is typically formulated into a pharmaceutical composition which is
formulated into a pharmaceutical dosage form which is administered
to a human or animal in need of AD treatment.
[0091] According to a third aspect of this invention there is
provided a method for reducing concentration of at least one AD
relevant protein selected from the group including, but not limited
to, APP, beta (.beta.) and gamma (.gamma.) secretases and A.beta.,
the method comprising contacting a cell surface protein, preferably
an extracellular matrix glycoprotein, with a nucleotide sequence,
preferably an RNA sequence, further preferably a short hairpin RNA
(shRNA) sequence or a short interfering RNA (siRNA) sequence or a
micro RNA (miRNA) sequence, such that binding occurs between mRNA
of the cell surface protein and the nucleotide sequence causing a
downregulation of the cell surface protein which in turn causes a
decrease in the concentration of the at last one AD relevant
proteins.
[0092] The reduction in concentration of the at least one AD
relevant protein is a reduction relative to the amount of AD
relevant proteins in a normal healthy human or animal, or relative
to a human or animal suffering from AD.
[0093] The cell surface protein may be a laminin receptor protein
and preferably LRP/LR as described above in the first and second
aspects of the invention. Preferably, when binding between the
nucleotide sequence and the mRNA occurs, such binding is between
the nucleotide sequence and LRP mRNA.
[0094] In a preferred embodiment of the invention the AD relevant
protein whose concentration is reduced via the method of this
invention is A.beta. as described above.
[0095] The nucleotide sequence is preferably shRNA, further
preferably at least one of shRNA1 and shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively.
[0096] In a preferred embodiment of the invention, the method for
reducing concentration of at least one AD relevant protein selected
from the group including, but not limited to, APP, beta (.beta.)
and gamma (.gamma.) secretases and A.beta., the method comprises
contacting LRP/LR with shRNA1 and/or shRNA7 having sequence listing
as set forth in SEQ ID NO: 1 and 2, respectively, such that binding
occurs between LRP mRNA and shRNA1 and/or shRNA7 causing a decrease
in the concentration of A.beta..
[0097] According to a fourth aspect of this invention there is
provided a method for reducing A.beta. shedding caused by the
proteolytic cleavage of amyloid precursor protein (APP) by beta
(.beta.) and gamma (.gamma.) secretases, the method comprising
contacting a cell surface protein, preferably an extracellular
matrix glycoprotein, with a nucleotide sequence, preferably an RNA
sequence, further preferably a short hairpin RNA (shRNA) sequence
or a short interfering RNA (siRNA) sequence or a mirco RNA (miRNA)
sequence, such that binding occurs between mRNA of the cell surface
protein and the nucleotide sequence causing a downregulation of the
cell surface protein which in turn causes a decrease in the
proteolytic cleavage of APP by beta (.beta.) and gamma (.gamma.)
secretases.
[0098] The cell surface protein may be a laminin receptor protein
and preferably LRP/LR as described above in the first and second
aspects of the invention. Preferably, when binding between the
nucleotide sequence and the mRNA occurs, such binding is between
the nucleotide sequence and LRP mRNA.
[0099] The reduction in A.beta. shedding may be a reduction
relative to A.beta. shedding in a normal healthy human or animal,
or it may be a reduction relative to A.beta. shedding in a human or
animal suffering from AD.
[0100] The nucleotide sequence is preferably a shRNA, further
preferably at least one of shRNA1 and shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively.
[0101] In a preferred embodiment of the invention, the method for
reducing A.beta. shedding caused by the proteolytic cleavage of APP
by beta (.beta.) and gamma (.gamma.) secretases, the method
comprises contacting LRP/LR with shRNA1 and/or shRNA7 having
sequence listing as set forth in SEQ ID NO: 1 and 2 respectively,
such that binding occurs between LRP mRNA and shRNA1 and/or shRNA7
causing a reduction in LRP/LR cell surface levels and hence a
reduction in A.beta. shedding.
[0102] According to a fifth aspect of this invention there is
provided a method for reducing concentration of at least one AD
relevant protein selected from the group including, but not limited
to, APP, beta (.beta.) and gamma (.gamma.) secretases and A.beta.,
the method comprising contacting a cell surface protein of the
first aspect of the invention with the antibody the first aspect of
the invention and the nucleotide sequence of the third aspect of
the invention. Essentially, the LRP/LR is downregulated by the
shRNAs (either shRNA 1 or 7 or both) and is substantially blocked
by the anti-LRP/LR specific antibody, in so doing, further reducing
the concentration of A.beta..
[0103] According to a sixth aspect of this invention there is
provided a method for reducing A.beta. shedding caused by the
proteolytic cleavage of APP by beta (.beta.) and gamma (.gamma.)
secretases, the method comprising the method comprising contacting
a cell surface protein of the second aspect of the invention with
the antibody the second aspect of the invention and the nucleotide
sequence of the fourth aspect of the invention. Essentially, the
LRP/LR is downregulated by the shRNAs (either shRNA 1 or 7 or both)
and is substantially blocked by the anti-LRP/LR specific antibody,
in so doing, further reducing A.beta. shedding.
[0104] According to a seventh aspect of this invention there is
provided for use of an anti-laminin receptor specific antibody in
the manufacture of a pharmaceutical composition to treat AD.
Typically, the anti-laminin receptor specific antibody is
IgG1-iS18.
[0105] According to an eighth aspect of this invention there is
provided an anti-laminin receptor specific antibody for use in
treating AD. Typically, the anti-laminin receptor specific antibody
is IgG1-iS18.
[0106] According to a ninth aspect of this invention there is
provided for use of a nucleotide sequence in the manufacture of a
pharmaceutical composition to treat AD. Typically, the nucleotide
sequence may be RNA, preferably a shRNA, further preferably at
least one of shRNA1 and shRNA7 having sequence listing as set forth
in SEQ ID NO: 1 and 2, respectively. The use may further include an
anti-laminin receptor specific antibody described above.
[0107] According to a tenth aspect of this invention there is
provided a nucleotide sequence for use in treating AD. Typically,
the nucleotide sequence may be RNA, preferably a shRNA, further
preferably at least one of shRNA1 and shRNA7 having sequence
listing as set forth in SEQ ID NO: 1 and 2, respectively. There is
provided for the nucleotide sequence to be used together with an
anti-laminin receptor specific antibody described above for use in
treating AD.
[0108] According to an eleventh aspect of this invention there is
provided a method of treating AD comprising administering an
anti-laminin receptor specific antibody, preferably an anti-LRP/LR
specific antibody, further preferably IgG1-iS18 to a human or
animal in need thereof.
[0109] According to a twelfth aspect of this invention there is
provided a method of treating AD comprising administering a
nucleotide sequence, preferably a RNA sequence, further preferably
shRNA, further preferably at least one of shRNA1 and shRNA7 having
sequence listing as set forth in SEQ ID NO: 1 and 2, respectively,
to a human or animal in need thereof. The method may further
include administering an anti-laminin receptor specific antibody,
preferably an anti-LRP/LR specific antibody, further preferably
IgG1-iS18 to the human or animal.
EXAMPLES
[0110] A representative example of the invention is described
and/or illustrated and/or exemplified below and should not be
viewed as limiting to the scope of the invention. The sequence
listing attached hereto is incorporated by reference.
[0111] In the examples of the invention illustrated and exemplified
below, it is shown that 37 kDa/67 kDa laminin receptor (LRP/LR) is
associated with the Alzheimer's Disease (AD) relevant proteins
amaloid precursor protein (APP), .beta.- and .gamma.-secretase as
well as amaloid beta peptide (A.beta.). It was found that A.beta.
binds to LRP/LR and this interaction may contribute to
A.beta.-induced cytotoxicity. Furthermore, antibody blockage (or
substantial blockage) and shRNA downregulation of LRP/LR was shown
to reduce A.beta. shedding, due to impediment of .beta.-secretase
activity, rather than alteration of APP, .beta.- and
.gamma.-secretase levels. These findings indicate that LRP/LR may
be implicated in AD pathogenesis and could lead to novel
therapeutic interventions for use in modulating LRP/LR and/or
modulating the concentration of AD relevant proteins APP, .beta.-
and .gamma.-secretases and A.beta. in a human or animal in the
treatment of AD.
[0112] The extracellular matrix glycoprotein, laminin, exhibits an
A.beta. binding site, namely the IKAV peptide sequence located on
the alpha (.alpha.) chain of the tri-peptide.sup.15. However, the
association between laminin and A.beta. is reported to promote
neurite outgrowth.sup.16 and inhibit fibrillogenesis.sup.15 and
thereby thwart A.beta. pathogenesis. Prior research does not
suggest LRP/LR functioning being important in the determining
concentrations of the abovementioned AD relevant proteins,
specifically A.beta., nor does the prior art suggest that LRP/LR
could play any role in impeding .beta.-secretase activity.
[0113] LRP/LR (also known as LAMR, RPSA and p40) is a
multifunctional protein located within the cholesterol-rich lipid
raft domains of the plasma membrane, in the cytoplasm as well as in
the nucleus.sup.17. Associations between LRP/LR and a multitude of
extracellular (laminin and elastin) and intracellular (cytoskeletal
proteins, histones, heparan sulfate proteoglycans (HSPGs))
components have been described, and are of physiological
significance.sup.18.
[0114] The experimental protocols described hereunder show a nexus
between LRP/LR and the amyloidgenic pathway in AD, more
specifically a nexus between LRP/LR and A.beta. shedding into the
extracellular space.
[0115] To explore the above mentioned nexus indirect
immunofluorescence microscopy was employed to assess the cellular
distribution of AD relevant proteins, namely the APP, .beta.- and
.gamma.-secretases and A.beta.. LRP/LR was shown to co-localise
with APP (FIG. 1a, i-iv), .beta.-secretase (FIG. 1a, v-viii),
.gamma.-secretase (FIG. 1a, ix-xii) and A.beta. (FIG. 1a, xiii-xvi)
on the surface of non-permeabilised human embryonic kidney cells
(HEK293). An alternative laminin binding receptor, Very Late
Antigen 6 (VLA6), was employed as a negative control (FIG. 1a,
xvii-xx). Analogous results were obtained for murine neuroblastoma
(N2a) cells (FIG. 1b).
[0116] In general, FIG. 1a shows co-localisation of LRP/LR with the
AD relevant proteins APP, .beta.-secretase, .gamma.-secretase and
A.beta. on the surface of human embryonic kidney cells (HEK293
and/or HEK 293 FT) cells. FIG. 1a shows cell surface receptors on
HEK293 cells having been indirectly immunolabelled to allow for
detection using the Olympus IX71 Immunofluorescence Microscope and
Analysis Get It Research Software. In particular, FIG. 1a shows (i)
APP (detected by anti-APP (rabbit polyclonal IgG) (Abcam), (v),
.beta.-secretase (detected using anti-BACE (M-83) (rabbit
polyclonal IgG) (Santa Cruz Biotechnology)), (ix),
.gamma.-secretase (detected by anti-PEN-2 (FL-101) (rabbit
polyclonal IgG) (Santa Cruz Biotechnology)), (xii), A.beta.
(detected using anti-.beta.-amyloid (22-35) (Sigma)) and (xvii),
VLA6 (detected by anti-very late antigen-6 (VLA6) CD49-f (rabbit
monoclonal IgG) (Immunotech) were indirectly labelled with
Alexaflour 633, while an anti-human FITC coupled antibody (Cell
Lab) was used to label LRP/LR (ii, vi, x, xiv, xviii).
[0117] The merges between LRP/LR and AD relevant proteins are shown
(iii, vii, xi, xv, xix) in FIG. 1a and the corresponding
2D-cytofluorograms (acquired using CellSens Software) have been
included to confirm the degree of co-localisation (iv, viii, xii,
xvi, xx).
[0118] FIG. 1b shows the same as FIG. 1a above, but as seen on the
surface of N2a cells. Scale bars on the figures are 10 .mu.m.
[0119] The proximity of the AD relevant proteins on the cell
surface thereby suggested that an association/interaction between
LRP/LR and AD relevant proteins is feasible and that the receptor
may indeed be implicated in AD pathogenesis. 2D-cytofluorograms
(FIGS. 1a and b, iv, viii, xii, xvi) show the joint distribution of
the red and green fluorescence, with a diagonal indicating
co-localisation between the cell surface proteins of interest. FIG.
1aa and 1bb show a black and white reproducible version of FIG. 1a
and 1b, where a diagonal again indicates co-localisation between
the cell surface proteins of interest. Pearson's Correlation
co-efficients for co-localisation were employed to further confirm
the observed results as shown in Table 1 below.
[0120] Table 1 Shows Pearson's Correlation Co-efficient for
Co-localisation between LRP/LR and AD relevant proteins
TABLE-US-00001 human embryonic kidney Murine neuronal cells
(HEK293) cells (N2a) LRP/LR + APP 0.862 0.948 LRP/LR + A.beta.
0.926 0.969 LRP/LR + .beta.-secretase 0.915 0.900 LRP/LR +
.gamma.-secretase 0.938 0.914 LRP/LR + VLA6 0.583 0.563
[0121] The Pearson's Correlation co-efficient was employed to
determine the degree of co-localisation between proteins of
interest, where 1 indicates complete co-localisation and 0 is
indicative of no co-localisation between proteins of interest. The
co-efficient was calculated for LRP/LR and AD relevant proteins
APP, A.beta., .beta.- and .gamma.-secretase respectively, as well
as the negative control VLA6.
[0122] To investigate whether the LRP/LR is involved in the
amyloidogenic pathway, and more specifically A.beta. shedding into
the extracellular space, cells were treated with the anti-LRP/LR
specific antibody IgG1-iS18.sup.21 and anti-cluster of
differentiation (CD19) antibody IgG1-HD37.sup.21 (negative
control).
[0123] Essentially, cellular incubation with IgG1-iS18 resulted in
a significant reduction (47.6% in human embryonic kidney cells
(HEK293FT) and 28.5% in human neuronal cells (SH-SY5Y)) in A.beta.
concentration when compared to the no antibody control (FIG. 2a).
To assess the optimal concentration of IgG1-iS18 for A.beta.
shedding impairment, dose dependency assays were conducted and a
noteworthy reduction in A.beta. concentration was observed for 25
.mu.g/ml, 50 .mu.g/ml, 75 .mu.g/ml and 100 .mu.g/ml (FIG. 2b). The
distinction between 50 .mu.g/ml-100 .mu.g/ml was nominal and thus
the of choice 50 .mu.g/ml IgG1-iS18 for further experimental
procedures was warranted.
[0124] In general, FIG. 2 shows the effects of IgG1-iS18 on A.beta.
concentration. FIG. 2a shows A.beta. concentrations in HEK293 and
SH-SY5Y cells after treatment with IgG1-iS18 and IgG1-HD37 as
detected by an A.beta. ELISA (Human Amyloid .beta.(1-x) Assay Kit
(IBL)) after 18 hours of antibody incubation. Data shown
(mean.+-.s.e.m) are representative of three independent experiments
(performed in triplicate) per cell line. *p<0.05, **p<0.01,
***p<0.001, NS not significant; Student's t-test.
[0125] FIG. 2b shows A.beta. concentrations after SH-SY5Y cells
were treated with varying doses of IgG1-iS18 for 18 hours, as
determined by an A.beta. ELISA. Data shown (Mean.+-.s.d.) comparing
A.beta. levels of untreated cells (0 .mu.g/ml) and IgG1-iS18
treated cells (25-100 .mu.g/ml), ***p<0.001; n=3; Student's
t-test.
[0126] FIG. 2c shows flow cytometric analysis of APP,
.beta.-secretase and .gamma.-secretase levels on the surface of
HEK293FT and SH-SY5Y cells post treatment with IgG1-iS18
(mean.+-.s.d., NS not significant, n=3, Student's t-test).
[0127] FIG. 2d shows a Western blot analysis depicting sAPP.beta.
(the shedded cleavage product after cleavage of APP by
.beta.-secretase) levels from cell culture medium after SH-SY5Y
cells were treated with varying concentrations (0-100 .mu.g/ml) of
IgG1-iS18 for 18 hours. Western blot band intensities from three
independent experiments were quantified using Quantity One 4.6
software.
[0128] Owing to the ability of IgG1-iS18 to decrease A.beta.
concentration, it is thought that LRP/LR mediates this process. To
further confirm this role in the amyloidogenic pathway, RNA
interference technology, specifically short hairpin RNA (shRNA)
(see FIG. 3), was employed to downregulate LRP/LR expression as
shown in FIG. 4.
[0129] FIG. 3 shows LRP/LR target sequences and structure of shRNA1
and shRNA7. It is to be understood that the references to thymine
should are to be read as uracil since the nucleotide sequences are
RNA. The sequence data for both shRNA1 and shRNA7 are attached
hereto as SEQ ID NO: 1 and 2 respectively. The complete shRNA
expression cassettes were designed with the guide strand on the
3'arm, a poly T termination signal, and to include a full H1 RNA
polymerase III promoter sequence. To prepare the shRNA cassettes,
the H1 RNA Pol III promoter was used as a template in a nested
polymerase chain reaction (PCR), whereby the sequences
corresponding to the shRNAs were incorporated into two reverse
primers (one for the primary PCR and one for the secondary PCR).
The same forward primer, which is complementary to the start of the
H1 promoter, was used in both. The PCR products coding for the
shRNA expression constructs were sub-cloned into the pTZ57R/T
vector (Fermentas). A scrambled shRNA (shRNAscr) that does not
target any gene product was used as a negative control.
[0130] In general, FIG. 4 shows the effects of shRNA on
downregulation of LRP/LR. FIG. 4a shows a Western Blot analysis of
HEK293 cells that were transfected with LRP-specific shRNA1 and
shRNA7 (as well as a scrambled control, shRNAscr). 72 hours
post-transfection, cells were lysed and LRP levels assessed by
Western blotting. .beta.-actin was used as a loading control.
Western blot band intensities from three independent experiments
were quantified using Quantity One 4.6 Software.
[0131] FIG. 4b shows the A.beta. concentration of the cell culture
medium of shRNA-transfected HEK293 cells analysed using an A.beta.
ELISA. Data shown (Mean.+-.s.d.) compare A.beta. levels of shRNA1
and shRNA7 to shRNAscr, *p<0.05, **p<0.01; n=3; Student's
t-test. Thus FIG. 4b shows the effects of shRNA on downregulation
of LRP/LR.
[0132] FIG. 4c shows flow cytometric analysis of APP,
.beta.-secretase and .gamma.-secretase levels on the surface of
shRNA-transfected HEK293 cells. Data shown (Mean.+-.s.d.); n=3;
Student's t-test.
[0133] FIG. 4d shows sAPP.beta. levels in shRNA-transfected HEK293
cells analysed by Western blotting.
[0134] These experiments, illustrated by FIGS. 4a-d, showed that
shRNA1 and shRNA7 resulted in a significant 42.85% and 16.42%
decrease in LRP/LR expression levels, respectively, compared to the
scrambled control (shRNAscr) (FIG. 4e). This downregulation
correlated to a significant 16.88% and 11.95% decrease in A.beta.
shedding in HEK293 cells (for shRNA1 and shRNA7 respectively) (FIG.
4b).
[0135] No significant difference was observed between
mock-transfected and shRNAscr control transfected HEK293 cells (as
can be seen in FIG. 4e). FIG. 4e shows A.beta. concentration of the
cell culture medium of shRNAscr-transfected and mock-transfected
HEK293 cells. HEK293 cells were either transfected with the
scrambled control (shRNAscr) or mock-transfected with no plasmid.
72 hours post transfection, the A.beta. concentration of the cell
culture medium was analysed using an A.beta. ELISA. Data shown
(Mean.+-.s.d); n=3; Student's t-test; p>0.05.
[0136] To investigate whether the receptor influences the
amyloidogenic pathway through altering cell surface protein
expression levels of APP, .beta.-secretase and .gamma.-secretase,
flow cytometric analysis of the cell surface levels of APP,
.beta.-secretase and .gamma.-secretase was performed post antibody
(FIG. 2c) and shRNA treatment (FIG. 4c). Blockage and/or
downregulation of LRP/LR did not significantly alter cell surface
expression levels of the aforementioned proteins in comparison to
controls as shown in FIG. 5. This suggests that the involvement of
LRP/LR in the amyloidogenic process may be independent of gene
expression modulation and possibly entails receptor interactions
with the AD relevant proteins.
[0137] FIG. 5 shows flow cytometry histogram overlay plots for
.beta.-secretase, .gamma.-secretase and APP after antibody or shRNA
treatments. In particular, FIG. 5a shows flow cytometry histogram
overlay plots after HEK293 cells were incubated with either 50
.mu.g/ml IgG1-iS18, IgG1-HD37 or no antibody for 18 hours after
which APP, .beta.- and .gamma.-secretase cell surface levels were
ascertained by flow cytometry (Coulter EPICS.RTM. XL-MCL).
.beta.-secretase levels was detected using anti-BACE (M-83) (rabbit
polyclonal IgG) (Santa Cruz Biotechnology) and goat anti-rabbit
FITC secondary antibody (Cell labs). .gamma.-secretase levels on
the surface of the cells was detected by a primary antibody
directed against the PEN-2 subunit of the .gamma.-secretase complex
(anti-PEN-2 (FL-101) (rabbit polyclonal IgG) (Santa Cruz
Biotechnology)), and the corresponding goat anti-rabbit FITC
secondary antibody. Cell surface APP levels were ascertained using
an anti-APP (rabbit polyclonal IgG) (Abcam) and the corresponding
goat anti-rabbit FITC secondary antibody. Images shown are averages
of 3 independent experiments.
[0138] FIG. 5b shows the same as above in FIG. 5a but with SH-SY5Y
cells.
[0139] FIG. 5c shows flow cytometry histogram overlay plots after
HEK293 cells having been transfected with either shRNA1, shRNA7 or
shRNAscr. 72 hours post transfection, the cell surface levels of
APP, .beta.- and .gamma.-secretase were ascertained by flow
cytometry (BD Accuri C6) using the methodology described above.
[0140] The fact that blockage and/or downregulation of LRP/LR did
not significantly alter cell surface expression levels of the AD
relevant proteins suggests that the involvement of LRP/LR in the
amyloidogenic process may be independent of gene expression
modulation and possibly entails receptor interactions with the said
proteins.
[0141] In an attempt to elucidate the mechanism by which LRP/LR
influences the amyloidogenic pathway, sAPP.beta. levels were
assessed post antibody (FIG. 2d) and shRNA treatment (FIG. 4d).
Upon a dose dependent administration of IgG1-iS18, a significant
reduction in sAPP.beta. levels was observed across all antibody
concentrations (56.29%, 69.35%, 92.42% and 99.76% for 25 .mu.g/ml,
50 .mu.g/ml, 75 .mu.g/ml and 100 .mu.g/ml respectively). Similar
results were obtained for shRNA1 mediated LRP/LR downregulated
HEK293 cells (FIG. 4d).
[0142] Following the above, pull down assays were conducted to
examine whether LRP/LR (recombinantly expressed fused to a FLAG
tag) and 100 ng/ml exogenously applied synthetic A.beta..sub.42
(Sigma-Aldrich) (which mimics augmented concentrations of soluble
A.beta..sub.42 present in AD brains) form stable interactions. The
presence of both proteins in eluted samples (FIG. 6a, i-lane 6)
implies that such an association exists. The identity of LRP/LR was
further confirmed by immunoblotting (FIG. 6a, ii). Relevant
controls are shown in FIG. 7 discussed below.
[0143] In general, FIG. 6 shows LRP/LR as an A.beta. interacting
protein and the cell rescuing effects of anti-LRP/LR antibody
IgG1-iS18. FIG. 6a (i) shows that a FLAG.RTM. Immunoprecipitation
kit (Sigma Aldrich) was employed to perform a pull down assay of
cell lysates containing recombinantly expressed LRP/LR::FLAG
co-incubated with exogenous A.beta.. Lane 1: Molecular weight
marker; lane 2: unbound sample; lanes 3-4: washes; lane 5: eluted
sample and lane 6: 2 .mu.g of synthetic A.beta.42 (positive
control).
[0144] FIG. 6a (ii) shows experimental results where Immunoblot was
employed to validate the position of LRP::FLAG (.about.38 kDa).
[0145] FIG. 6b shows cellular viability of HEK293 cells, as
determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (1 mg/ml) assay, post exogenous treatment with
synthetic A.beta..sub.42 and upon co-incubation with anti-LRP/LR
IgG1-iS18 or IgG1-HD37 (negative control). The cell viability was
assessed 48 h post treatment and the no antibody control was set to
100%. SH-SY5Y (as shown in FIGS. 6c) and N2a cells (as shown in
FIG. 6d) were exposed to similar treatments. It can be seen that
the co-incubation with anti-LRP/LR IgG1-iS18 significantly enhanced
cell viability.
[0146] FIG. 6e shows cellular proliferation of N2a cells as
determined by colorimetric 5-bromo-2'-deoxyuridine (BrdU)
non-isotopic immunoassay (Calbiochem.RTM.), allowing 4 h for BrdU
incorporation into cultured cells. Error bars represent sd.
**p<0.01; Student's t-test.
[0147] FIG. 7 shows the results of pull down assay protein
detection of control samples. Pull down assays were employed to
investigate the proteins detectable in unbound samples (lane 2),
wash steps (lanes 3 and 4) and eluted samples (FIG. a-c, lane 5,
FIG. d, lane 6). Figures represent anti-FLAG.RTM. M2 beads
incubated with (a) lysis buffer, (b) non-transfected HE293 cell
lysates, (c) HEK293 cell lysates of cells transfected with
pCIneo::FLAG as well as (d) pure synthetic A.beta..sub.42 in the
absence of cell lysate. Lane 7 of (d) represents 2 .mu.g of
synthetic A.beta..sub.42 which serves as a positive control.
Samples were resolved on 16% Tris-tricine SDS PAGE gels and stained
with Coomassie Brilliant Blue. Blue, red and green arrows are
indicative of A.beta..sub.42, LRP::FLAG and anti-FLAG M2 agarose
beads, respectively.
[0148] In summary, exogenous application of 200 nM and 500 nM
A.beta..sub.42 significantly reduced cell viability in HEK293 cells
(FIG. 6b). Co-incubation of cells with 50 .mu.g/ml anti-LRP/LR
specific antibody IgG1-iS18 and 500 nM A.beta..sub.42 (the
concentration at maximal cytotoxic effects were observed)
significantly enhanced cell viability (FIG. 6b). Similar results,
albeit at different A.beta..sub.42 concentrations were observed for
SH-SY5Y (FIG. 6c) and N2a (FIG. 6d) cells. The reduction in cell
viability observed in N2a cells (FIG. 6d) was later shown to be as
a result of reduced cellular proliferation (FIG. 6e). It is shown
that LRP/LR may be implicated in A.beta..sub.42 mediated
cytotoxicity and that the identified association (direct or
indirect) may be pathological in nature.
[0149] Animal Trials for Validation of Anti-LRP/LR Specific
Antibodies in the Treatment Alzheimer's Disease (AD)
[0150] LRP/LR plays a definitive role in the in A.beta. mediated
pathogenesis in AD, as proposed by the above in vitro data above.
It has been shown in vitro that IgG1-iS18 (LRP/LR specific
antibody) plays an important role in modulation of LRP/LR and the
modulation of the concentrations of Alzheimer's Disease (AD)
relevant proteins amyloid precursor protein (APP), beta (.beta.)
and gamma (.gamma.) secretases and amyloid beta peptide (A.beta.).
It is further proposed that the antibodies and/or shRNAs described
above may rescue neurons from A.beta. mediated cell death or impede
their proliferation.
[0151] Animal trials probing the potential of these antibodies
and/or shRNAs as an AD therapeutic will be initiated and conducted.
Transgenic AD mice harbouring human transgenes with 5 AD related
mutations (3 mutations in the APP protein and 2 in the PSEN1
enzymatic subunit of .gamma.-secretase) (The Jackson Laboratory,
strain: B6SJL-Tg (APPSwF|Lon,PSEN1*M146L*L286V)6799Vas.uparw.Mmjax)
will be employed. Transgenic animals (caged in individual cages in
a temperature controlled environment) will be divided into six
groups (5 mice/group). Ten wild-type mice per treatment will serve
as phenotype controls.
[0152] These 5x-Tg-AD mice develop pathological features mimicking
the human condition within 4 months, namely: plaque deposition,
synaptic and neuronal loss as well as cognitive deficits.
[0153] The antibodies and shRNA described above will be utilized in
any such animal experimentation. Particularly, anti-LRP/LR
antibody, IgG1-iS18, or IgG1-HD37 (negative control) (50 .mu.g/ml)
will be stereotaxically administered as a single
intracerebroventricular (ICV) injection either prior to plaque
deposition (<4 months) or post plaque deposition (>4 months).
Antibodies will be administered into the third ventricle (due to
its proximity to the hippocampus). Age matched transgenic mice
receiving ICV injections of the vehicle shall serve as controls. At
varying weekly time intervals, groups of mice will be tested for
deficits in spatial learning by means of the Morris Water Maze
Test. Two days prior to euthanization, the final Morris Water Maze
Test shall be performed. Mice will be euthanized by transcardial
perfusion with ice-cold saline followed by 4% buffered
paraformaldehyde (in saline solution). Approximately 18-20
hippocampal sections (35 .mu.m thickness) per animal (from both
hemispheres) will be collected and immunohistochemical methods
(anti-A.beta. antibodies) employed to assess total A.beta. levels
and Congo Red staining used to detect plaque deposits on these
sections (as detailed by Chauhan and Siegel, 2003). To ensure
antibody administration did not cause cerebral damage and
haemorrhaging sections shall be stained for haemosiderin using
Prussian blue.
CONCLUSION
[0154] The results for the in vitro experiments indicate that
LRP/LR co-localises with all the relevant AD proteins (APP, .beta.
and .gamma.-secretase as well as A.beta.) and consequently implies
that an association between these proteins and the receptor may
exist, as was further validated by pull down assay methodology with
respect to the neurotoxic A.beta..sub.42 peptide. In addition,
receptor blockage and/or downregulation of LRP/LR effectively
impeded A.beta. shedding affirming the importance of the receptor
in the amyloidogenic process. Interestingly, LRP/LR blockage did
not result in modulation of cell surface proteins central to the
amyloidogenic process, thereby inferring that the influence of
LRP/LR may rather be as a result of protein interactions. The
observed decrease in sAPP.beta. levels post antibody and shRNA
treatment suggests that LRP/LR exerts its affects via
.beta.-secretase. LRP/LR was further implicated in A.beta. induced
cytotoxicity and the interaction may possibly result in aberrant
proliferative cell signalling pathways. In conclusion, our findings
suggests that the LRP/LR is implicated in AD's pathogenesis and
recommends anti-LRP/LR specific antibodies and shRNAs as possible
alternative therapeutic tools for AD treatment.
[0155] While the invention has been described and/or illustrated
and/or exemplified in detail with respect to specific embodiments
and/or examples thereof, it will be appreciated that those skilled
in the art, upon attaining an understanding of the foregoing, may
readily conceive of alterations to, variations of and equivalents
to these embodiments. Accordingly, the scope of the present
invention should be assessed as that of the appended claims and
equivalents thereto.
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Sequence CWU 1
1
2121RNAHomo sapiens 1gcucgugcaa uuguugccau u 21221RNAHomo sapiens
2ggcagugacc aaggaggaau u 21
* * * * *