U.S. patent application number 12/667450 was filed with the patent office on 2010-11-04 for compounds and methods for the treatment of renal disease.
This patent application is currently assigned to OPSONA Therapeutics Ltd.. Invention is credited to Mark Heffernan, Brian Keogh, Christopher Locher, Peter McGuirk, Luke O'Neill.
Application Number | 20100278817 12/667450 |
Document ID | / |
Family ID | 39869930 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100278817 |
Kind Code |
A1 |
Heffernan; Mark ; et
al. |
November 4, 2010 |
COMPOUNDS AND METHODS FOR THE TREATMENT OF RENAL DISEASE
Abstract
The present invention provides compounds and methods for the
treatment and prophylaxis of renal disease and inflammation. In
particular the invention provides methods for the treatment of
kidney disease and failure through the administration of compounds
which function as inhibitors of TLR2 function and expression.
Inventors: |
Heffernan; Mark; (Dublin,
IE) ; O'Neill; Luke; (Dublin, IE) ; McGuirk;
Peter; (Dublin, IE) ; Keogh; Brian; (Dublinie,
IE) ; Locher; Christopher; (Lexington, MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
OPSONA Therapeutics Ltd.
Dublin 8
IE
|
Family ID: |
39869930 |
Appl. No.: |
12/667450 |
Filed: |
July 4, 2008 |
PCT Filed: |
July 4, 2008 |
PCT NO: |
PCT/EP2008/058732 |
371 Date: |
June 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61038304 |
Mar 20, 2008 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/139.1; 424/172.1; 435/325; 435/352; 435/366 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/70596 20130101; G01N 2500/02 20130101; A61K 2039/505
20130101; A61K 45/06 20130101; A61P 1/16 20180101; A61P 3/10
20180101; C07K 16/2896 20130101; A61P 37/02 20180101; A61P 37/06
20180101; A61P 31/04 20180101; A61P 13/12 20180101; A61P 43/00
20180101; A61P 31/14 20180101 |
Class at
Publication: |
424/133.1 ;
424/139.1; 435/366; 435/352; 435/325; 424/172.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/071 20100101 C12N005/071; C12N 5/07 20100101
C12N005/07; A61P 13/12 20060101 A61P013/12; A61P 1/16 20060101
A61P001/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2007 |
IE |
2007/0486 |
Claims
1. A method of reducing one or more biological activities of Toll
like receptor 2 (TLR2) in a TLR2 expressing cell or tissue
implicated in renal disease or inflammation, comprising: contacting
the cell or tissue with an antibody which acts as an antagonist of
TLR2 activity, in an amount sufficient to reduce one or more
biological activities of TLR2 in the cell or tissue.
2. The method as claimed in claim 1, wherein the TLR2 expressing
cell is a cell selected from the group consisting of a renal
tubular epithelial cell, an epithelial cell of the Bowman's
capsule, a kidney glomerulus parietal cell, kidney glomerulus
podocyte, kidney proximal tubule brush border cell, loop of henle
thin segment cell, kidney distal tubule cell, kidney collecting
duct cell.
3. The method as claimed in claim 1 wherein the contacting step
occurs in a cell lysate, a reconstituted system or cells in
culture.
4. The method as claimed in claim 1 wherein the contacting step
occurs on cells or a tissue present in a subject.
5. The method as claimed in claim 4, wherein the subject is a human
patient having, or at risk of renal failure or a renal
disorder.
6. The method as claimed in claim 1 wherein the TLR2 is human or
murine TLR2.
7-8. (canceled)
9. The method as claimed in claim 1, wherein the antibody has
binding specificity to human TLR2.
10. The method as claimed in claim 1, wherein the antibody is
selected from the group consisting of a human, humanised, chimeric,
synthetic, camelid, shark or in-vitro antibody which has binding
specificity to TLR2, or a binding fragment derived from any of the
same.
11. The method as claimed in claim 1, wherein the antibody is an
antibody binding fragment selected from the group consisting of a
Fab, scFv, Fv, and dAb.
12. The method as claimed in claim 1, wherein the antibody
comprises two complete heavy chains, and two complete light chains,
or an antigen-binding fragment thereof.
13. The method as claimed in claim 1 wherein the antibody binds to
an epitope defined by the extracellular domain of human TLR2.
14. The method as claimed in claim 1 wherein the antibody binds to
a non-continuous epitope comprising amino acid residues derived
from the amino and carboxyl terminals of the amino acid sequence of
human TLR2.
15. The method as claimed in claim 13, wherein the antibody binds
to an epitope on TLR2 comprising amino acid residues 19 to 39, or
538 to 549 of SEQ ID NO:2.
16-17. (canceled)
18. A method for the treatment and/or prophylaxis of a renal
disorder, the method comprising the steps of: providing a
therapeutically effective amount of an antibody or a binding
fragment thereof which is an agonist of the function of Toll-like
Receptor 2, and administering said compound to a subject in need of
such treatment.
19-21. (canceled)
22. The method as claimed in claim 18 wherein the antibody is
selected from the group consisting of: a monoclonal antibody, a
polyclonal antibody or a synthetic antibody.
23. The method of claim 18, wherein the antibody is selected from
the group comprising a human, humanised, camelid, in vitro
generated antibody to human TLR2.
24. The method as claimed in claim 18 wherein the antibody is of an
isotype selected from the group consisting of IgG, IgA, IgM, and
IgE.
25. The method as claimed in claim 18 wherein the antibody binds to
an inhibitory epitope present on TLR2 with a dissociation constant
(Kd) of from about 10.sup.-7M to about 10.sup.-11M.
26. The method of claim 18, wherein the antibody binds to an
epitope defined by the extracellular domain of human TLR2.
27. The method of claim 18, wherein the antibody binds to a
non-continuous epitope comprising amino acid residues derived from
the amino and carboxyl terminals of the amino acid sequence of
human TLR2.
28. The method of claim 18, wherein the antibody binds to an
epitope on TLR2 comprising amino acid residues 19 to 39, or 538 to
549 of SEQ ID NO:1.
29-34. (canceled)
35. The method as claimed in claim 18 wherein the antibody is
administered to the subject in order to reduce or inhibit one or
more TLR2 biological activities in a TLR2 expressing cell, wherein
the cell is a cell selected from the group consisting of a renal
tubular epithelial cell, an epithelial cell of the Bowman's
capsule, a kidney glomerulus parietal cell, kidney glomerulus
podocyte, kidney proximal tubule brush border cell, loop of henle
thin segment cell, kidney distal tubule cell, kidney collecting
duct cell.
36. The method as claimed in claim 18 wherein the renal disorder is
selected from the group consisting of renal disease, chronic renal
failure, acute renal failure heterologous nephrotoxic nephritis,
glomerulonephritis, sclerosis of the glomerulus, systemic lupus
erythematosus (SLE), diabetic nephropathy, and diabetic
nephropathy.
37. The method as claimed in claim 18 wherein the renal disorder is
selected from the group consisting of Immunoglobulin A nephropathy,
membranoproliferative glomerulonephritis, mesangial proliferative
glomerulonephritis, nonproliferative glomerulonephritis, membranous
glomerulonephritis, minimal-change disease, primary focal segmental
glomerulosclerosis, fibrillary glomerulonephritis, immunotactoid
glomerulonephritis, proliferative glomerulonephritis, progressive
glomerulonephritis, anti-GBM disease, kidney ischemia, delayed
graft function, kidney vasculitis, including disease associated
with anti-neutrophil cytoplasmic antibodies, lupus nephritis
cryoglobulinemia-associated glomerulonephritis, bacterial
endocarditis, Henoch-Schonlein purpura, postinfectious
glomerulonephritis, Hepatitis C disease, diabetic nephropathy,
myloidosis, hypertensive nephrosclerosis, light-chain disease from
multiple myeloma, secondary focal glomerulosclerosis, and
hypertensive nephrosclerosis.
38. The method as claimed in claim 18 wherein the TLR2 is human
TLR2 or murine TLR2.
39. The method as claimed in claim 18 further comprising the step
of administering a therapeutically effective amount of at least one
secondary therapeutic compound, said secondary therapeutic compound
being an immunosuppressant compound.
40. The method as claimed in claim 39 wherein the secondary
therapeutic compound is selected from the group consisting of: a
glucocorticoid, a cytostatic, an anti-metabolite, an anti-CD2
antibody or related binding fragment, an anti-CD20 antibody, an
anti-TNF-alpha antibody, cyclosporine, tacrolimus, sirolimus or
FTY720.
41. The method as claimed in claim 39 wherein the agent is
administered simultaneously with the secondary therapeutic
compound.
42. The method as claimed in claim 39 wherein the agent is
administered sequentially to the administration of the secondary
therapeutic compound.
43. (canceled)
44. A pharmaceutical composition for use in the treatment and
prophylaxis of a renal disorder comprising an antibody which is an
antagonist of the function of Toll-like Receptor 2 along with at
least one pharmaceutically acceptable carrier, diluent,
solubilizer, emulsifier, preservative and/or adjuvant.
45. The pharmaceutical composition as claimed in claim 44 wherein
the antibody is selected from the group consisting of: a polyclonal
antibody, a monoclonal antibody, a humanized antibody, a chimeric
antibody or an antibody fragment.
46-47. (canceled)
48. The pharmaceutical composition as claimed in claim 44 wherein
the composition further comprises a secondary therapeutic
compound.
49. The pharmaceutical composition as claimed in claim 48 wherein
the secondary therapeutic compound is an immunosuppressant selected
from the group consisting of: a glucocorticoid, a cytostatic, an
anti-metabolite, an anti-CD2 antibody or related binding fragment,
an anti-CD20 antibody, an anti-TNF-alpha antibody, cyclosporine,
tacrolimus, sirolimus or FTY720.
50-78. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
for the treatment and prophylaxis of renal disease. In particular,
there is provided methods for the treatment of conditions such as
glomerulonephritis which are characterised by inflammation of the
glomeruli of the kidney, said methods comprising the use of
compounds which antagonise the function of the receptor Toll-like
Receptor 2.
Background to the Invention
[0002] Each kidney is composed of around one million nephrons. The
glomerulus, a component of the nephron, is comprised of a bundle of
parallel capillaries, this structure being referred to as the
Bowman's capsule. the function of the glomerulus is to filter water
and solutes from the blood.
[0003] Glomerulonephritis is a disease of the kidney which is
characterised by the inflammation of the glomeruli. Inflammation of
the glomerulus compromises glomerular function, this resulting in a
narrowing or blocking of the capillaries which results in a
disruption to normal glomerular filtration.
[0004] Glomrulonephritis can result in large amounts of protein
being passed into the urine, in a condition known as proteinuria.
Further symptoms of glomerulonephritis may include excessive blood
urea nitrogen (uremia), haematuria, hyperlipidemia, a reduction in
glomerular filtration rate, hypoalbuminaemia and lipiduria.
[0005] Current treatments for glomerulonephritis include the
administration of corticosteroids and/or non-steroidal
anti-inflammatory drugs. In more severe cases, blood dialysis or
organ transplantation may be required. Such therapies are however
unsatisfactory for a number of reasons. In the case of
corticosteroid use, this being the most commonly employed therapy
for the treatment of glomerulonephritis, the treatment has to be
managed carefully in order to limit the immune suppression of the
patient which can result from prolonged steroid administration.
Further, the burden or long term dialysis, or the risks associated
with transplant surgery and organ rejection also make these
therapies less desirable.
[0006] Accordingly, there is a pressing need for a therapeutic
treatment which can be targeted to the inflamed glomerulus in order
to prevent or treat the inflamed glomerulus during
glomerulonephritis and which does not suffer from the disadvantages
and side effects of the hitherto known treatments.
[0007] Toll-like Receptors (TLRs) form a family of pattern
recognition receptors which have a key role in activating the
innate immune response. 11 Toll-like Receptors have been identified
in humans to date. The members of the Toll-like Receptor family are
highly conserved, with most mammalian species having between 10 to
15 Toll-like Receptors. Each Toll-like Receptor recognises specific
pathogen-associated molecular signatures. For example, Toll-like
Receptor 2 (TLR2, CD282, TLR-2) can be activated by peptidoglycan,
lipoproteins and lipoteichoic acid. Toll-like Receptors are known
to form either homodimers or heterodimers wherein each dimer has a
different ligand specificity. TLR2 forms a heterodimer with either
TLR1 or TLR6 and possibly also TLR10, in some instances, to form a
membrane bound receptor complex. In addition, the ectodomain of
Toll-like Receptor 2 is known to form a soluble heterodimer with
the CD14 protein in the circulatory system and also in mammalian
milk.
[0008] Ligand binding to Toll-like Receptor 2 results in downstream
signalling involving cytoplasmic adaptor proteins such as MyD88 and
Mal (MyD88-adaptor like), which is also known as TIRAP
(Toll-Interleukin-1 receptor domain containing adaptor
protein).
[0009] The involvement of Toll-like Receptor 2 and Toll-like
Receptor 2-induced signalling and immune system activation has
implicated Toll-like Receptor 2 as an important mediator in the
development of inflammation and immune-mediated disease.
Accordingly there has been significant therapeutic interest in
relation to the modulation of the Toll-like Receptor 2 signalling
pathway. Toll-like Receptor 2 mediated intracellular signaling
results in activation of the innate immune system. However, in some
instances, the activation of the innate immune system which results
following Toll-like Receptor 2 activation has been shown to be a
factor in causing inflammation and immune-mediated disease. As
such, a number of therapeutic approaches have sought to block TLR-2
ligand binding, or which suppress the functional activity of
Toll-like Receptor 2 following ligand binding, for example by
suppressing Toll-like Receptor 2 medaited intracellular signalling
which results following Toll-like Receptor 2 activation.
[0010] In relation to the pathology of kidney disease, Toll-like
Receptor 2 has been shown to be involved in the induction of the
antigen-specific adaptive immune response associated with the
development and recurrence of immune-mediated kidney disease such
as glomerulonephritis. Specifically, it has been shown that a
Toll-like Receptor 2 ligand, lipopeptide, can exacerbate
nephrotoxic nephritis in a Toll-like Receptor 2-dependent manner.
Toll-like Receptor 2 activation showed an increased amount of
antigen specific IgG isotypes such as IgG.sub.1, IgG.sub.2b,
IgG.sub.3 in the serum and IgG.sub.2b and IgG.sub.3 are seen to be
deposited within the glomerulus. Elevated CD4+ positive T cell
infiltration was also observed in the glomerulus. Nevertheless,
there has been no evidence to date to suggest that blocking the
Toll-like Receptor 2 pathway would provide a basis for a treatment
for immune-mediated kidney disorders.
[0011] Following extensive experimentation, the inventors have
surprisingly identified that compounds which antagonise Toll-like
Receptor 2 function, for example by blocking Toll-like Receptor 2
ligand binding, or by blocking intracellular signalling mediated by
activated Toll-like Receptor 2, cause a reduction in the level of
inflammation in the kidney. The inventors have identified that such
compounds have utility in methods for the treatment of inflammatory
conditions of the kidney, and in particular for the treatment of
conditions such as glomerulonephritis. In particular, the inventors
have surprisingly shown that blocking Toll-like Receptor 2 ligand
binding, through the use of an antibody, or a functional binding
fragment thereof, which has binding specificity to at least one
binding epitopes present on Toll-like Receptor 2, and which
inhibits ligand binding to Toll-like Receptor 2, can suppress the
production of neutrophils, and suppress albuminuria and thrombosis
which are typically elevated during onset of glomerulonephritis. As
such, compounds which mediate such an effect downregulate Toll-like
Receptor 2 mediated activation and downstream TLR-2 induced
intracellular signalling.
SUMMARY OF THE INVENTION
[0012] According to a first aspect of the present invention there
is provided a method of reducing one or more biological activities
of Toll-like receptor 2 (TLR2) in a TLR2 expressing cell or tissue
implicated in renal inflammation and disease, comprising: [0013]
contacting the cell or tissue with an antagonist of TLR2 activity
or TLR2 expression, in an amount sufficient to reduce one or more
biological activities of TLR2 in the cell or tissue.
[0014] In certain embodiments the TLR2 expressing cell or tissue is
a cell or tissue of the kidney. In certain embodiments of the
invention, the TLR2 expressing cell may be at least one cell type
selected from the group comprising, but not limited to: renal
tubular epithelial cells (TECs), an epithelial cell of the Bowman's
capsule, a kidney glomerulus parietal cell, a kidney glomerulus
podocyte, a kidney proximal tubule brush border cell, a loop of
henle thin segment cell, a kidney distal tubule cell, or a kidney
collecting duct cell.
[0015] In certain embodiments, the contacting step occurs in a cell
lysate, a reconstituted system or to cells in culture. In certain
embodiments, the contacting step occurs on cells or a tissue
present in a subject. In certain embodiments the TLR2 may be a
human TLR2, a murine TLR2, or a TLR2 derived from any other
mammalian species.
[0016] In certain embodiments the method is performed on a human
subject having, or at risk of having renal inflammation or renal
disease.
[0017] As herein defined, the term "renal inflammation and disease"
extends to all conditions which are substantially characterised by
the occurrence of inflammation within the kidney, or where the
occurrence of inflammation in the kidney is caused by a disease or
an inflammatory condition which primarily affects a site in the
body other than the kidney. In particular, inflammation may occur
at a site including, but not limited to; the glomerulus, Bowman's
capsule or Bowman's space. Typically, the inflammation results in
at least partial impairment of kidney function and/or kidney
failure.
[0018] Furthermore, the term "renal inflammation and disease" may
further include "kidney disease", wherein the term kidney disease
generally refers to a disorder of at least one kidney in a human,
wherein the disorder compromises or impairs the function of the
kidney(s), this being characterised physiologically by, for
example, the leakage of protein into the urine, or by the excretion
of nitrogenous waste. The Kidney disease may also result from a
primary pathology of the kidney, such as injury to the glomerulus
or tubule, or from damage to another organ, such as the pancreas,
which adversely affects the ability of the kidney to perform
biological functions, such as the retention of protein. Thus,
Kidney disease in the human can be the direct or indirect effect of
a disease condition which may affect other organs. Examples of
diseases which affect the kidneys, but which do not specifically
target the kidneys are diabetes and systemic lupus. The terms renal
disease and kidney disease are used interchangeably herein with the
phrase "diseases of the kidney". The kidney disease can, for
example, result from, or be a consequence of any change, damage, or
trauma to the glomerulus, tubules or interstitial tissue in either
the kidney cortex or kidney medulla.
[0019] The kidney disease may also be a progressive kidney disease.
The term "progressive kidney disease" as used herein refers to any
disease of the kidney that over time (e.g., days, weeks, months,
years) leads to a loss of kidney function. As herein defined, the
term "kidney function" generally refers to a physiological property
of the kidney, such as the ability to retain protein thereby
preventing proteinuria (e.g., albuminuria). Kidney function can be
assessed, for example, by glomerular filtration rate (e.g.,
creatinine clearance), excretion of protein in urine e.g,
albuminuria, blood urea nitrogen, serum or plasma creatinine, or
any combination thereof.
[0020] Examples of specific conditions which fall within the
meaning of the term "renal inflammation and disease" include, but
are not limited to: renal disorders which include, but are not
limited to: chronic renal failure, acute renal failure,
heterologous nephrotoxic nephritis, glomerulonephritis, sclerosis
of the glomerulus, systemic lupus erythematosus (SLE), diabetic
nephropathy, diabetic nephropathy wherein the diabetic nephropathy
accompanies sclerosis of the liver, and glomerulonephritis wherein
the glomerulonephritis is accompanied by sclerosis of the
liver.
[0021] In certain further embodiments, renal inflammation and
disease may relate to an immune-mediated disease which affects the
cells of the kidney and/or kidney function. Such conditions may
include, but are not limited to: Immunoglobulin A nephropathy,
membranoproliferative glomerulonephritis, mesangial proliferative
glomerulonephritis, nonproliferative glomerulonephritis, membranous
glomerulonephritis, minimal-change disease, primary focal segmental
glomerulosclerosis (FSGS), fibrillary glomerulonephritis,
immunotactoid glomerulonephritis, proliferative glomerulonephritis,
progressive glomerulonephritis, anti-GBM disease, kidney ischemia,
kidney vasculitis, including disease associated with
anti-neutrophil cytoplasmic antibodies (ANCA) (e.g., Wegener
granulomatosis), lupus nephritis cryoglobulinemia-associated
glomerulonephritis, bacterial endocarditis, Henoch-Schonlein
purpura, postinfectious glomerulonephritis, Hepatitis C, diabetic
nephropathy, myloidosis, hypertensive nephrosclerosis, light-chain
disease from multiple myeloma, secondary focal glomerulosclerosis,
and hypertensive nephrosclerosis.
[0022] The term "renal inflammation and disease" also encompasses
acute renal failure. Acute renal failure ("ARF") refers to the
clinical conditions associated with rapid, steadily increasing
azotemia, with or without oliguria (<500 mL/day). The cause of
ARF can be grouped into three diagnostic categories: prerenal
(inadequate renal perfusion); postrenal (obstruction); and renal.
The pathophysiology of ARF is complex and multifactorial. Current
concepts suggest that ARF may result from direct renal tubular
injury, renal ischemia or intra-tubular obstruction. Clinically,
ARF results in diminished glomerular filtration and reduced
secretion of metabolic waste products, water, and electrolytes.
Fluid overload, electrolyte imbalances and the uremic syndrome
result in organ dysfunction. Organ dysfunction may ultimately
result in death.
[0023] The antagonist of TLR2 activity or TLR2 expression may also
be referred to as TLR2 modulator agent. The TLR2 modulator agents
of the invention inhibit TLR2 function and therefore act to prevent
TLR2 mediated or dependent patho-physiological mechanisms
contributing to renal failure. Examples of such pathophysiological
mechanisms include direct renal tubular injury, renal ischemia, and
intra-tubular obstruction.
[0024] In certain embodiments, the TLR2 modulator agent is
administered to the subject such that it can specifically target
cellular populations of the kidney such as the renal tubular
epithelial cells (TECs) and/or the epithelial cells of the Bowman's
capsule. Direct targeting of the Toll-like Receptor 2 modulator
agent to the kidney or cells thereof avoids global suppression of
Toll-like Receptor 2 function in a subject. This targeted approach
to therapy can be particularly effective where it is desirable to
suppress Toll-like Receptor 2-mediated inflammation in the kidney,
but where it is not desirable to down-regulate Toll-like Receptor 2
activity at sites other than the kidney, this resulting in
potential immunosuppression in sites other than the kidney.
Compounds, such as labelling compounds or targeting molecules which
can be used to direct the TLR2 modulator compound of the invention
to the cells of the kidney will be known to the person skilled in
the art. Such targeting means typically comprise a binding agent
which has binding specificity for a ligand or cell surface marker
which is specific to the cell which is to be targeted.
[0025] In certain embodiments the TLR2 antagonist is selected from
the group comprising, but not limited to: a protein, a peptide, a
peptidomimetic, a nucleic acid, a carbohydrate, a lipid, an aptamer
and a small molecule compound.
[0026] In certain embodiments the TLR2 antagonist is an antibody
molecule. Typically the antibody has binding specificity to an
epitope present on human TLR2. In certain embodiments, the antibody
molecule, or a binding fragment derived therefrom has binding
specificity to an epitope comprising amino acid residues of the
extracellular domain of TLR2. In certain embodiments, the TLR2
antagonist binds to a non-continuous epitope comprising amino acid
residues derived from the amino and carboxyl terminals of the amino
acid sequence of human TLR2. In certain embodiments the TLR2
antagonist binds to an epitope on TLR2 comprising amino acid
residues 19 to 39, or 538 to 549 of the extracellular domain of
TLR2 are defined in SEQ ID NO:2.
[0027] In certain embodiments the antibody is selected from the
group consisting of a human, humanised, chimeric, synthetic,
camelid, shark or in-vitro antibody which has binding specificity
to TLR2. In further embodiments, the invention extends to an
antibody binding fragment which is derived from any of the
aforesaid antibodies. In certain embodiments, the antibody is an
antibody binding fragment selected from the group consisting of a
Fab, scFv, Fv, or dAb fragment. In certain embodiments the antibody
molecule comprises two complete heavy chains, and two complete
light chains, or an antigen-binding fragment thereof. In certain
embodiments, the antibody is of the isotype IgG, IgA, IgM. In
embodiments where the antibody is of the isotype IgG, the antibody
may be of the subtype IgG1, IgG2 or IgG3.
[0028] In certain embodiments, the antibody is a murine IgG1
anti-TLR2 antibody (mouse Toll-like Receptor 2 (TLR2) antibody,
derived from hybridoma clone T2.5, HyCult Biotechnology b.v., Cell
Sciences, Canton, USA: catalogue number 1054), or a humanised
version thereof.
[0029] In certain embodiments the TLR2 antagonist inhibits the
expression of nucleic acid encoding the TLR2 protein. In such
embodiments the TLR2 antagonist may be selected from the group
comprising, but not limited to: an anti-sense oligonucleotides,
triple helix molecules, anti-sense DNA, anti-sense RNA, ribozyme,
iRNA, miRNA, sRNA, and an shRNA molecule.
[0030] According to a further aspect of the invention there is
provided a method for the treatment and/or prophylaxis of a renal
disorder, the method comprising the steps of: [0031] providing a
therapeutically effective amount of an agent which modulates the
function or expression of Toll-like Receptor 2 (TLR2), and [0032]
administering said compound to a subject in need of such
treatment.
[0033] In certain embodiments, the renal disorder is at least one
of the group consisting of: renal disease, a renal inflammatory
condition, chronic renal failure, acute renal failure, heterologous
nephrotoxic nephritis, glomerulonephritis, sclerosis of the
glomerulus, systemic lupus erythematosus (SLE), diabetic
nephropathy, diabetic nephropathy wherein the diabetic nephropathy
accompanies sclerosis of the liver, and glomerulonephritis wherein
the glomerulonephritis is accompanied by sclerosis of the
liver.
[0034] In certain further embodiments, the renal disorder relates
to an immune-mediated disease which affects the cells of the kidney
and/or kidney function. Such disorders may include, but are not
limited to: Immunoglobulin A nephropathy, membranoproliferative
glomerulonephritis, mesangial proliferative glomerulonephritis,
nonproliferative glomerulonephritis, membranous glomerulonephritis,
minimal-change disease, primary focal segmental glomerulosclerosis
(FSGS), fibrillary glomerulonephritis, immunotactoid
glomerulonephritis, proliferative glomerulonephritis, progressive
glomerulonephritis, anti-GBM disease, kidney ischemia, kidney
vasculitis, including disease associated with anti-neutrophil
cytoplasmic antibodies (ANCA) (e.g., Wegener granulomatosis), lupus
nephritis cryoglobulinemia-associated glomerulonephritis, bacterial
endocarditis, Henoch-Schonlein purpura, postinfectious
glomerulonephritis, Hepatitis C disease, diabetic nephropathy,
myloidosis, hypertensive nephrosclerosis, light-chain disease from
multiple myeloma, secondary focal glomerulosclerosis, and
hypertensive nephrosclerosis.
[0035] In certain further embodiments, the renal disorder may
relate to any renal inflammatory condition or disease as
hereinbefore described, or further to any kidney disease as
hereinbefore described.
[0036] As herein defined, the term `modulates the function` means
that the agent modulates one or more of the biological functional
activities of Toll-like Receptor 2. In certain embodiments, the
modulation of Toll-like Receptor 2 function relates to an
inhibition of the functional activation of Toll-like Receptor 2
and/or of the inhibition or suppression of downstream intracellular
signalling mediated by Toll-like Receptor 2. Modulation may further
extend to a suppression of the expression of Toll-like Receptor 2
protein, or the expression of a gene which encodes Toll-like
Receptor 2.
[0037] As defined herein, an `agent` which modulates the expression
or function of TLR2 is a compound which suppresses or blocks the
activation, expression or function of Toll-like Receptor 2. In
certain embodiments, the `agent` may be a TLR2 antagonist compound
which inhibits or blocks the binding of a ligand or binding
compound to Toll-like Receptor 2. For example, the `agent` may be a
Toll-like Receptor 2 binding agent, such as an antibody, which
binds to the extracellular domain of Toll-like Receptor 2, said
agent inhibiting the binding of TLR2 activating ligands which have
binding specificity for TLR2. Further, the `agent` may be a
compound which inhibits or suppresses intracellular signalling
mediated by Toll-like Receptor 2 following ligand binding and/or
Toll-like Receptor 2 activation.
[0038] The `agent` may further modulate Toll-like Receptor 2
expression, for example by inhibiting the expression of a gene
encoding a Toll-like Receptor 2 protein.
[0039] In certain embodiments, the `an agent which modulates the
function or expression of Toll-like Receptor 2` may be a binding
compound which has binding specificity or which specifically binds
Toll-like Receptor 2. In certain embodiments, the binding compound
is selected from the group comprising, but not limited to: a
protein, a peptide, a peptidomimetic, a nucleic acid, a
polynucleotide, a polysaccharide, an oligopeptide, a carbohydrate,
a lipid, an aptamer, a small molecule compound, and a naturally
occurring compound, such as a plant derived compound.
[0040] In certain embodiments, the agent which modulates the
function or expression of Toll-like Receptor 2 is a binding
compound which specifically binds to Toll-like Receptor 2 at a site
other than the ligand binding site, and which, upon binding, causes
a change in the confirmation of Toll-like Receptor 2, which leads
to an inhibition of Toll-like Receptor 2 agonist binding.
Typically, said binding site will be located at the extracellular
domain of TLR2.
[0041] The term "specifically binds" or "binding specificity"
refers to the ability of a TLR2 modulator agent or TLR2 binding
compound to bind to a target epitope present on TLR2 with a greater
affinity than it binds to a non-target epitope. In certain
embodiments, the term "specificaly binds" refers to the binding of
the agent to a target epitope present on TLR2 with a binding
affinity which is at least 10, 50, 100, 250, 500, or 1000 times
greater than the binding affinity for a non-target epitope. In
certain embodiments, binding affinity is determined by an affinity
ELISA assay. In alternative embodiments, affinity is determined by
a BIAcore assay. Alternatively, binding affinity may be determined
by a kinetic method. In certain embodiments, affinity is determined
by an equilibrium/solution method.
[0042] According to one embodiment, TLR2 modulators, including TLR2
binding agents, such as TLR2 antagonists, bind to TLR2 with high
affinity, for example, with an affinity constant of at least about
10.sup.7 M.sup.-1, typically about 10.sup.8 M.sup.-1, and more
typically, about 10.sup.9 M.sup.-1 to 10.sup.10 M.sup.-1 or
stronger; and modulate, e.g., reduce and/or inhibit, one or more
TLR2 biological activities in a TLR2 responsive cell and/or
tissue.
[0043] In certain embodiments, the TLR2 modulator agent is targeted
to Toll-like Receptor 2 expressed on the cells of the kidney, in
particular the glomerulus or Bowman's capsule. Exemplary TLR2
activities that can be modulated, e.g., inhibited or reduced, using
the methods and compositions of the invention include, but are not
limited to, one or more of the following: (i) inhibiting or
suppressing TLR2 expression, (ii) inhibiting TLR2 ligand binding
and associated TLR2 activation, and (iii) inhibiting or suppressing
intracellular signalling mediated by TLR2.
[0044] Accordingly, in a further aspect, the invention provides a
method of modulating a function (e.g., modulating one or more
biological activities) of Toll-like Receptor 2 (TLR2) in a
TLR2-responsive cell and/or tissue (e.g., a cell or tissue of the
kidney). The method includes contacting the TLR2-responsive cell
and/or TLR2-responsive tissue with a TLR2 modulator agent, e.g., a
TLR2 binding compound, such as an antagonist of human TLR2 activity
or expression, in an amount sufficient to modulate the function of
the TLR2-responsive cell or tissue, or the biological activity of
TLR2 in the cell or tissue. In one embodiment, the contacting step
between the TLR2 modulator and the TLR2-responsive cell can be
effected in vitro, for example in a cell lysate or in a
reconstituted system. Alternatively, said method can be performed
on cells in culture, e.g., in-vitro or ex-vivo. For example, cells,
such as purified or recombinant cells can be cultured in-vitro and
the contacting step can be effected by adding the TLR2 modulator to
the culture medium. Typically, the TLR2-responsive cell is a
mammalian cell, such as a human cell. In some embodiments, the
TLR2-responsive tissue is a tissue of the kidney, or a cellular
population associated therewith. In certain further embodiments,
the method can be performed on cells present in a subject, for
example as part of an in-vivo protocol, or in an animal subject,
such as a human, or further in an in-vivo animal model. Said
in-vivo protocol can be therapeutic or prophylactic, and the
inflammatory model can be, for example, a genetically modified
model, such as an animal model having renal inflammation or
disease, overexpressed TLR2, or a mutation or deletion in a TLR2
receptor. For in vivo methods, the TLR2 modulator, alone or in
combination with another agent, can be administered to a subject
suffering from renal disease or inflammation, or a condition which
results in kidney disease or failure of normal kidney physiological
function, in an amount sufficient to modulate TLR2 expression, or
one or more TLR2 mediated activities or functions in a subject. In
certain embodiments, the dosage of the TLR2 modulator that is
administered to the subject may be determined prior to
administration by testing in-vitro or ex-vivo, the amount of TLR2
modulator required to alter, e.g., decrease or inhibit, one or more
of TLR2 activities (e.g., one or more TLR2 biological activities
described herein).
[0045] In certain embodiments where inhibition, reduction or
diminution of one or more TLR2 biological activities is desired,
the TLR2-responsive cell and/or tissue is contacted with a TLR2
antagonist, e.g., by administering the TLR2 antagonist to the
subject. In one embodiment, the TLR2 antagonist interacts with,
e.g., binds to, a TLR2 polypeptide or mRNA, and reduces or inhibits
one or more TLR2 activities. Typically, the TLR2 antagonized is a
mammalian TLR2 (or a functional variant thereof), e.g., human TLR2
or murine TLR2. In certain embodiments, the TLR2 antagonized
includes the human TLR2 sequence as defined in FIG. 19 (SEQ ID
NO:1) (comprising the 784 amino acid full length human Toll-like
Receptor sequence as defined as Genbank Accession Number AAC 34133
(URL www.ncbi.nlm.nih.gov)) or of the murine TLR2 sequence
comprising the amino acid sequence defined in FIG. 20 (SEQ ID NO:2)
(Genbank Accession Number NP.sub.--036035 (Mus musculus)), or a
portion thereof, and/or a sequence substantially homologous
thereto, or encoded by a nucleotide sequence and/or a sequence
substantially homologous thereto which encodes the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:2.
[0046] As herein defined, the term "Toll-like Receptor 2
activation" means the binding of Toll-like Receptor 2 by a ligand,
wherein the ligand acts as an agonist and activates Toll-like
Receptor 2 in order to induce an intracellular signalling cascade.
Intracellular signalling mediated following Toll-like Receptor 2
activation and signalling results in the activation of
transcription factors and the expression of genes which mediate a
pro-inflammatory immune response.
[0047] In certain embodiments the agent which modulates the
function or expression of Toll-like Receptor 2 inhibits the
interaction between Toll-like Receptor 2 and a Toll-like Receptor 2
agonist.
[0048] In certain embodiments, the modulator agent that suppresses
Toll-like Receptor 2 activation and/or signalling is a compound
which acts as a Toll-like Receptor 2 antagonist. Typically,
antagonism of Toll-like Receptor 2 function is achieved by the
binding of the Toll-like Receptor 2 binding agent to Toll-like
Receptor 2 in such a way that ligand binding to Toll-like Receptor
2 is prevented. This inhibition of Toll-like Receptor 2 ligand
binding may be achieved by a number of means, for example, through
partially or fully blocking the Toll-like Receptor 2 ligand binding
site, or by inducing a conformational change upon binding to or
association with Toll-like Receptor 2 which results in the
Toll-like Receptor 2 ligand binding site being altered in a manner
which prevents Toll-like Receptor 2 ligand binding, for example due
to a conformational change of the tertiary structure of the
Toll-like Receptor 2 ligand binding site which precludes
binding.
[0049] In certain embodiments, the TLR2 modulator agent binds to at
least one epitope present on TLR2, wherein binding to this epitope
results in an inhibition of TLR2 function, typically activation or
TLR2 mediated downstream signalling. As herein defined, an
"epitope" refers to a plurality of amino acid residues derived from
the TLR2 protein which are capable of being recognised by, and
bound to by, a binding compound such as a small molecule, antibody
or the like. Epitopes generally comprise chemically active surface
groups and have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0050] Typically, the TLR2 modulator agent antagonises the
functional activity of TLR2 and as such binds to an epitope known
as an inhibiting epitope or an inhibitory epitope. An "inhibiting"
or "inhibitory" epitope means an epitope present on TLR2 that, when
bound by a binding compound such as a small molecule or an
antibody, results in the loss of biological activity of TLR2, for
example due to the binding compound preventing the binding of TLR2
by a TLR2 agonist. The epitope that is present on TLR2, and which
is bound by the binding compounds in order to antagonise TLR2
function, may comprise 5 or more amino acid residues.
[0051] In certain embodiments, the TLR2 modulator agents of the
invention recognise a continuous epitope. In further embodiments,
the epitope is a discontinuous epitope which comprises residues
derived from both the N-terminal (amino terminal) and C-terminal
(carboxy terminal) portions of the mature Toll-like receptor 2
(TLR2) protein as defined in SEQ ID NO:1. In certain embodiments,
the epitope may comprise residues 19 to 39 as determined from the
586 amino acid sequence of Toll-like Receptor 2, said amino acids
being KEESSNQASLSCDRNGICKGS (SEQ ID NO:3). Further, the binding
epitope may further comprise amino acids residues 538 to 549 of
Toll-like Receptor 2 as present at the C-terminal region of the
amino acid sequence of SEQ ID NO:1, this sequence comprising the
amino acids CSCEFLSFTQEQQ (SEQ ID NO:4). The TLR2 modulatory agent
binding site may further be defined by amino acid residues 19 to
39, or 538 to 549 of SEQ ID NO:1, or by amino acid residues 19 to
39, or 538 to 549 of SEQ ID NO:1.
[0052] The reduction, inhibition or antagonism of Toll-like
Receptor 2 functional activity may occur regardless of whether
Toll-like Receptor 2 forms a heterodimer with Toll-like Receptor 1,
Toll-like Receptor 6 or another Toll-like Receptor such as
Toll-like Receptor 4 or Toll-like Receptor 10. By the term
"Toll-like Receptor 2 activation and downstream mediated signaling"
it is meant any intracellular signaling pathway which is induced by
activated TLR2. The signaling pathway may be a TLR2 specific
pathway, or may be a "shared" signalling pathway, for example where
the signalling pathway may be activated by other sources, for
example, by means of the activation of receptors other than TLR2
which contribute to the activation of mediators of the immune
response such as the transcription factor NF-kappaB.
[0053] TLR2 is known to dimerise into at least 2 functional
heterodimers. Most commonly, TLR2 is known to form a heterodimer
with either Toll-like Receptor 1 or Toll-like Receptor 6. Further
heterodimers may be formed between TLR2 and Toll-like Receptor 4
(TLR4, TLR-4) and Toll-like Receptor 10 (TLR10, TLR-10). It is
thought that this dimerisation is associated with a discrimination
that results in the binding of TLR2 by different microbial-derived
ligands. In addition, the ectodomain of TLR2 may form a soluble
heterodimer with CD14 in the circulatory system and in mammalian
milk.
[0054] The inventors have surprisingly identified that, in order to
provide a therapeutic approach which is comprehensive in
suppressing TLR2-mediated inflammation of the kidney, it is
desirable to provide a binding compound which has binding
specificity for TLR2, regardless of whether a heterodimer is formed
with TLR1, TLR6, TLR4 or TLR10. In this regard, following extensive
experimentation, the inventors have identified a conformational,
discontinuous epitope comprised of amino acid residues present at
both the N-terminal and C-terminal of the TLR2 protein which, when
bound, results in the global suppression of TLR2 functional
activity, that is the function of TLR2 is inhibited irrespective of
which TLR TLR2 associates with to form a heterodimer. Accordingly,
binding of this epitope by a TLR2 antagonist suppresses the
function of TLR2 irrespective of whether TLR2 forms a heterodimer
with TLR1, TLR4, TLR6 or TLR10.
[0055] As such, in certain further embodiments, the agent which
modulates the function or expression of Toll-like Receptor 2 as
provided by the present invention may have at least one of the
following characteristics: (i) it is a monoclonal antibody, (ii) it
is a human-derived or in-vitro generated antibody, (iii) it binds
to a conformational, discontinuous epitope comprising the amino
acids of SEQ ID NO:3 and/or 4 and mediates TLR2 functional
suppression irrespective of whether a heterodimer is formed between
TLR2 and TLR1, TLR6, TLR4 or TLR10, (iv) it binds to the epitope
defined by the amino acids of the extracellular domain of TLR2,
with an affinity constant (Ka) of at least 10.sup.-6M.
[0056] In certain embodiments, the agent which modulates the
function or expression of Toll-like Receptor 2 is selected from at
least one of the group comprising, but not limited to: proteins,
peptides, peptidomimetics, nucleic acids, polynucleotides,
polysaccharides, oligopeptides, carbohydrates, lipids, small
molecule compounds, and naturally occurring compounds.
[0057] In certain embodiments, the agent which modulates TLR2
function is a TLR2 antagonist, such as any of the TLR2 agonists or
binding compounds described hereinbefore.
[0058] In certain embodiments the TLR2 modulatory agent may be a
soluble form of recombinant Toll-like Receptor 2. In particular the
soluble form of TLR2 may be a fusion protein which substantially
comprises the amino acid residues of the extracellular domain on
the TLR2 protein conjoined to a secondary protein. In certain
embodiments, the secondary protein may be an Fc domain derived from
an immunoglobulin, or a fragment thereof.
[0059] In certain further embodiments, the TLR2 modulatory agent is
an inhibitory nucleic acid which inhibits expression of the TLR2
protein. In certain embodiments the inhibitory nucleic acid protein
is selected from the group consisting of: anti-sense
oligonucleotides, triple helix molecules, anti-sense DNA,
anti-sense RNA, ribozyme, iRNA, miRNA, sRNA, and shRNA.
[0060] In certain embodiments, the methods of the invention are
used to administer a TLR2 modulatory agent to a subject in order to
reduce or inhibit one or more TLR2 biological activities in a TLR2
expressing cell or tissue of the kidney.
[0061] In certain embodiments, the methods of the invention may be
used for the treatment or prevention of ischemia reperfusion injury
which may result from chronic kidney failure or kidney organ
transplantation in a subject. In certain embodiments, the methods
of the invention may comprise the further step of administering a
therapeutically effective amount of at least one secondary
therapeutic compound along with, the agent which modulates the
function or expression of Toll-like Receptor 2. Said secondary
therapeutic compound may be, for example, an immunosuppressant
compound selected from the group consisting of: a glucocorticoid, a
cytostatic, an anti-metabolite, an anti-CD2 antibody or related
binding fragment, an anti-CD20 antibody, an anti-TNF-alpha
antibody, cyclosporine, tacrolimus, sirolimus or FTY720.
[0062] In certain embodiments, the secondary therapeutic agent may
be administered simultaneously, sequentially or separately to the
administration of the agent which modulates the function or
expression of Toll-like Receptor 2.
[0063] According to a yet further aspect of the invention there is
provided a pharmaceutical composition for use in the treatment and
prophylaxis of renal inflammation or disease said composition
comprising an agent which modulates the function or expression of
Toll-like Receptor 2 along with at least one pharmaceutically
acceptable carrier, diluent, solubilizer, emulsifier, preservative
and/or adjuvant.
[0064] In certain embodiments the renal inflammation is a condition
selected from the group comprising: renal disease, including kidney
disease, acute renal failure, chronic renal failure or another
associated inflammatory condition of the kidney such as
glomerulonephritis, or any other condition as hereinbefore
described.
[0065] In certain embodiments the TLR2 modulatory agent is a
compound which is a TLR2 antagonist selected from the group
consisting of, but not limited to: a polyclonal antibody, a
monoclonal antibody, a humanized antibody, a chimeric antibody or
antibody fragment, an aptamer, a fusion protein or a
peptidomimetic.
[0066] In certain embodiments, the antibody is a murine IgG1
anti-TLR2 antibody (mouse Toll-like Receptor 2 (TLR2) antibody,
derived from hybridoma clone T2.5, HyCult Biotechnology b.v., Cell
Sciences, Canton, USA: catalogue number 1054), or a humanised
version thereof.
[0067] In certain embodiments, the TLR2 modulatory agent is a
soluble form of the TLR2 receptor, or a fragment thereof. Said
soluble form of TLR2 may be produced by recombinant means. In
certain embodiments, said soluble form of TLR2 may comprise the
extracellular domain of TLR2 or a fragment thereof.
[0068] In certain embodiments the TLR2 modulatory agent is an
inhibitory nucleic acid based compound which inhibits the
expression of TLR2.
[0069] In certain embodiments, the pharmaceutical composition may
further comprise, or be administered along with at least one
secondary therapeutic agent, such as, but not limited to: an
immunosuppressant, compound which may be at least one of the group
consisting of, but not limited to: a glucocorticoid, in particular
a glucocorticoid which suppresses the expression of a cytokine; a
cytostatic such as an alkylating agent, an anti-metabolite such as
methotrexate; an antibody or related binding fragment, such as an
anti-CD3 antibody such as OKT-3, an anti-CD20 antibody, the
anti-TNF-alpha antibody infliximab (REMICADE.TM.), etanercept
(ENBREL.TM.) or adalimumab (HUMIRA.TM.); a drug compound which acts
on immunophilins such as cyclosporine, tacrolimus or sirolimus; or
a small molecule, such as FTY720 or a therapeutic cardiovascular
compound comprising at least one or more of; an HMG-CoA reductase
inhibitor, a vasodilatory agent, a diuretic, an angiotensin
converting enzyme inhibitor, a beta-blocker, an angiotensin II
receptor antagonist, a calcium channel blocker, an anticoagulant,
an adenosine diphosphate receptor antagonist such as ticlopidine or
clopidogrel bisulfate, a glycoprotein IIb/IIIa receptor antagonist
such as bivalirudin, argatroban or heparin, a beta adrenergic
receptor agonist, an antithrombolytic agent, an antioxidant, and an
alpha blocker.
[0070] In certain embodiments, the Toll-like Receptor 2 modulator
agent is orally administered to the subject at a dose of from about
1 mg/kg to about 10 mg/kg of the subject's body weight per day. In
certain embodiments, the dose of the Toll-like Receptor 2 modulator
agent is from about 100 mg per day to about 1000 mg per day. In
certain further embodiments, the dose of the Toll-like Receptor 2
modulator agent is from about 200 mg per day to about 300 mg per
day. In certain embodiments, the Toll-like Receptor 2 modulator
agent is administered to the subject parenterally with a dosage
range of between about 0.001 mg/kg to 1.0 mg/kg of the mammal's
body weight.
[0071] In certain embodiments, the Toll-like Receptor 2 modulator
agent is administered to the subject for a time, and under
conditions sufficient to down regulate the level and/or activity of
Toll-like Receptor 2.
[0072] In certain embodiments the TLR2 modulatory agent is an
inhibitory nucleic acid based compound which inhibits the
expression of TLR2 by way of blocking the expression of a TLR2
gene, or allowing the expression of mRNA encoding for the TLR2
protein. In certain embodiments, the inhibitory nucleic acid may be
selected from the group consisting of: anti-sense oligonucleotides,
triple helix molecules, anti-sense DNA, anti-sense RNA, ribozyme,
iRNA, miRNA, sRNA, and shRNA.
[0073] As herein defined, the terms "blocks" and "blocking" when
used in relation to Toll-like Receptor 2 gene expression mean the
silencing of the expression of at least one gene which results in
the expression of the Toll-like Receptor 2 protein. Gene silencing
is the switching off of the expression of a gene by a mechanism
other than genetic modification. Gene silencing can be mediated at
the transcriptional level or at the post-transcriptional level.
Transcriptional gene silencing can results in a gene being
inaccessible to transcriptional machinery, and can be mediated, for
example, by means of histone modifications. Post-transcriptional
gene silencing results from the mRNA of a gene being destroyed,
thus preventing an active gene product, such as a protein, in the
present case the TLR2 protein.
[0074] Accordingly, in one embodiment of this aspect of the
invention, there is provided a method for the administration to a
subject of an effective amount of an inhibitory nucleic acid
molecule such as an RNAi (RNA interference) agent, for example an
interfering ribonucleic acid (such as sRNA or shRNA) or a
transcription template thereof, such as a DNA encoding an shRNA to
at least one cell type, tissue or organ present in the subject in
order to block the expression of the TLR2 gene and therefore
expression of the TLR2 protein.
[0075] In certain further embodiments, the inhibitory nucleic acid
molecule may be an antisense RNA molecule. Antisense causes
suppression of gene expression and involves single stranded RNA
fragments which physically bind to mRNA, this blocking mRNA
translation. Techniques for the preparation of appropriate nucleic
acids for use as an inhibitory nucleic acid are well known to the
person skilled in the art.
[0076] According to a further aspect of the invention there is
provided the use of an inhibitory nucleic acid which blocks the
expression of the Toll-like Receptor 2 protein in the preparation
of a medicament for the treatment of renal disease and
inflammation.
[0077] A further aspect of the present invention provides a
pharmaceutical composition for the treatment of renal disease and
inflammation, the composition comprising a therapeutically
effective amount of an inhibitory nucleic acid which blocks the
expression of Toll-like Receptor 2, along with at least one
pharmaceutically acceptable carrier, diluent, solubiliser,
emulsifier, preservative and/or adjuvant.
[0078] In certain embodiments the inhibitory nucleic acid is
selected from the group consisting of: anti-sense oligonucleotides,
anti-sense DNA, anti-sense RNA, ribozyme, iRNA, miRNA, sRNA, shRNA.
In certain embodiments the pharmaceutical composition may further
comprise at least one further immunosuppressor compound, as defined
hereinbefore.
[0079] Techniques for the preparation of appropriate nucleic acids
for use as inhibiting nucleic acids which block the expression of
Toll-like Receptor 2 are well known to the person skilled in the
art.
[0080] In a further aspect, the invention extends to the provision
of at least one aptamer with binding specificity to Toll-like
Receptor 2, which causes blocking or suppression of the functional
activity of Toll-like Receptor 2. Techniques for the selection of
suitable aptamers will be well known to the person skilled in the
art, for example, using SELEX technology. Accordingly, in various
further embodiments, the present invention extends to a method of
identifying and isolating nucleic acid ligands which have binding
specificity for Toll-like Receptor 2 or a Toll-like Receptor 2 gene
product, the method comprising the steps of: [0081] (a) providing a
candidate mixture of nucleic acids [0082] (b) contacting a cell
expressing Toll-like Receptor 2 with the candidate nucleic acid
mixture [0083] (c) selecting nucleic acids which have an increased
affinity to Toll-like Receptor 2 relative to the other candidate
nucleic acids, [0084] (d) amplifying the selected nucleic acids in
order to provide at least one nucleic acid with affinity for
Toll-like Receptor 2, and [0085] (e) selecting at least one nucleic
acid therefrom which has a high affinity and specificity for
Toll-like Receptor 2.
[0086] The inventors have further identified that suppression of
the function of Toll-like Receptor 2 can be achieved by way of
reducing the amount of TLR2 binding ligand which is available to
bind to and activate membrane bound Toll-like Receptor 2. A
reduction in the amount of ligand which is available to bind
membrane bound Toll-like Receptor 2 results in a downregulation of
Toll-like Receptor 2 mediated signalling and thus of TLR2-mediated
activation of the pro-inflammatory immune response. In particular,
the inventors have identified the utility of a soluble peptide
which is either a soluble form of Toll-like Receptor 2 or a
functional fragment thereof, in the suppression of Toll-like
Receptor 2 mediated activation of a pro-inflammatory response. Said
suppression results from the soluble form of Toll-like Receptor 2
or truncated non-membrane form of Toll-like Receptor 2 competing
with the membrane bound form of TLR2 for the binding of TLR2
binding ligands. This competitive binding results in the soluble or
truncated forms of TLR2 effectively "mopping up" available
Toll-like Receptor 2 ligand, that is, due to the binding of TLR2
ligands to the soluble forms of TLR2, the pool of TLR2 ligands
which is available to bindi to and activate membrane bound TLR2 is
depleted. An associated reduction in the binding and activation of
membrane bound Toll-like Receptor 2 results in a downregulation of
the Toll-like Receptor 2 mediated pro-inflammatory immune
response.
[0087] Accordingly, the administration of a soluble form of
Toll-like Receptor 2 has utility in methods for suppressing the
pro-inflammatory immune response which contributes to tissue injury
during renal inflammation and disease.
[0088] The amino acid sequence of the extracellular domain
(ectodomain) of human Toll-like Receptor 2 is provided herein as
SEQ ID NO:5 (FIG. 21). The extracellular domain of the human form
of Toll-like Receptor 2 comprises 587 amino acid resides,
specifically amino acids 1-587 of the defined 784 amino acid full
length human Toll-like Receptor sequence as defined in SEQ ID NO:1
and also defined as Genbank Accession Number AAC 34133 (URL
www.ncbi.nlm.nih.gov). As herein defined, the ectodomain of TLR2 is
the portion of the membrane bound form of TLR2 which extends into
the extracellular space.
[0089] In certain embodiments of the invention, where the TLR2
modulatory agent is a soluble form of TLR2, the soluble form of
TLR2 may be prepared by a recombinant technique. A soluble form of
Toll-like Receptor 2 typically comprises the extracellular domain
of TLR2 only, and hence the intracellular and transmembrane domains
of Toll-like Receptor 2 as defined in Genbank Accession Number AAC
34133 are absent. In certain embodiments, the soluble form of
Toll-like Receptor 2 may comprise amino acids 1 to 587 of the
defined human Toll-like Receptor 2 sequence as shown in SEQ ID
NO:1. The soluble Toll-like Receptor 2 sequence may be modified by
means of the addition, deletion or substitution of 1 or more amino
acid residues. Accordingly, in certain embodiments, the soluble
form of the Toll-like Receptor 2 is derived from the extracellular
domain of the determined membrane bound form of Toll-like Receptor
2 as defined herein in SEQ ID NO:1. In further embodiments, the
soluble form of the Toll-like Receptor 2 is derived from a
truncated form of the full length membrane bound Toll-like Receptor
2 amino acid sequence as defined herein in SEQ ID NO:1, wherein
said truncated form exhibits the functional characteristics of (i)
being soluble, and (ii) being capable of being bound by a ligand
which has binding specificity to at least one epitope which is
present on the membrane bound form of Toll-like Receptor 2.
[0090] In certain embodiments, in addition to a substitution of the
amino acid residues relating to the intracellular and/or
transmembrane domains derived from the membrane bound form of TLR2,
at least one deletion and/or substitution may further be made to
the amino acid residues of the extracellular domain of the TLR2 to
form a homologous protein. Any number of deletions and/or
substitutions to the amino acid residues of the extracellular
domain of the TLR2 may be made so long as the resultant peptide is
capable of binding a ligand which is capable of binding to at least
one epitope present on the membrane bound form of TLR2. The soluble
form of the Toll-like Receptor 2 may also comprise a sequence which
has at least a 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology
to the amino acid sequence of SEQ ID NO:5. Such homology may be
determined by a number of ways which are well known to the person
skilled in the art, such as the use of an aligned algorithm. In
particular, the percentage identity between two amino acid
sequences may be determined, for example, by comparing the amino
acid sequences of the 2 proteins using the GAP computer program,
version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387,
1984) and available from the University of Wisconsin Genetics
Computer Group (UWGCG). The GAP program utilises the alignment
method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as
revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981).
[0091] In certain embodiments, the soluble form of Toll-like
Receptor 2 (sTLR2) may be targeted to the kidney, or in particular
to at least one specific cell type which is involved with disease
pathogenesis of in an inflamed state, for example the renal tubular
epithelial cells (TECs) and/or the epithelial cells of the Bowman's
capsule. The targeting of sTLR2 in this way is advantageous as
systemic administration of sTLR2 may result in global
immunosuppression of the TLR2 receptor and accordingly TLR2
mediated signalling which may be undesirable in some instances.
[0092] Targeting of soluble forms of sTLR2 may be provided through
the formation of a fusion protein, wherein said fusion protein is
comprised of the soluble TLR2 protein, most typically the
extracellular domain of TLR2 as described hereinbefore, or a
portion thereof, which is linked to a secondary peptide, typically
the Fc domain of an immunoglobulin, such as the heavy chain of an
immunoglobulin, typically a human immunoglobulin. The Fc domain has
been extensively used to prolong the circulatory half-life of
therapeutic proteins. The fusion protein may be further modified to
improve its half life, or efficacy. A number of techniques for
mediating such modifications are known to the person in the art,
such a PEGylation, or refolding of the tertiary protein
structure.
[0093] A yet further aspect of the present invention provides a kit
comprising a pharmaceutical preparation comprising an agent which
suppresses the function, expression or signalling mediated by a
membrane bound form of Toll-like Receptor 2 along with instructions
for the administration of said preparation, said kit having utility
in the treatment of subjects with kidney disease or inflammation.
Typically said agent is a soluble form the Toll-like Receptor
2.
[0094] The present invention further extends to screening assays
for use in identifying compounds which are capable of preventing
TLR2 activation and TLR2 mediated immune responses which contribute
to renal inflammation and disease by means of suppressing the
function of Toll-like Receptor 2.
[0095] A yet further aspect of the present invention provides a
screening method for the identification of compounds which suppress
Toll-like Receptor 2 mediated inflammation associated with a renal
disorder or disaese the method comprising: [0096] providing
membrane bound Toll-like Receptor 2 receptor along with a ligand
which has binding specificity thereto, [0097] bringing a candidate
compound into contact with the Toll-like Receptor 2, [0098]
exposing the Toll-like Receptor 2 to a Toll-like Receptor 2 ligand
agonist, [0099] determining the binding of the Toll-like Receptor 2
ligand agonist to Toll-like Receptor 2, wherein the inhibition of
binding of Toll-like Receptor 2 by the Toll-like Receptor 2 ligand
indicates that said candidate compound is a modulator of Toll-like
Receptor 2 activation and signalling.
[0100] A further aspect of the present invention provides for the
use of a modulator identified according to the previous aspect of
the invention in the preparation of a medicament for suppressing a
TLR2-mediated inflammatory response for the treatment of renal
inflammation and disease.
[0101] A further aspect of the present invention provides for a
modulator identified by the foregoing assay method for the
invention for use in the treatment of renal inflammation and
disease.
[0102] In certain embodiments the agent which modulates the
function or expression of Toll-like Receptor 2 for use in treating
renal disease. In certain embodiments, the renal disease is
selected from the group consisting of renal disease, chronic renal
failure, acute renal failure heterologous nephrotoxic nephritis,
glomerulonephritis, sclerosis of the glomerulus, systemic lupus
erythematosus (SLE), diabetic nephropathy, and diabetic
nephropathy.
[0103] In certain embodiments the agent is selected from the group
consisting of a protein, a peptide, a peptidomimetic, a nucleic
acid, a carbohydrate, a lipid, and a small molecule compound. In
certain embodiments, the agent is an antibody which has binding
specificity to human TLR2. In certain embodiments the antibody is
selected from the group consisting of a human, humanised, chimeric,
synthetic, camelid, shark or in-vitro antibody which has binding
specificity to TLR2, or a binding fragment derived from any of the
same.
[0104] In certain embodiments, the agent is a TLR2 antagonist which
binds to an epitope defined by the extracellular domain of human
TLR2. In certain embodiments, the agent is a TLR2 antagonist which
binds to a non-continuous epitope comprising amino acid residues
derived from the amino and carboxyl terminals of the amino acid
sequence of human TLR2.
[0105] In certain embodiments of this aspect of the invention, the
agent inhibits the expression of nucleic acid encoding the TLR2
protein. In certain embodiments, the agent is selected from the
group consisting of an anti-sense oligonucleotides, triple helix
molecules, anti-sense DNA, anti-sense RNA, ribozyme, iRNA, miRNA,
sRNA, shRNA molecule.
[0106] In certain embodiments of this aspect of the invention, the
agent is a soluble form of Toll-like Receptor 2. Typically, the
soluble form of Toll-like Receptor 2 comprises an amino acid
sequence as defined in SEQ ID NO:5, or a fragment thereof.
[0107] The inventors have identified that TLR2 mediated signalling
and activation of the immune response results in the upregulation
of levels of the cytokine IL-8. The expression of this cytokine has
been identified by the inventors as being one of the key
inflammatory mediators in stimulating renal inflammation, and the
progress of disease conditions caused by renal inflammation.
[0108] Accordingly, a further aspect of the present invention
provides a method of suppressing Toll-like Receptor 2-mediated IL-8
production by at least one cell type of the kidney, the method
comprising the step of: [0109] providing a therapeutically
effective amount of an agent which modulates the functional
activity or expression of Toll-like Receptor 2 (TLR2), and [0110]
administering the same to a subject in need of such treatment.
[0111] In certain embodiments the TLR2 modulator agent is a TLR2
antagonist selected from the group comprising, but not limited to:
a protein, a peptide, a peptidomimetic, a nucleic acid, a
carbohydrate, a lipid, an aptamer and a small molecule compound. In
certain embodiments the TLR2 antagonist is an antibody molecule. In
further embodiments, the TLR2 antagonist is a fusion protein, for
example a fusion protein comprising a soluble fragment of TLR2. In
certain further embodiments, the TLR2 modulator agent is an
inhibitory nucleic acid which includes, but is not limited to;
anti-sense oligonucleotides, anti-sense DNA, anti-sense RNA,
ribozyme, iRNA, miRNA, sRNA, shRNA.
BRIEF DESCRIPTION OF THE FIGURES
[0112] FIG. 1 shows a dot plot graph illustrating the number of
neutrophils per glomerula in a glomerular cross section obtained
from extracted kidneys of C57BL/6 Toll-like Receptor 2 deficient
mice treated with either 30 mg/kg intraperitoneal (i.p.) prednisol
(pred), PBS control (PBS), 10 mg/kg intravenous (i.v.) isotype
control antibody (cont) or 10 mg/kg intravenous (i.v.) anti-TLR2
antibody OPN301 (opn301) followed by immunization with 10 ug of the
lipopeptide Pam3CysSK4,
[0113] FIG. 2 shows histological sections of the kidney from
C57BL/6 Toll-like Receptor 2 deficient mice treated with either 30
mg/kg intraperitoneal (i.p.) prednisol (pred), PBS control (PBS),
10 mg/kg intravenous (i.v.) isotype control antibody (Control Ab)
or 10 mg/kg intravenous (i.v.) anti-TLR2 antibody OPN301 (opn301)
followed by immunization with bug of the lipopeptide Pam3CysSK4 for
2 hours,
[0114] FIG. 3 shows a dot plot graph of circulating neutrophils
from C57BL/6 Toll-like Receptor 2 deficient mice treated with
either 30 mg/kg intraperitoneal (i.p.) prednisol (pred), PBS
control (PBS), 10 mg/kg intravenous (i.v.) isotype control antibody
(cont) or 10 mg/kg intravenous (i.v.) anti-TLR2 antibody OPN301
(opn301) followed by immunization with bug of the lipopeptide
Pam3CysSK4,
[0115] FIG. 4 shows a dot plot graph of total white blood cells
from C57BL/6 Toll-like Receptor 2 deficient mice treated with
either 30 mg/kg intraperitoneal (i.p.) prednisol (pred), PBS
control (PBS), 10 mg/kg intravenous isotype control antibody (cont)
or 10 mg/kg intravenous (i.v.) anti-TLR2 antibody OPN301 (opn301)
followed by immunization with 10 ug of the lipopeptide
Pam3CysSK4,
[0116] FIG. 5 shows a dot plot graph of albuminuria/24 hours from
C57BL/6 Toll-like Receptor 2 deficient mice treated with either 30
mg/kg intraperitoneal (i.p.) prednisol (pred), PBS control (PBS),
10 mg/kg intravenpus (i.v) isotype control antibody (cont) or 10
mg/kg intravenous (i.v.) anti-TLR2 antibody OPN301 (opn301)
followed by immunization with bug of the lipopeptide
Pam3CysSK4,
[0117] FIG. 6 shows a dot plot graph of thrombosis score from
C57BL/6 Toll-like Receptor 2 deficient mice treated with either 30
mg/kg intraperitoneal (i.p.) prednisol (pred), PBS control 9PBS),
10 mg/kg intravenous (i.v.) isotype control antibody (cont) or 10
mg/kg intravenous anti-TLR2 antibody OPN301 (opn301) followed by
immunization with bug of the lipopeptide Pam3CysSK4,
[0118] FIG. 7 shows histological sections of the kidney from
C57BL/6 Toll-like Receptor 2 deficient mice treated with either 30
mg/kg intraperitoneal (i.p.) prednisol (Pred), PBS control (PBS),
10 mg/kg intravenous (i.v.) isotype control antibody (Control Ab)
or 10 mg/kg intravenous (i.v.) anti-TLR2 antibody OPN301 (opn301)
followed by immunization with bug of the lipopeptide Pam3CysSK4 at
24 hours,
[0119] FIG. 8 shows dot plot graphs of measurements of urea
(mmol/L) (A) and creatinine (mmol/L) (B) in mice kidneys from
treatment groups (i) Sham-operated, (ii) PBS-vehicle, (iii) 10
mg/kg isotype control antibody (Control Ab), (iv) 10 mg/kg
anti-TLR2OPN301 monoclonal antibody, and (v) 6 mg/kg pargyline,
measured 1 and 5 days after renal ischemia/reperfusion (n=6 per
group),
[0120] FIG. 9 shows (A) representative histological kidney sections
showing tubular injury and (b) a dot plot graph of semi
quantitative scoring of the percentage of damage tubules of mice
from treatment groups (i) Sham-operated, (ii) PBS-vehicle, (iii) 10
mg/kg isotype control antibody (Control Ab), (iv) 10 mg/kg
anti-TLR2 OPN301 monoclonal antibody, and (v) 6 mg/kg pargyline
measured 1 day after renal ischaemia/repefusion (n=6),
[0121] FIG. 10 shows (A) dot plot graphs of percentage of
macrophage infiltration and (B) accumulation of myeloperoxidase
(MPO) (U/mg proteins) in mice kidney from treatment groups (i)
Sham-operated, (ii) PBS-vehicle, (iii) 10 mg/kg isotype control
antibody (Control Ab), (iv) 10 mg/kg anti-TLR2OPN301 monoclonal
antibody, and (v) 6 mg/kg pargyline measured 1 day after renal
ischaemia/repefusion (n=6 per group),
[0122] FIG. 11 shows a dot plot graph indicating malondialdehyde
(MDA) production (nmol/mg proteins) used as an index of lipid
peroxidation in mice kidney 1 day after renal ischemia/reperfusion
in the following treatment groups: (i) Sham-operated, (ii)
PBS-vehicle, (iii) 10 mg/kg isotype control antibody (Control Ab),
and (iv) 10 mg/kg anti-TLR2 OPN301 monoclonal antibody and (v) 6
mg/kg pargyline measured 1 day after renal ischaemia/repefusion.
(n=6 per group),
[0123] FIG. 12 shows dot plot graphs of measurements of (A) Uremia
(urea (mmol/L)) and (B) Creatininemia (creatinine (mmol/L)) in mice
kidney from Step 1 treatment groups 1 and 5 days after renal
ischemia/reperfusion (n=4 per group),
[0124] FIG. 13 shows (A) representative histological tubular kidney
sections showing tubular injury, and (B) a dot plot graph of semi
quantitative scoring of the percentage of damage tubules of mice
from Step 1 treatment groups 1 and 5 days after renal
ischaemia/repefusion (n=4 per group),
[0125] FIG. 14 shows dot plot graphs of measurements of (A) Uremia
(urea (mmol/L)), and (B) Creatininemia (creatinine (mmol/L)) in
mice kidney from Step 2 treatment groups as follows: (i)
Sham-operated, (ii) PBS-vehicle, (iii) 10 mg/kg isotype control
antibody (Control Ab), (iv) 10 mg/kg anti-TLR2 monoclonal antibody
OPN301, and (v) 6 mg/kg pargyline measured 1 and 5 days after renal
ischemia/reperfusion (n=6 per group),
[0126] FIG. 15 shows a dot plot graph of accumulation of
myeloperoxidase (MPO) (U/mg proteins) accumulation from neutrophils
in mice from Step 2 treatment groups as follows: (i) Sham-operated,
(ii) PBS-vehicle, (iii) 10 mg/kg isotype control antibody (Control
Ab), (iv) 10 mg/kg anti-TLR2 monoclonal antibody OPN301, and (v) 6
mg/kg pargyline measured 1 and 5 days after renal
ischaemia/repefusion (n=6 per group),
[0127] FIG. 16 shows a dot plot graph indicating malondialdehyde
(MDA) production which is used as an index of lipid peroxidation
(nmol/mg proteins) in mice kidney in Step 2 treatment groups as
follows: (i) Sham-operated, (ii) PBS-vehicle, (iii) 10 mg/kg
isotype control antibody (Control Ab), (iv) 10 mg/kg anti-TLR2
monoclonal antibody OPN301, and (v) 6 mg/kg pargyline measured 1
and 5 days after renal ischaemia/repefusion (n=6 per group),
[0128] FIG. 17 shows a dot plot graph of semi quantitative scoring
of the damage to tubules of mice from Step 2 treatment groups as
follows: (i) Sham-operated, (ii) PBS-vehicle, (iii) 10 mg/kg
isotype control antibody (Control Ab), (iv) 10 mg/kg anti-TLR2
monoclonal antibody OPN301, and (v) 6 mg/kg pargyline measured 1
and 5 days after renal ischaemia/repefusion (n=6 per group),
[0129] FIGS. 18(a), (b) and (c) show a dot plots of accumulation of
p-GST .mu.g/L in mice kidney from Step 2 treatment groups (i)
Sham-operated (ii) PBS-vehicle iii) 10 mg/kg isotype control
antibody (Control Ab), (iv) 10 mg/kg OPN301 mAb and (v) 6 mg/kg
pargyline measured 1 day after renal ischemia/reperfusion at day 0
(FIG. 18(a)), day 1 (FIG. 18(b)) and day 5 (FIG. 18(c)) after renal
ischemia/reperfusion (n=6 per group),
[0130] FIG. 19 shows the amino acid sequence of human Toll-like
Receptor 2 (SEQ ID NO:1),
[0131] FIG. 20 shows the amino acid sequence of human Toll-like
Receptor 2 (SEQ ID NO:2), and
[0132] FIG. 21 shows the amino acid sequence of the extracellular
domain of human Toll-like Receptor 2 (SEQ ID NO:3).
DETAILED DESCRIPTION OF THE INVENTION
[0133] The present invention relates to agents which inhibit the
function or expression of Toll-like Receptor 2 (TLR2), or to
methods which use such agents for the treatment of renal disease
and inflammation, and in particular for the treatment or
prophylaxis of inflammation of the glomeruli as well as kidney
diseases mediated by Toll-like Receptor 2 signaling.
[0134] TLR2 modulator agents such as TLR2 antagonistic binding
compounds, which have affinity and binding specificity to a binding
epitope of TLR2 have utility in the inhibition of a number of
immune mediated and disease conditions which affect the kidney. As
such, the present invention provides compositions and methods for
the treatment and prophylaxis of immune-mediated, inflammatory and
pathogenic conditions which target the kidney, or which affect
kidney function.
[0135] In certain embodiments, the pathogenic condition is an
infectious condition mediated by a bacterial pathogen. The
bacterial pathogen may be a gram positive bacteria, or a gram
negative bacteria. In certain embodiments, the bacterial pathogenic
is a sepsis-causing bacteria, said compositions and methods having
utility in the treatment of kidney related inflammation arising
from conditions such as sepsis or septic shock, for example gram
negative sepsis.
[0136] The term "epitope" as used herein relates to a portion of a
macromolecule, in this case the TLR2 protein, or a related protein,
which is capable of being bound by a specific binding ligand, in
this case a Toll-like Receptor 2 ligand agonist. Epitopes may be
defined from contiguous or non-contiguous sequences of amino acid
residues comprised within a polypeptide sequence. The term
"contiguous epitope" defines an epitope comprised of a linear
series of amino acid residues within a polypeptide which define the
epitope. A "non-contiguous epitope" is an epitope which is
comprised of a series of amino acid residues which are non-linear
in alignment, that is that the residues are spaced or grouped in a
non-continuous manner along the length of a polypeptide sequence. A
non-continuous epitope can be a discontinuous epitope wherein the
amino acid residues are grouped into 2 linear sequences, or
alternatively the non-continuous epitope can be a discontinuous
scattered epitope wherein the residues which contribute to the
epitope are provided in 3 or more groups of linear amino acid
sequences arranged along the length of the polypeptide.
Antibodies
[0137] An "antibody" is an immunoglobulin, whether natural or
partly or wholly synthetically produced. The term also covers any
polypeptide, protein or peptide having a binding domain that is, or
is homologous to, an antibody binding domain. These can be derived
from natural sources, or they may be partly or wholly synthetically
produced. Examples of antibodies are the immunoglobulin isotypes
and their isotypic subclasses and fragments which comprise an
antigen binding domain such as Fab, scFv, Fv, dAb, Fd, and a
bi-specific antibody.
[0138] In further embodiments, the antibody may be a Camelid
antibody, in particular a Camelid heavy chain antibody. Further the
antibody fragment may be a domain antibody or a nanobody derived
from a Camelid heavy chain antibody. In a further embodiment the
antibody may be a shark antibody or a shark derived antibody.
[0139] In certain embodiments, the antibody is an "isolated
antibody", this meaning that the antibody is (1) free of at least
some proteins with which it would normally be found, (2) is
essentially free of other proteins from the same source, e.g., from
the same species, (3) is expressed by a cell from a different
species, or (4) does not occur in nature.
[0140] As antibodies can be modified in a number of ways, the term
"antibody" should be construed as covering any binding member or
substance having a binding domain with the required specificity.
The antibody of the invention may be a monoclonal antibody, or a
fragment, derivative, functional equivalent or homologue thereof.
The term includes any polypeptide comprising an immunoglobulin
binding domain, whether natural or wholly or partially synthetic.
Chimeric molecules comprising an immunoglobulin binding domain, or
equivalent, fused to another polypeptide are therefore included.
Cloning and expression of chimeric antibodies are described in
European Patent Application Publication Number EP 0,120,694 and
European Patent Application Publication Number EP 0,125,023.
[0141] The constant region of the antibody may be of any suitable
immunoglobulin subtype, however it is preferred that the antibody
subtype is IgG1. However, in alternative embodiments, the subtype
of the antibody may be of the class IgA, IgM, IgD and IgE where a
human immunoglobulin molecule is used. Such an antibody may further
belong to any subclass e.g. IgG1, IgG2a, IgG2b, IgG3 and IgG4.
[0142] Fragments of a whole antibody can perform the function of
antigen binding. Examples of such binding fragments are; a Fab
fragment comprising of the VL, VH, CL and CH1 antibody domains; an
Fv fragment consisting of the VL and VH domains of a single
antibody; a F(ab')2 fragments, a bivalent fragment comprising two
linked Fab fragments; a single chain Fv molecule (scFv), wherein a
VH domain and a VL domain are linked by a peptide linker which
allows the two domains to associate to form an antigen binding
site; or a bi-specific antibody, which may be multivalent or
multispecific fragments constructed by gene fusion.
[0143] A fragment of an antibody or of a polypeptide for use in the
present invention, for example, a fragment of a TLR2 specific
antibody, generally means a stretch of amino acid residues of at
least 5 to 7 contiguous amino acids, often at least about 7 to 9
contiguous amino acids, typically at least about 9 to 13 contiguous
amino acids, more preferably at least about 20 to 30 or more
contiguous amino acids and most preferably at least about 30 to 40
or more consecutive amino acids.
[0144] A "derivative" of such an antibody or polypeptide, or of a
fragment of a TLR2 specific antibody means an antibody or
polypeptide modified by varying the amino acid sequence of the
protein, e.g. by manipulation of the nucleic acid encoding the
protein or by altering the protein itself. Such derivatives of the
natural amino acid sequence may involve insertion, addition,
deletion and/or substitution of one or more amino acids, preferably
while providing a peptide having TLR2 binding activity. Preferably
such derivatives involve the insertion, addition, deletion and/or
substitution of 25 or fewer amino acids, more preferably of 15 or
fewer, even more preferably of 10 or fewer, more preferably still
of 4 or fewer and most preferably of 1 or 2 amino acids only.
[0145] The term "antibody" includes antibodies which have been
"humanised". Methods for making humanised antibodies are known in
the art. Methods are described, for example, in Winter, U.S. Pat.
No. 5,225,539. A humanised antibody may be a modified antibody
having the hypervariable region of a monoclonal antibody such as a
TLR2 specific antibody and the constant region of a human antibody.
Thus the binding member may comprise a human constant region.
[0146] The variable region other than the hypervariable region may
also be derived from the variable region of a human antibody and/or
may also be derived from a monoclonal antibody such as a TLR2
specific antibody. In such case, the entire variable region may be
derived from murine monoclonal antibody a TLR2 specific antibody
and the antibody is said to be chimerised. Methods for making
chimerised antibodies are known in the art. Such methods include,
for example, those described in U.S. patents by Boss (Celltech) and
by Cabilly (Genentech). See U.S. Pat. Nos. 4,816,397 and 4,816,567,
respectively.
[0147] It is possible to take monoclonal and other antibodies and
use techniques of recombinant DNA technology to produce other
antibodies or chimeric molecules which retain the specificity of
the original antibody. Such techniques may involve introducing DNA
encoding the immunoglobulin variable region, or the complementarity
determining regions (CDRs), of an antibody to the constant regions,
or constant regions plus framework regions, of a different
immunoglobulin. See, for instance, the teachings of EP 0,184,187,
GB 2,188,638A or EP 0,239,400. A hybridoma or other cell producing
an antibody may be subject to genetic mutation or other changes,
which may or may not alter the binding specificity of antibodies
produced.
[0148] In certain embodiments, where the TLR2 inhibitory compound
or TLR2 binding compound of the invention is an antibody, the
antibody is administered to a subject in a therapeutically
effective amount. In certain embodiments, the therapeutically
effective amount comprises the antibody in a range chosen from 1
.mu.g/kg to 20 mg/kg, 1 g/kg to 10 mg/kg, 1 .mu.g/kg to 1 mg/kg, 10
.mu.g/kg to 1 mg/kg, 10 .mu.g/kg to 100 .mu.g/kg and 500 .mu.g/kg
to 1 mg/kg.
Production of Antibodies
[0149] The antibodies provided by the present invention may be
provided by a number of techniques. For example, a combinatorial
screening technique such as a phage display-based biopanning assay
may be used to in order to identify amino acid sequences which have
binding specificity to the binding epitopes of the invention. Such
phage display biopanning techniques involve the use of phage
display libraries, which are utilised in methods which identify
suitable epitope binding ligands in a procedure which mimics immune
selection, through the display of antibody binding fragments on the
surface of filamentous bacteria. Phage with specific binding
activity are selected. The selected phage can thereafter be used in
the production of chimeric, CDR-grafted, humanised or human
antibodies.
[0150] In further embodiments, the antibody is a monoclonal
antibody may be produced using any suitable method which produces
antibody molecules by continuous cell lines in culture. Suitable
methods will be well known to the person skilled in the art and
include, for example, the method of Kohler and Milstein (Kohler et
al. Nature, 256, 495-497. 1975), Chimeric antibodies or CDR-grafted
antibodies are further provided within the scope of the present
invention. In further embodiments, the antibodies of the invention
may be produced by the expression of recombinant DNA in host
cell.
[0151] In further embodiments, humanized antibodies are also
provided. Humanized antibodies may be produced by the method of
Winter as described in U.S. Pat. No. 5,585,089.
[0152] In further certain embodiments, the monoclonal antibodies
may be human antibodies, produced using transgenic animals, for
example, transgenic mice, which have been genetically modified to
delete or suppress the expression of endogenous murine
immunoglobulin genes, with loci encoding for human heavy and light
chains being expressed in preference, this resulting in the
production of fully human antibodies.
[0153] In certain further embodiments, the binding compound is a
binding fragment which is derived from an antibody, for example, an
antibody binding fragment, such as a Fab, F(ab').sub.2, Fv or a
single chain Fv (scFV).
[0154] In certain further embodiments, the binding compound
comprises a polyclonal antibody, a chimeric antibody, a synthesized
or synthetic antibody, a fusion protein or fragment thereof, or a
natural or synthetic chemical compound or a peptidomimetic.
[0155] Certain methodologies for producing antibodies which have an
affinity and binding specificity for the Toll-like Receptor 2
epitope of the present invention are described hereinbefore.
[0156] The antibodies or antibody fragments of and for use in the
present invention may also be generated wholly or partly by
chemical synthesis. The antibodies can be readily prepared
according to well-established, standard liquid or, preferably,
solid-phase peptide synthesis methods, general descriptions of
which are broadly available and are well known by the person
skilled in the art. Further, they may be prepared in solution, by
the liquid phase method or by any combination of solid-phase,
liquid phase and solution chemistry.
[0157] Another convenient way of producing antibodies or antibody
fragments suitable for use in the present invention is to express
nucleic acid encoding them, by use of nucleic acid in an expression
system.
[0158] Nucleic acid for use in accordance with the present
invention may comprise DNA or RNA and may be wholly or partially
synthetic. In a preferred aspect, nucleic acid for use in the
invention codes for antibodies or antibody fragments of the
invention as defined above. The skilled person will be able to
determine substitutions, deletions and/or additions to such nucleic
acids which will still provide an antibody or antibody fragment of
the present invention.
[0159] Nucleic acid sequences encoding antibodies or antibody
fragments for use with the present invention can be readily
prepared by the skilled person using the information and references
contained herein and techniques known in the art (for example, see
Sambrook et al. (1989), and Ausubel et al, (1992)), given the
nucleic acid sequences and clones available. These techniques
include (i) the use of the polymerase chain reaction (PCR) to
amplify samples of such nucleic acid, e.g. from genomic sources,
(ii) chemical synthesis, or (iii) preparing cDNA sequences. DNA
encoding antibody fragments may be generated and used in any
suitable way known to those of skill in the art, including by
taking encoding DNA, identifying suitable restriction enzyme
recognition sites either side of the portion to be expressed, and
cutting out said portion from the DNA. The portion may then be
operably linked to a suitable promoter in a standard commercially
available expression system. Another recombinant approach is to
amplify the relevant portion of the DNA with suitable PCR primers.
Modifications to the sequences can be made, e.g. using site
directed mutagenesis, to lead to the expression of modified peptide
or to take account of codon preferences in the host cells used to
express the nucleic acid.
[0160] The nucleic acid may be comprised as constructs in the form
of a plasmid, vector, transcription or expression cassette which
comprises at least one nucleic acid as described above. The
construct may be comprised within a recombinant host cell which
comprises one or more constructs as above. Expression may
conveniently be achieved by culturing under appropriate conditions
recombinant host cells containing the nucleic acid. Following
production by expression the antibody or antibody fragments may be
isolated and/or purified using any suitable technique, then used as
appropriate.
[0161] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, mammalian cells, yeast, insect and baculovirus
systems. Mammalian cell lines available in the art for expression
of a heterologous polypeptide include Chinese hamster ovary (CHO)
cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma
cells. A common, preferred bacterial host is E. coli. The
expression of antibodies and antibody fragments in prokaryotic
cells such as E. coli is well established in the art. Expression in
eukaryotic cells in culture is also available to those skilled in
the art as an option for production of a binding member.
[0162] General techniques for the production of antibodies are well
known to the person skilled in the field, with such methods being
discussed in, for example, Kohler and Milstein (1975) Nature 256:
495-497; U.S. Pat. No. 4,376,110; Harlow and Lane, Antibodies: a
Laboratory Manual, (1988) Cold Spring Harbor, the contents of which
are incorporated herein by reference.
[0163] Techniques for the preparation of recombinant antibody
molecules are described in the above references and also in, for
example, EP 0,623,679 and EP 0,368,684 which are incorporated
herein by reference.
[0164] In certain embodiments of the invention, recombinant nucleic
acids comprising an insert coding for a heavy chain variable domain
and/or for a light chain variable domain of antibodies are
employed. By definition such nucleic acids comprise coding single
stranded nucleic acids, double stranded nucleic acids consisting of
said coding nucleic acids and of complementary nucleic acids
thereto, or these complementary (single stranded) nucleic acids
themselves.
[0165] Furthermore, nucleic acids encoding a heavy chain variable
domain and/or a light chain variable domain of antibodies can be
enzymatically or chemically synthesised nucleic acids having the
authentic sequence coding for a naturally-occurring heavy chain
variable domain and/or for the light chain variable domain, or a
mutant thereof.
[0166] Recombinant DNA technology may be used to improve the
antibodies of the invention. Thus, chimeric antibodies may be
constructed in order to decrease the immunogenicity thereof in
diagnostic or therapeutic applications. Moreover, immunogenicity
within, for example, a transgenic organism such as a pig, may be
minimised, by altering the antibodies by CDR grafting in a
technique analogous to humanising antibodies. Examples of such
techniques are described in EP 0,239,400 to Winter. In order to
reduce immunogenicity within a recipient, the invention may employ
recombinant nucleic acids comprising an insert coding for a heavy
chain variable domain of an antibody fused to a human constant
domain. Likewise the invention concerns recombinant DNAs comprising
an insert coding for a light chain variable domain of an antibody
fused to a human constant domain kappa or lambda.
[0167] Antibodies may moreover be generated by mutagenesis of
antibody genes to produce 5 artificial repertoires of antibodies.
This technique allows the preparation of antibody libraries.
Antibody libraries are also available commercially. Hence, the
present invention advantageously employs artificial repertoires of
immunoglobulins, preferably artificial scFv repertoires, as an
immunoglobulin source in order to identify binding molecules which
have specificity for the epitope of the present invention.
Antibody Selection Systems
[0168] Immunoglobulins which are able to bind to the epitope of the
present invention and which accordingly may be used in the methods
of the invention can be identified using any technique known to the
skilled person. Such immunoglobulins may be conveniently isolated
from libraries comprising artificial repertoires of immunoglobulin
polypeptides. A "repertoire" refers to a set of molecules generated
by random, semi-random or directed variation of one or more
template molecules, at the nucleic acid level, in order to provide
a multiplicity of binding specificities. Methods for generating
repertoires are well characterised in the art.
[0169] Any library selection system may be used in conjunction with
the invention. Selection protocols for isolating desired members of
large libraries are known in the art, as typified by phage display
techniques. Such systems, in which diverse peptide sequences are
displayed on the surface of filamentous bacteriophage, have proven
useful for creating libraries of antibody fragments (and the
nucleotide sequences that encode them) for the in vitro selection
and amplification of specific antibody fragments that bind a target
antigen. The nucleotide sequences encoding the VH and VL regions
are linked to gene fragments which encode leader signals that
direct them to the periplasmic space of E. coli and as a result the
resultant antibody fragments are displayed on the surface of the
bacteriophage, typically as fusions to bacteriophage coat proteins
(e.g., pIII or pVIII). Alternatively, antibody fragments are
displayed externally on lambda phage capsids (phage bodies). An
advantage of phage-based display systems is that, because they are
biological systems, selected library members can be amplified
simply by growing the phage containing the selected library member
in bacterial cells. Furthermore, since the nucleotide sequence that
encodes the polypeptide library member is contained on a phage or
phagemid vector, sequencing, expression and subsequent genetic
manipulation is relatively straight forward.
[0170] Methods for the construction of bacteriophage antibody
display libraries and lambda phage expression libraries are well
known in the art (for example, McCafferty et al. (1990) Nature 348
552-554. One particularly advantageous approach has been the use of
scFv phage-libraries (see for example Huston et al., 1988, Proc.
Natl. Acad. Sci. USA).
[0171] An alternative to the use of phage or other cloned libraries
is to use nucleic acid, preferably RNA, derived from the B cells of
an animal which has been immunised with the selected target, e.g.
the TLR2 epitope of the present invention.
[0172] Isolation of V-region and C-region mRNA permits antibody
fragments, such as Fab or Fv, to be expressed intracellularly.
Briefly, RNA is isolated from the B cells of an immunised animal,
for example from the spleen of an immunised mouse or the
circulating B cells of a llama, and PCR primers used to amplify VH
and VL cDNA selectively from the RNA pool. The VH and VL sequences
thus obtained are joined to make scFv antibodies. PCR primer
sequences may be based on published VH and VL sequences.
Peptidomimetics
[0173] Peptide analogues, such as peptidomimetics or peptide
mimetics are non-peptide compounds with properties representative
of a template peptide. Such peptide analogues are typically
developed using computerised molecular modelling. Peptidomimetics
which are structurally similar to peptides which have affinity and
binding specificity to the TLR2 binding epitope of the present
invention may be used to mediate similar diagnostic, prophylactic
and therapeutic effects.
[0174] Peptidomimetics are typically structurally similar to a
template peptide, but have one or more peptide linkages replaced by
an alternative linkage, by methods which are well known in the art.
For example, a peptide which has a binding specificity for the TLR2
epitope of the invention may be modified such that it comprises
amide bond replacement, incorporation of non peptide moieties, or
backbone cyclisation. Suitably if cysteine is present the thiol of
this residue is capped to prevent damage of the free sulphate
group. A peptide may further be modified from the natural sequence
to protect the peptides from protease attack.
[0175] Suitably a peptide of and for use in the present invention
may be further modified using at least one of C and/or N-terminal
capping, and/or cysteine residue capping.
[0176] Suitably, a peptide of and for use in the present invention
may be capped at the N terminal residue with an acetyl group.
Suitably, a peptide of and for use in the present invention may be
capped at the C terminal with an amide group. Suitably, the thiol
groups of cysteines are capped with acetamido methyl groups.
[0177] Expression, isolation and purification of polypeptides
defining the epitope of the invention and fragments thereof may be
accomplished by any suitable technique.
[0178] A method for producing polypeptides comprises culturing host
cells transformed with a recombinant expression vector encoding a
polypeptide under conditions that promote expression of the
polypeptide, then recovering the expressed polypeptides from the
culture. The skilled man will recognise that the procedure for
purifying the expressed polypeptides will vary according to such
factors as the type of host cells employed, and whether the
polypeptide is intracellular, membrane-bound or a soluble form that
is secreted from the host cell.
[0179] Any suitable expression system may be employed. The vectors
include a DNA encoding a polypeptide or fragment of the invention,
operably linked to suitable transcriptional or translational
regulatory nucleotide sequences, such as those derived from a
mammalian, avian, microbial, viral, bacterial, or insect gene.
Nucleotide sequences are operably linked when the regulatory
sequence functionally relates to the DNA sequence. Thus, a promoter
nucleotide sequence is operably linked to a DNA sequence if the
promoter nucleotide sequence controls the transcription of the DNA
sequence. An origin of replication that confers the ability to
replicate in the desired (E. coli) host cells, and a selection gene
by which transformants are identified, are generally incorporated
into the expression vector.
[0180] In addition, a sequence encoding an appropriate signal
peptide (native or heterologous) can be incorporated into
expression vectors. A DNA sequence for a signal peptide (secretory
leader) may be fused in frame to the nucleic acid sequence of the
invention so that the DNA is initially transcribed, and the mRNA
translated, into a fusion protein comprising the signal peptide. A
signal peptide that is functional in the intended host cells
promotes extracellular secretion of the polypeptide. The signal
peptide is cleaved from the polypeptide during translation, but
allows secretion of polypeptide from the cell.
[0181] Suitable host cells for expression of polypeptides include
higher eukaryotic cells and yeast. Prokaryotic systems are also
suitable. Mammalian cells, and in particular Chinese hamster ovary
(CHO) cells are particularly preferred for use as host cells.
Appropriate cloning and expression vectors for use with mammalian,
prokaryotic, yeast, fungal and insect cellular hosts are described,
for example, in Pouwels et al. Cloning Vectors: A Laboratory
Manual, Elsevier, New York, (1986) (ISBN 0444904018).
Small Molecules
[0182] In various further aspects, the present invention relates to
screening and assay methods for use in identifying compounds which
antagonise TLR2 activity. Certain further aspects extend to the
compounds identified thereby, wherein said binding compounds have
affinity and binding specificity for the epitope of the
invention.
[0183] A substance identified as a ligand of the TLR2 receptor may
be a peptide or may be non-peptide in nature, for example a
peptidomimetic as described hereinbefore. However, non-peptide
"small molecules" are often preferred for many in-vivo
pharmaceutical uses. Accordingly, a mimetic or mimic of a TLR2
binding compound for use in the present invention may be designed
for pharmaceutical uses.
[0184] The designing of mimetics to a known pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound. This might be desirable where the
active compound is difficult or expensive to synthesise, or where
it is unsuitable for a particular method of administration. For
example, peptides are not well suited as active agents for oral
compositions and administration as they are degraded by proteases
present in the alimentary canal. Mimetic design, synthesis and
testing may be used to avoid randomly screening large number of
molecules for a target property.
[0185] There are several steps commonly taken in the design of a
mimetic from a compound having a given target property. Firstly,
the particular parts of the compound that are critical and/or
important in determining the target property are determined. In the
case of a peptide, this can be done by systematically varying the
amino acid residues in the peptide, for example by substituting
each amino acid residue in turn. These parts or residues
constituting the active region of the compound are known as its
"pharmacophore".
[0186] Once the pharmacophore has been determined, its structure is
modelled according to its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data from a
range of sources, e.g. spectroscopic techniques, X-ray diffraction
data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the
bonding between atoms) and other techniques can also be used in
this modelling process.
[0187] In a variant of this approach, the three-dimensional
structure of the TLR2 binding compound is modelled. This can be
especially useful where the ligand and/or binding partner change
conformation on binding, allowing the model to take account of the
design of the mimetic.
[0188] A template molecule is then selected onto which chemical
groups which mimic the pharmacophore can be grafted. The template
molecule and the chemical groups grafted on to it can conveniently
be selected so that the mimetic is easy to synthesise, is likely to
be pharmacologically acceptable, and does not degrade in-vivo,
while retaining the biological activity of the lead compound. The
mimetic or mimetics found by this approach can then be screened to
see whether they have the target property, or to what extent they
exhibit it. Further optimisation or modification can then be
carried out to arrive at one or more final mimetics for in-vivo or
clinical testing.
[0189] In certain embodiments, the mimetic binding compound may be
a natural or synthetic chemical compound used in drug screening
programmes. Extracts of plants which contain several characterised
or uncharacterised components may also be used.
[0190] A candidate binding compound which has affinity and binding
specificity to TLR2 may be isolated and/or purified, manufactured
and/or used to modulate TLR2 functional activity.
[0191] In yet further aspects, the invention extends to the use of
combinatorial library technology (Schultz, J S (1996) Biotechnol.
Prog. 12:729-743) which provides an efficient way of testing a
potentially vast number of different substances for ability their
ability to bind to an epitope or to modulate the activity of a
ligand which binds to an epitope. Prior to, or as well as, being
screened for modulation of activity, test substances may be
screened for ability to interact with the polypeptide, e.g. in a
yeast two-hybrid system (which requires that both the polypeptide
and the test substance can be expressed in yeast from encoding
nucleic acid). This may be used as a coarse screen prior to testing
a substance for actual ability to modulate activity of the
polypeptide.
[0192] The amount of test substance or compound which may be added
to an assay of the invention will normally be determined by trail
and error depending upon the type of compound used. Typically, from
about 0.01 to 100 nM concentrations of putative inhibitor compound
may be used, for example from 0.1 to 10 nM. Greater concentrations
may be used when a peptide is the test substance.
Combination Medicaments
[0193] As described hereinbefore, the present invention extends to
combinational therapies wherein compositions or methods relates to
the administration of a binding compound which inhibits the
functional activity of TLR2 are administered in combination with at
least one further therapeutic compound which serves to suppress the
immune response which may contribute to acute renal failure,
chronic renal failure or renal disease.
[0194] Typically the primary and secondary therapeutic compositions
are given contemporaneously. In certain embodiments, the primary
therapeutic composition (i.e. the binding compound which
antagonises the functional activity of TLR2) and the secondary
therapeutic compounds are administered simultaneously. In certain
further embodiments, they are administered sequentially.
[0195] In certain embodiments, the combination therapy may comprise
a TLR2 functional inhibitor which is co-administered to a subject
along with at least one of: a cytokine inhibitor, such as, but not
limited to an inhibitor of IL-1, IL-6, IL-8 and IL-15, and
inhibitor of tumour necrosis factor, a growth factor inhibitor, an
immunosuppressor, an anti-inflammatory, an enzymatic inhibitor, a
metabolic inhibitor, a cytotoxic agent or a cytostatic agent.
[0196] A person of relevant skill in the field will recognise that
the administration to a subject of a combination therapy can be
advantageous in that it permits administration of a lower dose of
therapeutic to a subject in order to achieve and associated
therapeutically effective effect. The administration of a lower
combined dose also results in the subject being exposed to a lower
toxicity level. Furthermore, as the secondary therapeutic compounds
which are administered as part of the combination therapy provided
by the invention target different pathways, there is likely to be a
synergistic improvement in the overall efficacy of the therapy. An
improvement in efficacy would again result in the need for a lower
dose to be administered and as such an associated reduction in
toxicity.
[0197] In identifying and selecting suitable secondary therapeutic
compounds for administration along with the TLR2 inhibitory
compounds of the present invention, said secondary therapeutic
compounds may be selected on the basis of such compounds modulating
the immune response at a different stage of the inflammatory
response which results in renal inflammation and disease. Such
secondary compounds may include, but are not limited to; soluble
receptors, peptide inhibitor compound, small molecule, fusion
proteins or ligands, antibodies, and cytokines which mediate an
anti-inflammatory effect.
Administration
[0198] The monoclonal antibody or fusion protein of the present
invention may be administered alone but will preferably be
administered as a pharmaceutical composition, which will generally
comprise a suitable pharmaceutically acceptable excipient, diluent
or carrier selected depending on the intended route of
administration. Examples of suitable pharmaceutical carriers
include; water, glycerol, ethanol and the like.
[0199] The monoclonal antibody or fusion protein of the present
invention may be administered to a patient in need of treatment via
any suitable route. As detailed herein, it is preferred that the
composition is administered parenterally by injection or infusion.
Examples of preferred routes for parenteral administration include,
but are not limited to; intravenous, intracardial, intraarterial,
intraperitoneal, intramuscular, intracavity, subcutaneous,
transmucosal, inhalation or transdermal.
[0200] Routes of administration may further include topical and
enteral, for example, mucosal (including pulmonary), oral, nasal,
rectal.
[0201] In preferred embodiments, the composition is deliverable as
an injectable composition. For intravenous, intradermal or
subcutaneous application, the active ingredient will be in the form
of a parenterally acceptable aqueous solution which is pyrogen-free
and has suitable pH, isotonicity and stability. Those of relevant
skill in the art are well able to prepare suitable solutions using,
for example, isotonic vehicles such as sodium chloride injection,
Ringer's injection or, Lactated Ringer's injection.
[0202] Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
[0203] The composition may also be administered via microspheres,
liposomes, other microparticulate delivery systems or sustained
release formulations placed in certain tissues including blood.
[0204] Examples of the techniques and protocols mentioned above and
other techniques and protocols which may be used in accordance with
the invention can be found in Remington's Pharmaceutical Sciences,
18th edition, Gennaro, A. R., Lippincott Williams & Wilkins;
20th edition ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and
Drug Delivery Systems; Ansel, H. C. et al. 7th Edition ISBN
0-683305-72-7, the entire disclosures of which is herein
incorporated by reference.
[0205] The composition is preferably administered to an individual
in a "therapeutically effective amount", this being sufficient to
show benefit to the individual to whom the composition is
administered. The actual dose administered, and rate and
time-course of administration, will depend on, and can be
determined with due reference to, the nature and severity of the
condition which is being treated, as well as factors such as the
age, sex and weight of the patient to be treated and the route of
administration. Further due consideration should be given to the
properties of the composition, for example, its binding activity
and in-vivo plasma life, the concentration of the fusion protein in
the formulation, as well as the route, site and rate of
delivery.
[0206] Dosage regimens can include a single administration of the
composition of the invention, or multiple administrative doses of
the composition. The compositions can further be administered
sequentially or separately with other therapeutics and medicaments
which are used for the treatment of the condition for which the
fusion protein of the present invention is being administered to
treat.
[0207] Examples of dosage regimens which can be administered to a
subject can be selected from the group comprising, but not limited
to; 1 .mu.g/kg/day through to 20 mg/kg/day, 1 .mu.g/kg/day through
to 10 mg/kg/day, 10 .mu.g/kg/day through to 1 mg/kg/day.
[0208] The TLR2 epitope binding compound of the present invention
is preferably administered to an individual in a "therapeutically
effective amount", this being sufficient to show benefit to the
individual.
[0209] The actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of what is
being treated. Prescription of treatment, e.g. decisions on dosage
etc, is ultimately within the responsibility and at the discretion
of general practitioners and other medical doctors, and typically
takes account of the disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners.
[0210] Unless otherwise defined, all technical and scientific terms
used herein have the meaning commonly understood by a person who is
skilled in the art in the field of the present invention.
[0211] Throughout the specification, unless the context demands
otherwise, the terms `comprise` or `include`, or variations such as
`comprises` or `comprising`, `includes` or `including` will be
understood to imply the inclusion of a stated integer or group of
integers, but not the exclusion of any other integer or group of
integers.
[0212] As used herein, terms such as "a", "an" and "the" include
singular and plural referents unless the context clearly demands
otherwise. Thus, for example, reference to "an active agent" or "a
pharmacologically active agent" includes a single active agent as
well as two or more different active agents in combination, while
references to "a carrier" includes mixtures of two or more carriers
as well as a single carrier, and the like.
[0213] The nomenclature used to describe the polypeptide
constituents of the fusion protein of the present invention follows
the conventional practice wherein the amino group (N) is presented
to the left and the carboxy group to the right of each amino acid
residue.
[0214] The expression "amino acid" as used herein is intended to
include both natural and synthetic amino acids, and both D and L
amino acids. A synthetic amino acid also encompasses chemically
modified amino acids, including, but not limited to salts, and
amino acid derivatives such as amides. Amino acids present within
the polypeptides of the present invention can be modified by
methylation, amidation, acetylation or substitution with other
chemical groups which can change the circulating half life without
adversely affecting their biological activity.
[0215] The terms "peptide", "polypeptide" and "protein" are used
herein interchangeably to describe a series of at least two amino
acids covalently linked by peptide bonds or modified peptide bonds
such as isosteres. No limitation is placed on the maximum number of
amino acids which may comprise a peptide or protein. Furthermore,
the term polypeptide extends to fragments, analogues and
derivatives of a peptide, wherein said fragment, analogue or
derivative retains the same biological functional activity as the
peptide from which the fragment, derivative or analogue is
derived
[0216] Furthermore the term "fusion protein" as used herein can
also be taken to mean a fusion polypeptide, fusion peptide or the
like, or may also be referred to as an immunoconjugate. The term
"fusion protein" refers to a molecule in which two or more subunit
molecules, typically polypeptides, are covalently or non-covalently
linked.
[0217] As used herein, the term "effective amount" or
"therapeutically effective amount" means the amount of an agent,
binding compound, small molecule, fusion protein or peptidomimetic
of the invention which is required to suppress TLR2-mediated
inflammation in the kidney or which reduces the severity of and/or
ameliorates a TLR2 mediated renal disease or at least one symptom
thereof or condition associated therewith.
[0218] As used herein, the term "prophylactically effective amount"
relates to the amount of a composition which is required to prevent
the initial onset, progression or recurrence of TLR2-mediated
inflammation of the kidney, or renal disease, or at least one
symptom thereof in a subject following the administration of the
compounds of the present invention.
[0219] As used herein, the term "treatment" and associated terms
such as "treat" and "treating" means the reduction of the
progression, severity and/or duration of a TLR2 mediated condition
of at least one symptom thereof, wherein said reduction or
amelioration results from the administration of a binding compound
which has specificity for the TLR2 binding epitope of the present
invention. The term `treatment` therefore refers to any regimen
that can benefit a subject. The treatment may be in respect of an
existing condition or may be prophylactic (preventative treatment).
Treatment may include curative, alleviative or prophylactic
effects. References herein to "therapeutic" and "prophylactic"
treatments are to be considered in their broadest context. The term
"therapeutic" does not necessarily imply that a subject is treated
until total recovery. Similarly, "prophylactic" does not
necessarily mean that the subject will not eventually contract a
disease condition.
[0220] As used herein, the term "subject" refers to an animal,
preferably a mammal and in particular a human. In a particular
embodiment, the subject is a mammal, in particular a human. The
term "subject" is interchangeable with the term "patient" as used
herein.
[0221] The present invention will now be described with reference
to the following examples which are provided for the purpose of
illustration and are not intended to be construed as being limiting
on the present invention.
EXAMPLES
Example 1
Effect of Toll-Like Receptor 2 Agonist on Glomerular
Inflammation
Methods
[0222] Methods for generating nephrotoxic antibody, blood counts,
processing histology, assessment of histology are disclosed in
Brown H J, Lock, H R, Sacks S H, Robson MG TLR2 stimulation of
intrinsic kidney cells in the induction of immune mediated
glomerulonephritis, J Immunol 2006, 177(3):1925-1931, the contents
of which are incorporated herein by reference. These methods were
followed in the performance of the present example.
Mice
[0223] Age, gender, and weight-matched wild-type C57BL/6 mice were
obtained from Harlan (Harlan UK Limited, Bicester, Oxon, UK). All
mice used were 9 to 10 weeks of age at commencement of the
experiments. Animal experiments were performed according to United
Kingdom Home Office regulations. The genotype of the TLR2-deficient
mice was confirmed by PCR.
Induction of Accelerated Nephrotoxic Nephritis
[0224] A mouse glomerular extract was made as described previously
in Brown et al., and serum was prepared in sheep by Micropharm
(Llandysul, UK). The nephrotoxic serum (NTS) was heat inactivated
at 56.degree. C. for 30 minutes and frozen in aliquots at
120.degree. C. until use. Mice were immunized intraperitoneally
with 200 1 g of normal sheep IgG that was given with 80 .mu.l of
aluminum hydroxide gel (Sigma, Poole, UK) in 310 .mu.l of PBS, with
100 1 g of Pam3CysSK4 (EMC Microcollections, Tubingen, Germany) in
10 .mu.l of DMSO or with 10 .mu.l of DMSO alone. All of these
components were given as a single intraperitoneal (i.p.) injection.
Five days later, mice received an injection of 160 .mu.l of NTS via
the tail vein to induce disease. The dose of lipopeptide and NTS
given were based on those used in other in vivo studies and on our
own pilot studies. At 14 days after disease induction, mice were
killed and exsanguinated for serum, and the kidneys were harvested.
Any mice that showed signs of ill health before 14 days were killed
humanely according to local regulations.
Histologic Analysis of Glomerular Injury
[0225] Kidney samples were fixed for 4 hours in Bouin's solution,
transferred into formalin, then processed and embedded in paraffin.
Sections were cut at 0.5 .mu.m thickness and stained with periodic
acid-Schiff (PAS) reagent. All samples then were assessed blindly
for glomerular thrombosis by identification of the amount of
PAS-positive material. In total, 50 glomeruli were assessed for
each mouse for signs of glomerular thrombosis, and a score was
assigned to each as follows: Grade 0, no PAS-positive material;
grade 1, 25%; grade 2, 25 to 50%, grade 3, 50 to 75%, and grade 4,
75 to 100%. The average glomerular thrombosis score for each mouse
then was calculated. Sections also were assessed blindly for
glomerular crescents. A total of 50 glomeruli were assessed, and
the number of crescents (two or more layers of cells in Bowman's
space) was expressed as a percentage.
Immunohistochemical Analysis of Glomerular Inflammation
[0226] The glomerular histology was assessed at 2 hours and 24
hours following disease induction with NTS. Kidneys were fixed in
Bouin's solution and stained with periodic acid-Schiff reagent. At
2 hours, the number of neutrophils (identified by their
characteristic nuclear morphology) per 50 glomerular cross sections
was counted. At 24 hours, the amount of glomerular thrombosis was
assessed by identification of the amount of periodic
acid-Schiff-positive material. In total, 50 glomeruli were assessed
for each animal for signs of glomerular thrombosis, and a score was
assigned as follows: grade 0, no periodic acid-Schiff-positive
material; grade 1, <25%; grade 2, 25-50%; grade 3, 50-75%; grade
4, 75-100%. All sections scored for neutrophils and glomerular
thrombosis were done so blindly.
Albuminuria
[0227] Mice were housed in metabolic cages for 24 hours for urine
collections. The urine albumin concentration was measured by radial
immunodiffusion. The sensitivity of the radial immunodiffusion
assay for albumin was 0.05 mg/ml. Urine samples were diluted to
1:40 so that values fell on the standard curve.
[0228] Female C57BL/6 mice were from Harlan. Weights were as
detailed in Table 1 below:
TABLE-US-00001 TABLE 1 Weights were as follows group wts (not
correlated with specific mice) mean 1 to 5 16.8, 16.7, 17.5, 17.4,
17.7 17.22 6 to 10 18.1, 17.3 17.3, 17, 17.1, 17.36 11 to 15 17.8,
16.9, 16.8, 17.2, 17.9 17.32 16 to 20 17, 17.4, 17.9, 16.7, 17.4,
17.28 21 to 24 18.9, 17.9, 18.7, 19.9 13.85 26 to 30 18.3, 18.4,
18.7, 19.4, 18.9 18.78 31 to 35 18.9, 19.3, 17.9, 19, 19 18.82 36
to 40 19, 19.1, 18.9, 18.7, 18.3 18.8
[0229] 200 ul of nephrotoxic serum containing 10 ug of the
lipopetide Pam3CysSK4 (EMC microcollections) was given
intravenously via the tail vein. 2 hours prior to this, mice were
treated as follows.
[0230] Group 1-5 were given prednisol 30 mg/kg via the
intraperitoneal (ip) route which was dissolved in pyrogen free
sterile PBS at 2.73 mg/ml. Therefore 190 ul=519 ug were given to
all mice based on a mean weight of 17.3 g.
[0231] Group 6-10 were given 190 .mu.l pyrogen free sterile PBS
intravenously.
[0232] Group 11-15 were given isotype control antibody 10 mg/kg
intravenously. The isotype control antibody is mouse IgG1 isotype
control purchased from RnD systems (catalogue number MAB002), this
isotype control antibody having binding specificity for Keyhole
Limpet Hemocyanin (KLH) antigen. OD was checked after
reconstitution as was shown to be 0.86 mg/ml, so 200 ul was equal
to 173 ug given to all mice (based on a mean weight of 17.3 g).
[0233] Group 16-20 were given the anti-TLR2 monoclonal antibody
OPN301 at a dose of 10 mg/Kg intravenously 0.91 mg/ml, so 190
ul=173 ug administered to all mice (based on a mean weight of 17.3
g). OPN301 (OPN-301) is a murine IgG1 anti-TLR2 antibody (mouse
Toll-like Receptor 2 (TLR2) antibody, clone T2.5, HyCult
Biotechnology b.v., Cell Sciences, Canton, USA: catalogue number
1054). The above mice were bled from the tail vein for total white
count and blood film, and then exsanguinated under anaesthesia.
[0234] Group 11-15 were given prednisol 30 mg/kg intraperitoneally
(i.p.). Dissolved in pyrogen free sterile PBS at 2.73 mg/ml, so 207
ul=564 ug given to all mice (based on a mean weight of 18.8 g).
[0235] Group 16-20 were given 207 .mu.l pyrogen free sterile PBS
intravenously.
[0236] Group 21-24 were given isotype control antibody 10 mg/kg
intravenously (RnD Systems, catalogue number MAB002). OD was
checked after reconstitution=0.86 mg/ml, so 219 .mu.l=188 .mu.g
given to all mice (based on a mean weight of 18.8 g).
[0237] Group 36-40 were given the anti-TLR2 monoclonal antibody
OPN301 at 10 mg/kg intravenously. 0.91 mg/ml, so 207 ul=188 ug
given to all mice (based on a mean weight of 18.8 g).
[0238] The above groups were place in metabolic cages immediately
after induction of disease and exsanguinated under anaesthesia 24
hours later. Statistics were performed with GraphPad prism
software. A one-way ANOVA was used with Tukey's post-test.
Albuminuria and thrombosis data was analysed after a logarithmic
transformation.
Results
Glomerular Neutrophils
[0239] There were less neutrophils in glomeruli at 2 hours in mice
pretreated with the anti-TLR2 monoclonal antibody OPN301 than in
PBS or control antibody treated mice (Table 1, FIG. 1). Neutrophil
numbers were similar to those seen in mice pretreated with
prednisolone, Representative histology at 2 hours is shown in FIG.
2.
Circulating Leukocytes
[0240] This decrease in neutrophil numbers seen in glomeruli in
opn301 treated mice was not due to a decrease in circulating
neutrophils since these were not lower in the anti-TLR2 monoclonal
antibody OPN301 treated mice than PBS or isotype control antibody
treated groups (Table 2, FIG. 3). In fact there was a trend towards
more neutrophils in the anti-TLR2 monoclonal antibody OPN301
treated mice, which would have reached significance if at test was
used to compare the anti-TLR2 monoclonal antibody OPN301 and the
isotype control antibody treated mice (p=0.03). The circulating
neutrophil numbers in steroid treated mice were much higher than in
other groups, though this did not lead to a significant increase in
glomerular number. The total blood leukocytes were higher in the
anti-TLR2 monoclonal antibody OPN301 treated mice than in any other
group (Table 2, FIG. 4).
TABLE-US-00002 TABLE 2 Circulating neutrophil numbers in treated
mice Mouse cells/ul % pmn pmn/ul pmn/glom Prednisone 1 47.7 70 33.4
0.28 2 55.8 64 35.7 0.42 3 49 0.24 4 60.3 65 39.2 0.34 5 35.1 71
24.9 0.54 PBS 6 12 3 7 47.7 28 13.4 2.8 8 54 34 18.4 3.36 9 42.3 23
9.7 3.02 10 50.4 10 5.0 2.96 Isotype Control Antibody 11 42.3 21
8.9 2.7 12 38.7 17 6.6 2.94 13 35.1 9 3.2 2.8 14 46.8 18 8.4 3.28
15 43.2 14 6.0 2.58 OPN301 Antibody 16 121.5 17 20.7 0.14 17 99 12
11.9 0.12 18 21 0.32 19 88.2 14 12.3 0.24 20 85.5 10 8.6 0.2
Albuminuria and Glomerular Thrombosis
[0241] There was less glomerular albuminuria and thrombosis in the
anti-TLR2 monoclonal antibody OPN301 treated mice than in PBS,
isotype control antibody or steroid treated mice (Table 3 and FIGS.
5 and 6). The reduction in albuminuria due to steroids did not
reach significance when compared with PBS or control antibody
treated mice. Steroid treated mice had significantly less
thrombosis than PBS but not control antibody treated mice. It
should be noted that glomerular thrombosis is not marked in this
model, as the numbers shown are the numbers of glomeruli showing
any thrombosis out of a total of 50. Representative histology at 24
hours is shown in FIG. 7.
TABLE-US-00003 TABLE 3 Glomerular albuminura and thrombosis in
treated mice Thromb/50 gloms alb, mg/ml Prednisone 21 7 2.54 22 7
4.54 23 7 4.71 24 3 4.98 PBS 26 12 4.62 27 12 9.95 28 12 7 29 16
13.56 30 9 7.52 OPN301 Antibody 31 0 0.92 32 0 0.5 33 0 1.26 34 0
0.63 35 0 0.27 Isotype Control Antibody 36 8 12.02 37 7 9.99 38 9
5.86 39 6 6.68 40 10 6.06
Conclusions
[0242] The anti-TLR2 monoclonal antibody OPN301 caused a decrease
in glomerular inflammation that was equivalent to high-dose
steroids. The effect on albuminuria and thrombosis was greater than
that seen with steroids.
Example 2
Renal Ischemia
[0243] All experiments reported were conducted as stated in the
National Institutes of Health Guide For The Care And Use Of
Laboratory Animals (Institute of Laboratory Animal Resources,
National Academy Press, Washington D.C., 1996) and were approved by
a local animal care and use committee, and in accordance with a
currently valid license for experiments on vertebrate animals,
issued by the French Ministry for Agriculture and Fisheries to Dr
Yara Barreira (N.degree. 31-125 dated 8 Jun. 2002, validity=5
years).
[0244] Nine Experimental groups were used as follows: [0245] 1)
Sham-operated mice sacrificed 1 day after surgery, n=6, [0246] 2)
PBS-vehicle-treated mice 15 min before ischemia/1 day reperfusion,
n=6, [0247] 3) Isotype control antibody 10 mg/kg-treated mice 15
minutes before ischemia/1 day reperfusion, n=6 (The isotype control
antibody is mouse IgG1 isotype control purchased from RnD systems
(catalogue number MAB002), this isotype control antibody having
binding specificity for Keyhole Limpet Hemocyanin (KLH) antigen),
[0248] 4) OPN301 anti-TLR2 monoclonal antibody 10 mg/kg-treated
mice 15 minutes before ischemia/1 day reperfusion, n=6, [0249] 5)
Pargyline 6 mg/kg-treated mice 15 minutes before ischemia/1 day
reperfusion, n=6, [0250] 6) PBS-vehicle-treated mice 15 minutes
before ischemia/5 days reperfusion, n=6, [0251] 7) Isotype control
antibody (RnD systems catalogue number MAB002) 10 mg/kg-treated
mice 15 minutes before ischemia/5 days reperfusion, n=6, [0252] 8)
OPN301 anti-TLR2 antibody 10 mg/kg-treated mice 15 minutes before
ischemia/5 days reperfusion, n=6, [0253] 9) Pargyline 6
mg/kg-treated mice 15 minutes before ischemia/5 days reperfusion,
n=6.
Experimental Warm Renal Ischemia/Reperfusion
[0254] Warm renal ischemia/reperfusion (I/R) experiments were
adapted from a previous study (Stokman et al, 2005). Briefly, a
wild type strain of C57BL/6 mice were anesthetized with 10 .mu.l/g
of a mix of 2.5 ml ketamine (Ketalar 50 mg/ml, Centravet;
Lapalisse, France), 0.5 ml xylazine (Rompun 2.5%, Centravet;
Lapalisse, France), and 7 ml NaCl 0.9% (Centravet; Lapalisse,
France). During surgery, animals were set on a 35.degree. C.
hotplate. After a 2 millimeters incision, the jugular vein was
exposed for intravenous injection (insulin syringe; VWR,
Fontenay-sous-Bois, France) of 300 .mu.l (1 min infusion) of the
considered treatment. The incision was closed after haemostasis
(5/0 Ethicrin thread, Ethicon; Auneau, France). Three centimetre in
length abdominal cutaneous and muscular incisions were performed;
renal pedicles were exposed and clamped for 30 minutes using
microaneurysm clamps (AREX; Palaiseau, France). At the time of
clamp installation and removal, kidneys were inspected for blood
flow arrest and restoration, respectively. Throughout renal
ischemia duration, the abdomen was wrapped up to avoid loss of heat
and to keep a relative intra-abdominal pressure. At the time of
abdomen closing in 2 layers (5/0 Ethicrin thread, Ethicon; Auneau,
France), mice were supplemented with 1 ml sterile NaCl 0.9% to
maintain fluid balance and volume status. Finally, all mice
received an intramuscular injection of 10 mg/kg profenid 50 mg/ml
(Centravet; Lapalisse, France) for analgesic purpose then allowed
to recover from surgery for 12 hours at 32.degree. C. in a
ventilated stove. Food and water were given ad libitum.
Sham-operated mice underwent the same procedure without
clamping.
[0255] At the end of the experiment, mice were sacrificed by lethal
injection of pentobarbital (100 .mu.l/10 g, Ceva Sante Animale;
Libourne, France) 1 and 5 days after surgery. Blood samples were
collected by abdominal aortic puncture, transferred into tubes for
coagulation (30 minutes, 4.degree. C.) then centrifuged (10
minutes, 1200 rpm, 4.degree. C.) for plasma collection and stored
at -80.degree. C. until use for renal function measurement. Kidneys
were removed and divided for preparation of histology (one-half
kidney transferred in Carnoy's solution--absolute ethanol 60%:
chloroform 30%: icy acetic acid 1%), for biochemical analyses (2
one-half kidneys snap-frozen in liquid nitrogen then stored at
-80.degree. C. until use for MPO and MDA evaluations), and
cytokine/chemokine measurements (one-half kidney snap-frozen in
liquid nitrogen then stored at -80.degree. C. until sending to the
Sponsor by CryoExpress shipment).
[0256] All the following analyses were made blindly.
Renal Function
[0257] The renal function determination was based on the evaluation
of plasmatic creatinine and urea concentrations on a Cobas Mira
biochemical analyzer (Horiba ABX; Montpellier, France), according
to standard diagnostic procedures followed by the Rangueil hospital
research services. Results were expressed as mmol/l for urea and
.mu.mol/L for creatinine.
Histology for Ischemia/Reperfusion Injury Estimation
[0258] After the 24 hour-fixation in Carnoy's solution, the
one-half kidneys were embedded in paraffin according to standard
procedures. Longitudinal 3-4-.mu.m-thick histological sections were
performed and stained with PAS. Briefly, sections were
deparaffinized and rehydrated to water, then oxidized in 0.5%
periodic acid solution for 10 minutes (DAKO; Trappes, France).
After rinsing, Schiff's reagent (DAKO; Trappes, France) was added
for 10 minutes then washed in lukewarm tap water for 5 minutes to
stop reduction of Schiff base. Counterstaining was proceeded in
Mayer's hematoxylin solution (DAKO; Trappes, France) for 2 minutes,
washed in tap water for 5 minutes, then rapidly dehydrated and
mounted.
[0259] Damaged tubules were assessed by semi-quantitative scoring
of the percentage of affected tubules per 10 non-overlapping fields
(.times.400 magnitude) in the cortico-medullary region. The injury
criteria such as tubular dilatation, epithelial necrosis, cast
deposition, and loss of brush border were followed and graded on a
5-point scale: 0 for 0% injury, 1 for <10% injury, 2 for 10 to
25% injury, 3 for 25 to 50% injury, 4 for 50 to 75% injury, and 5
for >75% injury.
Immunohistology for Macrophage Infiltration
[0260] Longitudinal sections were also used for F4/80
immunodetection. Briefly, deparaffinized sections were incubated
for 10 minutes with 3% hydrogen peroxide to block the endogenous
peroxidase activity. After the non-specific antigenic sites were
saturated, sections were sequentially incubated at room temperature
in a humidified atmosphere with: (i) the rat monoclonal antibody to
mouse F4/80 (1/400e--Caltag Laboratories, Invitrogen; Cergy
Pontoise, France) for 15 minutes, (ii) the rabbit polyclonal
antibody to rat IgG (1/800e--DAKO; Trappes, France) for 15 minutes;
and then (iii) the labeled HRP anti-rabbit Envision System (DAKO;
Trappes, France) for 20 minutes.
[0261] Specific antigenic sites were revealed with DAB (DAKO;
Trappes, France). Sections were counterstained with Mayer's
hematoxylin solution (DAKO; Trappes, France). Negative controls for
the immunohistochemical procedure included use of the control
isotype of primary antibody F4/80 (IgG2a, 1/400e--Caltag
Laboratories, Invitrogen; Cergy Pontoise, France). Analysis was
performed by an operator unaware of the origin of each kidney
section and removal of the histological codes was performed at the
end of recordings. Quantifications of the selected objects within a
given image were recorded into a Microsoft Excel file combined to
the Explorallova software. Results were given in percentage (%) of
specific coloured area per total studied area of the 10
non-overlapping fields (.times.200 magnitude), representing thus
the surface occupied by the analyzed marker in the
cortico-medullary junction.
Malondialdehyde Production Evaluation as an Index of Oxidative
Stress
[0262] Snap-frozen one-half kidneys were first homogenized in a mix
of PBS and PMSF 2 mM (Sigma Aldrich; St Quentin Fallavier, France),
then MDA concentration was indirectly determined by quantifying the
production of a specific chromogen compound from the reaction
between MDA and 2 molecules of thiobarbituric acid, as previously
described (Aruoma et al, 1989).
[0263] Briefly, homogenate aliquot was mixed with trichloracetic
acid 5% (Sigma Aldrich; St Quentin Fallavier, France) and
centrifuged (20 minutes, 4000 rpm, 4.degree. C.). The supernatant
was added to thiobarbituric acid 78 mM (Sigma Aldrich; St Quentin
Fallavier, France) and HCl 0.02M then heated for 10 minutes at
95.degree. C. The chromogen compound produced was then
spectrophotometrically measured at 535 nM wavelength (Helios .beta.
Unicam, Thermo Fisher Scientific; Courtaboeuf, France). MDA
bismethyl acetal (Sigma Aldrich; St Quentin Fallavier, France) was
used as an external standard. Results were expressed as nmol MDA/mg
proteins.
Myeloperoxidase Activity for Neutrophil Accumulation
[0264] Snap-frozen one-half kidneys were also homogenized in a mix
of potassium phosphate buffer 5 mM, pelleted (20,000 g, 45 minutes,
4.degree. C.) and put suspended in a mix of phosphate buffer 50 mM
and hexadecyltrimethyl ammonium bromide 0.5% (Fluke, Sigma Aldrich;
St Quentin Fallavier, France). A last centrifugation (20,000 g, 45
minutes, 4.degree. C.) finalized MPO release in the supernatant
that was kept for the enzyme activity measurement.
[0265] Briefly, MPO activity was indirectly determined by using
o-dianisidine dihydrochloride 0.167 mg/mL (Acros Organics; Noisy le
Grand, France) and hydrogen peroxide 0.1 mM in phosphate buffer 50
mM/pH 6. The absorbance of the product was spectrophotometrically
determined at 460 nm wavelength (Helios .beta. Unicam, Thermo
Fisher Scientific; Courtaboeuf, France). Results were expressed as
U MPO/mg proteins after normalization to protein content.
Analysis and Expression of Results
[0266] The results were given as mean values.+-.standard error of
the mean (SEM). A one-way ANOVA was used for comparison of
within-group difference followed by a Newman-Keuls test for
comparison of all pairs of columns. p<0.05 was considered
statistically significant.
Results
Effects of Warm Renal Ischemia/Reperfusion
[0267] The functional impact of I/R was assessed by the measurement
of key plasmatic parameters such as urea (FIG. 8A) and creatinine
(FIG. 8B). One day after I/R, creatininemia and uremia were greatly
increased as compared to sham-operated group, indicating a renal
function impairment (47.54.+-.3.49 mmol/L urea and 371.83.+-.27.34
.mu.mol/L creatinine vs. 5.21.+-.0.32 mmol/L urea and
43.02.+-.12.01 .mu.mol/L creatinine in PBS-vehicle and
sham-operated groups, respectively; n=6 per group, ***p<0.01,
ANOVA with Newman-Keuls test).
[0268] The loss of function was correlated to structural findings
as assessed by the percentage of damaged tubules (FIG. 9(B)). One
day after bilateral I/R, the degree of tubular injury in kidney
sections was severe and spread to the whole corticopapillar
gradient, and not only to the outer medulla (FIG. 9A); (n=6 per
group, ***p<0.01, ANOVA with Newman-Keuls test).
[0269] As one of the key features of I/R injury is acute
inflammation associated with phagocytes infiltration (macrophages
and neutrophils), F4/80 immunostaining (FIG. 10(A)) and MPO
activity (FIG. 10(B)) quantifications were performed. One day after
I/R, the increases in macrophage infiltration as well as MPO
activity in renal tissue were correlated to the ischemic injury
time course (3.24.+-.0.21% F4/80-positive staining and 63.2.+-.9.55
U MPO/mg protein vs. 0.91.+-.0.13% F4/80-positive staining and
20.72.+-.1.18 U MPO/mg protein in PBS-vehicle and sham-operated
groups, respectively; ***p<0.01 compared to Sham-operated group,
#p<0.05, ##p<0.01 and ###p<0.001 compared to PBS-vehicle
group, .sctn..sctn..sctn.p<0.001 compared to OPN301ContAb,
p<0.05 compared to OPN301 monoclonal antibody; n=6 per group,
ANOVA with Newman-Keuls test.
[0270] MDA production used as an index of oxidative stress injury,
was not significantly modified one day after I/R as compared to
basal value (0.082.+-.0.004 nmol MDA/mg protein vs. 0.080.+-.0.005
nmol/mg protein in PBS-vehicle and sham-operated groups,
respectively (FIG. 11); ***p<0.01 compared to Sham-operated
group, #p<0.05 and ###p<0.001 compared to PBS-vehicle group,
.sctn.p<0.001 compared to OPN301ContAb; n=6 per group, ANOVA
with Newman-Keuls test.
[0271] The second endpoint, 5 days after renal I/R, was not
assessed since mice mortality was about 80% at day 2 and 100% at
day 4. Considering the functional values, it was concluded that the
renal damage after 30-minute ischemia was too extended to allow the
mice survival and the recovery of renal function. The histological
analysis confirmed this hypothesis since the whole cortico-papillar
gradient was destroyed by consequences of I/R injury.
Effects of OPN301 Anti-TLR2 Monoclonal Antibody and Pargyline on
Acute Renal Failure One Day after Ischemia/Reperfusion
[0272] When the effects of OPN301 anti-TLR2 monoclonal antibody
were analyzed one day after I/R, the main obstacle encountered was
the severity of damages induced, and thus, the stringency of the
model. Indeed, from a functional and histological point of view, we
could not be able to discriminate between experimental groups
(FIGS. 8 and 9).
[0273] However, some parameters gave some encouraging suggestions
such as those related to inflammation i.e. macrophage infiltration
and neutrophil accumulation in renal tissue. Considering macrophage
infiltration (FIG. 10(A)), immunohistochemical analysis of kidney
sections demonstrated a decrease in F4/80 positive staining when
compared with PBS-vehicle group (1.38.+-.0.11% vs. 3.24.+-.0.21% in
OPN301 mAb and PBS-vehicle groups, respectively, n=6, p<0.001,
ANOVA with Newman-Keuls test). Considering neutrophil accumulation
(FIG. 10(B)), MPO activity levels demonstrated a decrease in enzyme
activity when compared with PBS-vehicle group (27.32.+-.1.95 U
MPO/mg protein vs. 63.2.+-.9.55 U MPO/mg protein in OPN301 mAb and
PBS-vehicle groups, respectively, n=6, p<0.01, ANOVA with
Newman-Keuls test).
[0274] It was noteworthy that the isotype control antibody seemed
to possess intrinsic properties since it was able to decrease
macrophage infiltration (2.69.+-.0.27% vs. 3.24.+-.0.21%
F4/80-positive staining in the isotype control antibody and
PBS-vehicle groups, respectively, n=6, p<0.05, ANOVA with
Newman-Keuls test; FIG. 10(3A)) as well as neutrophil accumulation
in renal tissue (38.1.+-.7.34 U MPO/mg protein and 63.2.+-.9.55 U
MPO/mg protein in the isotype control antibody and PBS-vehicle
groups, respectively, n=6, p<0.05, ANOVA with Newman-Keuls test;
FIG. 10(3B)). Whereas its activity was significantly distinct from
OPN301 mAb on the decrease in macrophage infiltration
(2.69.+-.0.27% vs. 1.38.+-.0.11% F4/80-positive staining in the
isotype control antibody and OPN301 anti-TLR2 monoclonal antibody
groups, respectively; n=6, p<0.0001, ANOVA with Newman-Keuls
test), its effect was not significantly different from the
experimental antibody on MPO activity (38.1.+-.7.34 U MPO/mg
protein vs. 27.32.+-.1.95 U MPO/mg protein in the isotype control
antibody and OPN301 anti-TLR2 monoclonal antibody groups,
respectively; n=6, p<0.0001, ANOVA with Newman-Keuls test).
[0275] The reference substance treatment (pargyline) had no effect
on neutrophil accumulation in renal tissue (53.48.+-.5.34 U MPO/mg
protein versus 63.20.+-.9.55 U MPO/mg protein in pargyline and
PBS-vehicle groups, respectively; n=6; FIG. 10(3B)). Its action on
macrophage infiltration was similar to the experimental mAb
(1.39.+-.0.15% and 1.38.+-.0.11% F4/80-positive staining in
pargyline and OPN301 anti-TLR2 monoclonal antibody groups,
respectively, n=6; FIG. 10(3A)).
[0276] Considering lipid peroxidation, the administration of OPN301
anti-TLR2 monoclonal antibody produced a significant reduction in
basal MDA level (0.048.+-.0.002 nmol/mg proteins vs. 0.082.+-.0.004
nmol/mg proteins in OPN301 anti-TLR2 monoclonal antibody and
PBS-vehicle groups, respectively; n=6, p<0.001, ANOVA with
Newman-Keuls test; FIG. 11). However, once again, it had to be
taken into account that the isotype control antibody had the same
effect on MDA level (0.046.+-.0.002 nmol/mg proteins vs.
0.082.+-.0.004 nmol/mg proteins in the isotype control antibody and
PBS-vehicle groups, respectively; n=6, p<0.001, ANOVA with
Newman-Keuls test; FIG. 11).
[0277] Regarding the reference substance treatment with pargyline,
it led to an expected decrease in the oxidative stress induced by
ischemia/reperfusion, as demonstrated by the MDA level
(0.051.+-.0.002 nmol/mg proteins vs. 0.082.+-.0.004 nmol/mg
proteins in pargyline and PBS-vehicle groups, respectively; n=6,
p<0.001, ANOVA with Newman-Keuls test; FIG. 11).
Conclusions
[0278] Example 2 shows that renal warm ischemia/reperfusion (I/R)
induced a huge loss of renal function characterized by increases in
the creatininemia and uremia that could be directly correlated with
marked damages along the whole cortico-papillar gradient mainly
characterized by tubular necrosis. The only criticism of this model
was its severity that did not allow discrimination of treatment
efficacies from a functional or structural point of view. Indeed,
as ischemic lesions of the initial acute renal failure were not
reversible, the tubular cell loss was not compensated and ischemic
acute renal failure quickly evolved towards terminal renal failure
and animal death. However, some biochemical markers used as index
of macrophage infiltration, neutrophil accumulation, and lipid
peroxidation represented convincing arguments in favour of the
usefulness of OPN301 anti-TLR2 monoclonal antibody treatment in
ischemic renal injury. Indeed, a beneficial effect of OPN301
anti-TLR2 monoclonal antibody was demonstrated on these parameters,
despite significant intrinsic properties of control antibody,
notably on MPO activity and MDA level.
Example 3
Renal Ischemia Repeat study
[0279] Five experimental groups were used in Step 1 as follows:
[0280] 1) Sham-operated mice sacrificed 1 day after surgery, n=4,
[0281] 2) 20 minutes of ischemia/1 day reperfusion, n=4, [0282] 3)
20 minutes of ischemia/5 day reperfusion, n=4, [0283] 4) 25 minutes
of ischemia/1 day reperfusion, n=4, [0284] 5) 25 minutes of
ischemia/1 day reperfusion, n=4.
[0285] Nine Experimental groups were used in Step 2: [0286] 1)
Sham-operated mice sacrificed 1 day after surgery, n=6, [0287] 2)
PBS-vehicle-treated mice 15 minutes before ischemia/1 day
reperfusion, n=6, [0288] 3) Isotype control antibody 10
mg/kg-treated mice 15 minutes before 25 minutes ischemia/1 day
reperfusion, n=6 (The isotype control antibody is mouse IgG1
isotype control purchased from RnD systems (catalogue number
MAB002), this isotype control antibody having binding specificity
for Keyhole Limpet Hemocyanin (KLH) antigen), [0289] 4) OPN301
anti-TLR2 monoclonal antibody 10 mg/kg-treated mice 15 minutes
before 25 minutes ischemia/1 day reperfusion, n=6, [0290] 5)
Pargyline 6 mg/kg-treated mice 15 minutes before 25 minutes
ischemia/1 day reperfusion, n=6, [0291] 6) PBS-vehicle-treated mice
15 minutes before 25 min ischemia/5 day reperfusion, n=6, [0292] 7)
Isotype control antibody (RnD Systems catalogue Number MAB002) 10
mg/kg-treated mice 15 minutes before 25 minutes ischemia/5 day
reperfusion, n=6, [0293] 8) OPN301 anti-TLR2 monoclonal antibody 10
mg/kg-treated mice 15 minutes before 25 minutes ischemia/5 day
reperfusion, n=6, [0294] 9) Pargyline 6 mg/kg-treated mice 15
minutes before 25 minutes ischemia/5 day reperfusion, n=6,
Experimental Warm Renal Ischemia/Reperfusion (Step 1 and 2)
[0295] Briefly, mice were anesthetized with 10 .mu.l/g of a mix of
2.5 ml ketamine (Ketalar 50 mg/mL, Centravet; Lapalisse, France),
0.5 ml xylazine (Rompun 2.5%, Centravet; Lapalisse, France), and 7
mL NaCl 0.9% (Centravet; Lapalisse, France). During surgery,
animals were set on a 35.degree. C. hotplate.
[0296] After a 2 millimetre incision was made at the right jugular
level, jugular vein was exposed for a 1 minute-intravenous
injection (insulin syringe; VWR, Fontenay-sous-Bois, France) of 70
.mu.l of the considered treatment then closed after haemostasis
(5/0 Ethicrin thread, Ethicon; Auneau, France). In Step 1, no
jugular injection was performed.
[0297] Three centimetre midline incisions were made to abdominal
cutaneous followed by muscular incisions were performed; renal
pedicles were exposed and clamped for 25 minutes using
microaneurysm clamps (AREX; Palaiseau, France). At the time of
clamp installation and removal, kidneys were inspected for blood
flow arrest and restoration, respectively. During renal ischemia
duration, the abdomen was wrapped up to avoid loss of heat and be
kept in relative intra-abdominal pressure. At the time of abdomen
closing in 2 layers (5/0 Ethicrin thread, Ethicon; Auneau, France),
mice were supplemented with 1 mL sterile NaCl (sodium chloride)
0.9% to maintain fluid balance and volume status. Finally, all mice
received an intramuscular injection of 10 mg/kg profenid 50 mg/mL
(Centravet; Lapalisse, France) for analgesic purpose then allowed
to recover from surgery for 12 hours at 32.degree. C. in a
ventilated stove. Food and water were given ad libitum.
Sham-operated mice underwent the same procedure without
clamping.
[0298] At the end of the protocol, mice were sacrificed by lethal
injection of pentobarbital (100 .mu.L/10 g, Ceva Sante Animale;
Libourne, France) 1 and 5 days after surgery.
[0299] Experimental methodology such as urea, creatinine, MPO and
MDA as described in Example 2.
Analysis and Expression of Results
[0300] The results were given as mean values.+-.standard error of
the mean (SEM). A one-way ANOVA was used for comparison of
within-group difference followed by a Newman-Keuls test for
comparison of all pairs of columns (GraphPad Prism, San Diego,
USA). A p<0.05 was accepted for statistical significance.
Results
[0301] Step 1: Determination of the optimal duration of warm renal
ischemia The functional impact of UR was assessed by the
measurement of key plasmatic parameters such as urea (FIG. 12(A))
and creatinine (FIG. 12(B), Table 4). No variation in the plasmatic
parameter levels during the minute ischemia duration was noticed.
However, it was demonstrated that when a 25 minute ischemia was
performed, the loss of renal function then recovery that
characterizes acute renal failure model was obtained. Indeed, one
day after 25 minutes of ischemia, uremia and creatininemia were
significantly increased as compared to sham-operated group,
indicating renal function impairment (39.58.+-.2.23 mmol/L urea and
142.18.+-.20.74 .mu.mol/L creatinine vs. 6.99.+-.0.55 mmol/L urea
and 23.20.+-.2.44 .mu.mol/L creatinine in 25 min ischemia day 1 and
Sham-operated groups, respectively; ***p<0.001 compared to 25
min ischemia day 1 group; n=4 per group; ANOVA followed by
Newman-Keuls test. Then, as expected, five days after ischemia, the
plasmatic parameter levels returned to basal value (16.14.+-.4.37
mmol/L urea and 36.83.+-.5.52 .mu.mol/L creatinine vs. 6.99.+-.0.55
mmol/L urea and 23.20.+-.2.44 .mu.mol/L creatinine in 25 min
ischemia day 5 and Sham-operated groups, respectively; n=4).
TABLE-US-00004 TABLE 4 Step 1: Individual data and mean results for
the uremia (top) and creatininemia (bottom) at day 1 and day 5. Day
1 Day 5 Sham-operated 20 min ischemia 25 min ischemia 20 min
ischemia 25 min ischemia 7.59 10.45 37.05 10.97 6.35 6.50 7.61
38.79 13.14 14.34 5.69 6.26 36.40 11.99 16.33 8.17 18.16 46.08 9.93
27.52 Mean .+-. SEM 6.99 .+-. 0.55 10.62 .+-. 2.66 39.58 .+-. 2.23
11.51 .+-. 0.69 16.14 .+-. 4.37 19 39.3 137.8 36.6 25.2 23.9 40.7
158.1 30.6 31.5 20.1 27.4 87.3 34.4 39.9 29.8 62.9 185.5 26 50.7
Mean .+-. SEM 23.20 .+-. 2.44 42.58 .+-. 7.40 142.18 .+-. 20.74
31.90 .+-. 2.33 36.83 .+-. 5.52
[0302] These functional modifications were correlated to structural
damage as assessed by the percentage of damaged tubules (FIG. 13,
Table 5). Data are expressed as the mean.+-.SEM; #p<0.05,
##p<0.01 compared to 25 min ischemia day 5 group, ***p<0.001
compared to 25 min ischemia day 1 group; n=4 per group; ANOVA
followed by Newman-Keuls test. One day after bilateral ischemia,
the degree of tubular injury in kidney sections was significant and
limited to the cortico-medullar junction. Five days after ischemia,
tubular injury was still distinguishable but limited to the
cortical area and associated with tubular regeneration. Lesions
noticed at day 5 were less severe than those at day 1; for example,
the losses of brush border or tubule cells as well as cast
deposition were less/not detected as compared with tubular
dilatation.
TABLE-US-00005 TABLE 5 Step 1: Individual data and mean results for
the scoring of the percentage of damage tubules. Day 1 Day 5
Sham-operated 20 min ischemia 25 min ischemia 20 min ischemia 25
min ischemia 0 2.75 3.75 2.5 2.75 0 2.5 3.5 2.5 3.25 0 2.75 4.25
2.5 3 0 2.5 3.75 2.25 3 Mean .+-. SEM 0 2.63 .+-. 0.07 3.81 .+-.
0.16 2.44 .+-. 0.06 3.00 .+-. 0.10
[0303] It is noteworthy that, despite any functional impairment at
both endpoints when 20 minute ischemia was performed, ischemic
injury consequences were observed in the renal tissue. Moreover, 25
minute ischemia was necessary and sufficient to combine the
reversibility of functional impairment with structural alterations,
characteristic from acute renal failure. Finally, a 5 minute
interval between 25 and 30 minutes was enough to convert an acute
renal failure model in end stage renal disease for which no
discrimination between treatments could be possible.
Step 2: Effects of OPN301 Anti-TLR2 Monoclonal Antibody and
Pargyline on Acute Renal Failure
[0304] Plasmatic parameter follow up after 25 minute ischemia
(FIGS. 14(A) and 14(B)) demonstrated that OPN301 anti-TLR2
monoclonal antibody had a preventive effect on renal function
impairment induced by ischemic insult since uremia and
creatininemia were not increased 1 day (D1) after ischemia when
compared with the positive control group (13.10.+-.3.15 mmol/L urea
and 48.47.+-.16.41 .mu.mol/L creatinine vs. 31.05.+-.5.70 mmol/L
urea and 99.97.+-.25.64 .mu.mol/L creatinine in OPN301 anti-TLR2
monoclonal antibody D1 and PBS-vehicle D1 groups, respectively
(Table 6 A & B). This effect was specific from OPN301 anti-TLR2
monoclonal antibody, as it was not observed for isotype control
antibody (31.22.+-.3.37 mmol/L urea and 95.55.+-.16.80 .mu.mol/L
creatinine versus 31.05.+-.5.70 mmol/L urea and 99.97.+-.25.64
.mu.mol/L creatinine in OPN301ContAb D1 and PBS-vehicle D1 groups,
respectively, n=6), and also for pargyline (33.72.+-.5.64 mmol/L
urea and 104.7.+-.25.20 .mu.mol/L creatinine versus 31.05.+-.5.70
mmol/L urea and 99.97.+-.25.64 .mu.mol/L creatinine in pargyline D1
and PBS-vehicle D1 groups, respectively; *p<0.05, **p<0.01,
***p<0.001 compared to PBS-vehicle D1 group, .sup.#p<0.05,
.sup.##p<0.01, .sup.###p<0.001 compared to the isotype
control antibody D1 group, .sup..sctn.p<0.05,
.sup..sctn..sctn.p<0.01, .sup..sctn..sctn..sctn.p<0.001
compared to Pargyline group; n=6 per group; ANOVA followed by
Newman-Keuls test.
TABLE-US-00006 TABLE 6 Step 2: Individual data and mean results for
the uremia (A) and creatininemia (B) 1 and 5 days after 25 minute
ischemia. A Day 1 Sham-operated PBS-vehicle OPN301ContAb OPN301mAb
Pargyline 4.47 43.83 46.83 8.13 47.99 5.9 49.25 26.95 23.23 48.92
0.54 16.37 33.73 15.08 40.01 4.76 26.33 24.39 20.67 27.83 3.47
34.62 26.92 6.51 19.35 5.34 15.89 28.47 4.95 18.22 Mean .+-. SEM
4.08 .+-. 0.78 31.05 .+-. 5.70 31.22 .+-. 3.37 13.10 .+-. 3.15
33.72 .+-. 5.64 Day 5 PBS-vehicle OPN301ContAb OPN301mAb Pargyline
7.62 10.87 6.65 7.63 7.16 11.69 7.2 9.48 9.99 9.48 10.03 7.37 6.82
11.43 5.86 7.53 10.82 7.83 6.71 8.78 10.27 6.99 8.8 7.29 Mean .+-.
SEM 8.78 .+-. 0.72 9.72 .+-. 0.80 7.54 .+-. 0.64 8.01 .+-. 0.37 B
Day 1 Sham-operated PBS-vehicle OPN301ContAb OPN301mAb Pargyline
20.3 130.9 175.6 29.2 185.9 19.8 199.1 98.3 120.6 150.7 24.6 43.5
90.2 28.9 134.1 21.2 93.4 66.5 71.1 77.6 28.1 108 72.5 25.2 53.3
23.8 24.9 70.2 15.8 26.6 Mean .+-. SEM 22.97 .+-. 1.29 99.97 .+-.
25.64 95.55 .+-. 16.80 48.47 .+-. 16.41 104.7 .+-. 25.20 Day 5
PBS-vehicle OPN301ContAb OPN301mAb Pargyline 20.6 22.6 14 16 15.5
24.4 19.8 27.2 25.2 21.8 27.5 30.7 26.4 26.9 24.9 19.2 25.8 34.1
29.5 22.9 18.3 32.7 26.9 22.9 Mean .+-. SEM 21.97 .+-. 1.84 27.08
.+-. 2.13 23.77 .+-. 2.37 23.15 .+-. 2.16
[0305] At day 5, there was no difference between treatments; as
expected, the regeneration step was functionally engaged.
Considering parameters related to inflammation, mainly neutrophil
accumulation in renal tissue (FIG. 15, Table 7), no modification in
MPO activity was evidenced when compared with PBS-vehicle group 1
day after ischemia (84.03.+-.4.10 U MPO/mg protein vs.
80.45.+-.8.66 U MPO/mg protein in OPN301 anti-TLR2 monoclonal
antibody D1 and PBS-vehicle D1 groups, respectively; .sup..English
Pound.p<0.05, .sup..English Pound..English Pound.p<0.01
compared to Pargyline D5 group. .sup..sctn.p<0.05 compared to
OPN301 anti-TLR2 monoclonal antibody D5 group. .sup.#p<0.05
compared to PBS-vehicle D5 group, *p<0.05, **p<0.01 compared
to OPN301 anti-TLR2 monoclonal antibody D1 group; n=6 per group;
ANOVA followed by Newman-Keuls test. At the same endpoint, the
reference substance treatment with pargyline had a strongest effect
on this parameter when compared to the OPN301 anti-TLR2 monoclonal
antibody (57.37.+-.5.78 U MPO/mg protein vs. 84.03.+-.4.10 U MPO/mg
protein in Pargyline D1 (day 1) and OPN301 anti-TLR2 monoclonal
antibody (OPN301 mAb) D1 (day 1) groups, respectively. Five days
after ischemia, MPO activity in renal tissue was decreased in the
four different groups when compared to the PBS-vehicle D1
group.
TABLE-US-00007 TABLE 7 Individual data and mean results for the MPO
activity as an index of neutrophil accumulation after 1 and 5 days.
Day 1 Sham-operated PBS-vehicle OPN301ContAb OPN301mAb Pargyline
124 76.6 68.4 91.3 48.8 41.5 67.1 71.6 76.1 45.4 70.9 119.6 79.8
83.7 47.9 76.3 83.3 109.5 88.6 52.7 99.6 57.9 58.7 95.7 68.7 77
78.2 60.7 68.8 80.7 Mean .+-. SEM 81.55 .+-. 11.39 80.45 .+-. 8.66
74.78 .+-. 7.61 84.03 .+-. 4.10 57.37 .+-. 5.78 Day 5 PBS-vehicle
OPN301ContAb OPN301mAb Pargyline 54.1 72.1 54.8 44.4 53.1 51.4 47.2
49.2 55.9 47.1 52.4 47.6 55 68 52.8 42.2 57.4 49 47.3 46.3 54.2
61.5 46.3 49.6 Mean .+-. SEM 54.95 .+-. 0.62 57.85 .+-. 4.16 50.13
.+-. 1.48 46.55 .+-. 1.17
[0306] Statistical analyses were performed using one-way ANOVA
followed by Newman-Keuls test.
TABLE-US-00008 TABLE 8 Individual data and mean results for the MDA
level as an index of lipid peroxidation. Day 1 Sham-operated
PBS-vehicle OPN301ContAb OPN301mAb Pargyline 0.178 0.179 0.179
0.197 0.249 0.183 0.116 0.132 0.176 0.202 0.181 0.108 0.196 0.222
0.285 0.204 0.145 0.225 0.175 0.163 0.153 0.181 0.177 0.180 0.212
0.141 0.189 0.266 0.169 0.242 Mean .+-. SEM 0.173 .+-. 0.009 0.153
.+-. 0.014 0.196 .+-. 0.019 0.187 .+-. 0.008 0.225 .+-. 0.017 Day 5
PBS-vehicle OPN301ContAb OPN301mAb Pargyline 0.160 0.513 0.303
0.290 0.159 0.187 0.121 0.231 0.166 0.252 0.283 0.218 0.182 0.194
0.241 0.261 0.179 0.328 0.235 0.234 0.279 0.203 0.232 0.299 Mean
.+-. SEM 0.188 .+-. 0.019 0.279 .+-. 0.051 0.236 .+-. 0.026 0.255
.+-. 0.014
[0307] FIG. 17 indicates the degree of tubular injury in the kidney
by measuring histological scores. One day after bilateral I/R, the
degree of tubular injury in control and PBS-vehicle samples where
severe compared with OPN301 mAb; (n=6 per group, ***p<0.01,
ANOVA with Newman-Keuls test). p-GST .mu.g/L, an indicator of
tubular damage in mice kidney was measured at day 0, day 1 and day
5 after renal ischemia/reperfusion (FIG. 18)
Conclusion
[0308] In the present study it was shown, for the first time in
mice, that the optimal duration of renal warm ischemia is 25
minutes. This protocol induced clear renal function impairment at
day 1 followed by recovery at day 5, as expected in an acute renal
failure model. From a structural point of view, when 25 minute
ischemia was performed, tubular damages were only restricted to the
cortico-medullar junction at day 1 before affecting the cortical
area and regenerating at day 5. Second, our functional results in
Step 2 demonstrated that the treatment with OPN301 anti-TLR2
monoclonal antibody 10 mg/kg had a protective and specific effect
against renal function alterations induced by ischemic injury 1 day
after ischemia. The results of neutrophil accumulation (MPO) and
oxidative stress (MDA) did not reveal any positive effects of
OPN301 mAb treatment. However, the fact that we also did not
observe significant differences between sham and
ischemia-reperfusion animals (which is unusual) suggests that
different experimental conditions (for instance, different
time-course of ischemia-reperfusion) may be necessary. Moreover,
the fact that the treatment with pargyline was without effects, in
sharp contrast to our previous studies using the I/R model in rats
(Kunduzova et al., 2002) suggest that species differences should be
taken into account. In conclusion, this study is the first proof of
concept for the use of OPN301 anti-TLR2 monoclonal antibody in the
treatment of acute renal failure, i.e. 1 day after ischemia.
Sequence CWU 1
1
51784PRTHomo sapiens 1Met Pro His Thr Leu Trp Met Val Trp Val Leu
Gly Val Ile Ile Ser1 5 10 15Leu Ser Lys Glu Glu Ser Ser Asn Gln Ala
Ser Leu Ser Cys Asp Arg 20 25 30Asn Gly Ile Cys Lys Gly Ser Ser Gly
Ser Leu Asn Ser Ile Pro Ser 35 40 45Gly Leu Thr Glu Ala Val Lys Ser
Leu Asp Leu Ser Asn Asn Arg Ile 50 55 60Thr Tyr Ile Ser Asn Ser Asp
Leu Gln Arg Cys Val Asn Leu Gln Ala65 70 75 80Leu Val Leu Thr Ser
Asn Gly Ile Asn Thr Ile Glu Glu Asp Ser Phe 85 90 95Ser Ser Leu Gly
Ser Leu Glu His Leu Asp Leu Ser Tyr Asn Tyr Leu 100 105 110Ser Asn
Leu Ser Ser Ser Trp Phe Lys Pro Leu Ser Ser Leu Thr Phe 115 120
125Leu Asn Leu Leu Gly Asn Pro Tyr Lys Thr Leu Gly Glu Thr Ser Leu
130 135 140Phe Ser His Leu Thr Lys Leu Gln Ile Leu Arg Val Gly Asn
Met Asp145 150 155 160Thr Phe Thr Lys Ile Gln Arg Lys Asp Phe Ala
Gly Leu Thr Phe Leu 165 170 175Glu Glu Leu Glu Ile Asp Ala Ser Asp
Leu Gln Ser Tyr Glu Pro Lys 180 185 190Ser Leu Lys Ser Ile Gln Asn
Val Ser His Leu Ile Leu His Met Lys 195 200 205Gln His Ile Leu Leu
Leu Glu Ile Phe Val Asp Val Thr Ser Ser Val 210 215 220Glu Cys Leu
Glu Leu Arg Asp Thr Asp Leu Asp Thr Phe His Phe Ser225 230 235
240Glu Leu Ser Thr Gly Glu Thr Asn Ser Leu Ile Lys Lys Phe Thr Phe
245 250 255Arg Asn Val Lys Ile Thr Asp Glu Ser Leu Phe Gln Val Met
Lys Leu 260 265 270Leu Asn Gln Ile Ser Gly Leu Leu Glu Leu Glu Phe
Asp Asp Cys Thr 275 280 285Leu Asn Gly Val Gly Asn Phe Arg Ala Ser
Asp Asn Asp Arg Val Ile 290 295 300Asp Pro Gly Lys Val Glu Thr Leu
Thr Ile Arg Arg Leu His Ile Pro305 310 315 320Arg Phe Tyr Leu Phe
Tyr Asp Leu Ser Thr Leu Tyr Ser Leu Thr Glu 325 330 335Arg Val Lys
Arg Ile Thr Val Glu Asn Ser Lys Val Phe Leu Val Pro 340 345 350Cys
Leu Leu Ser Gln His Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser 355 360
365Glu Asn Leu Met Val Glu Glu Tyr Leu Lys Asn Ser Ala Cys Glu Asp
370 375 380Ala Trp Pro Ser Leu Gln Thr Leu Ile Leu Arg Gln Asn His
Leu Ala385 390 395 400Ser Leu Glu Lys Thr Gly Glu Thr Leu Leu Thr
Leu Lys Asn Leu Thr 405 410 415Asn Ile Asp Ile Ser Lys Asn Ser Phe
His Ser Met Pro Glu Thr Cys 420 425 430Gln Trp Pro Glu Lys Met Lys
Tyr Leu Asn Leu Ser Ser Thr Arg Ile 435 440 445His Ser Val Thr Gly
Cys Ile Pro Lys Thr Leu Glu Ile Leu Asp Val 450 455 460Ser Asn Asn
Asn Leu Asn Leu Phe Ser Leu Asn Leu Pro Gln Leu Lys465 470 475
480Glu Leu Tyr Ile Ser Arg Asn Lys Leu Met Thr Leu Pro Asp Ala Ser
485 490 495Leu Leu Pro Met Leu Leu Val Leu Lys Ile Ser Arg Asn Ala
Ile Thr 500 505 510Thr Phe Ser Lys Glu Gln Leu Asp Ser Phe His Thr
Leu Lys Thr Leu 515 520 525Glu Ala Gly Gly Asn Asn Phe Ile Cys Ser
Cys Glu Phe Leu Ser Phe 530 535 540Thr Gln Glu Gln Gln Ala Leu Ala
Lys Val Leu Ile Asp Trp Pro Ala545 550 555 560Asn Tyr Leu Cys Asp
Ser Pro Ser His Val Arg Gly Gln Gln Val Gln 565 570 575Asp Val Arg
Leu Ser Val Ser Glu Cys His Arg Thr Ala Leu Val Ser 580 585 590Gly
Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu Thr Gly Val Leu 595 600
605Cys His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp
610 615 620Leu Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn
Ile Cys625 630 635 640Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp
Ala Tyr Trp Val Glu 645 650 655Asn Leu Met Val Gln Glu Leu Glu Asn
Phe Asn Pro Pro Phe Lys Leu 660 665 670Cys Leu His Lys Arg Asp Phe
Ile Pro Gly Lys Trp Ile Ile Asp Asn 675 680 685Ile Ile Asp Ser Ile
Glu Lys Ser His Lys Thr Val Phe Val Leu Ser 690 695 700Glu Asn Phe
Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser705 710 715
720His Phe Arg Leu Phe Glu Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu
725 730 735Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys
Lys Leu 740 745 750Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp
Pro Met Asp Glu 755 760 765Ala Gln Arg Glu Gly Phe Trp Val Asn Leu
Arg Ala Ala Ile Lys Ser 770 775 7802784PRTMus musculus 2Met Leu Arg
Ala Leu Trp Leu Phe Trp Ile Leu Val Ala Ile Thr Val1 5 10 15Leu Phe
Ser Lys Arg Cys Ser Ala Gln Glu Ser Leu Ser Cys Asp Ala 20 25 30Ser
Gly Val Cys Asp Gly Arg Ser Arg Ser Phe Thr Ser Ile Pro Ser 35 40
45Gly Leu Thr Ala Ala Met Lys Ser Leu Asp Leu Ser Phe Asn Lys Ile
50 55 60Thr Tyr Ile Gly His Gly Asp Leu Arg Ala Cys Ala Asn Leu Gln
Val65 70 75 80Leu Met Leu Lys Ser Ser Arg Ile Asn Thr Ile Glu Gly
Asp Ala Phe 85 90 95Tyr Ser Leu Gly Ser Leu Glu His Leu Asp Leu Ser
Asp Asn His Leu 100 105 110Ser Ser Leu Ser Ser Ser Trp Phe Gly Pro
Leu Ser Ser Leu Lys Tyr 115 120 125Leu Asn Leu Met Gly Asn Pro Tyr
Gln Thr Leu Gly Val Thr Ser Leu 130 135 140Phe Pro Asn Leu Thr Asn
Leu Gln Thr Leu Arg Ile Gly Asn Val Glu145 150 155 160Thr Phe Ser
Glu Ile Arg Arg Ile Asp Phe Ala Gly Leu Thr Ser Leu 165 170 175Asn
Glu Leu Glu Ile Lys Ala Leu Ser Leu Arg Asn Tyr Gln Ser Gln 180 185
190Ser Leu Lys Ser Ile Arg Asp Ile His His Leu Thr Leu His Leu Ser
195 200 205Glu Ser Ala Phe Leu Leu Glu Ile Phe Ala Asp Ile Leu Ser
Ser Val 210 215 220Arg Tyr Leu Glu Leu Arg Asp Thr Asn Leu Ala Arg
Phe Gln Phe Ser225 230 235 240Pro Leu Pro Val Asp Glu Val Ser Ser
Pro Met Lys Lys Leu Ala Phe 245 250 255Arg Gly Ser Val Leu Thr Asp
Glu Ser Phe Asn Glu Leu Leu Lys Leu 260 265 270Leu Arg Tyr Ile Leu
Glu Leu Ser Glu Val Glu Phe Asp Asp Cys Thr 275 280 285Leu Asn Gly
Leu Gly Asp Phe Asn Pro Ser Glu Ser Asp Val Val Ser 290 295 300Glu
Leu Gly Lys Val Glu Thr Val Thr Ile Arg Arg Leu His Ile Pro305 310
315 320Gln Phe Tyr Leu Phe Tyr Asp Leu Ser Thr Val Tyr Ser Leu Leu
Glu 325 330 335Lys Val Lys Arg Ile Thr Val Glu Asn Ser Lys Val Phe
Leu Val Pro 340 345 350Cys Ser Phe Ser Gln His Leu Lys Ser Leu Glu
Phe Leu Asp Leu Ser 355 360 365Glu Asn Leu Met Val Glu Glu Tyr Leu
Lys Asn Ser Ala Cys Lys Gly 370 375 380Ala Trp Pro Ser Leu Gln Thr
Leu Val Leu Ser Gln Asn His Leu Arg385 390 395 400Ser Met Gln Lys
Thr Gly Glu Ile Leu Leu Thr Leu Lys Asn Leu Thr 405 410 415Ser Leu
Asp Ile Ser Arg Asn Thr Phe His Pro Met Pro Asp Ser Cys 420 425
430Gln Trp Pro Glu Lys Met Arg Phe Leu Asn Leu Ser Ser Thr Gly Ile
435 440 445Arg Val Val Lys Thr Cys Ile Pro Gln Thr Leu Glu Val Leu
Asp Val 450 455 460Ser Asn Asn Asn Leu Asp Ser Phe Ser Leu Phe Leu
Pro Arg Leu Gln465 470 475 480Glu Leu Tyr Ile Ser Arg Asn Lys Leu
Lys Thr Leu Pro Asp Ala Ser 485 490 495Leu Phe Pro Val Leu Leu Val
Met Lys Ile Arg Glu Asn Ala Val Ser 500 505 510Thr Phe Ser Lys Asp
Gln Leu Gly Ser Phe Pro Lys Leu Glu Thr Leu 515 520 525Glu Ala Gly
Asp Asn His Phe Val Cys Ser Cys Glu Leu Leu Ser Phe 530 535 540Thr
Met Glu Thr Pro Ala Leu Ala Gln Ile Leu Val Asp Trp Pro Asp545 550
555 560Ser Tyr Leu Cys Asp Ser Pro Pro Arg Leu His Gly His Arg Leu
Gln 565 570 575Asp Ala Arg Pro Ser Val Leu Glu Cys His Gln Ala Ala
Leu Val Ser 580 585 590Gly Val Cys Cys Ala Leu Leu Leu Leu Ile Leu
Leu Val Gly Ala Leu 595 600 605Cys His His Phe His Gly Leu Trp Tyr
Leu Arg Met Met Trp Ala Trp 610 615 620Leu Gln Ala Lys Arg Lys Pro
Lys Lys Ala Pro Cys Arg Asp Val Cys625 630 635 640Tyr Asp Ala Phe
Val Ser Tyr Ser Glu Gln Asp Ser His Trp Val Glu 645 650 655Asn Leu
Met Val Gln Gln Leu Glu Asn Ser Asp Pro Pro Phe Lys Leu 660 665
670Cys Leu His Lys Arg Asp Phe Val Pro Gly Lys Trp Ile Ile Asp Asn
675 680 685Ile Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val
Leu Ser 690 695 700Glu Asn Phe Val Arg Ser Glu Trp Cys Lys Tyr Glu
Leu Asp Phe Ser705 710 715 720His Phe Arg Leu Phe Asp Glu Asn Asn
Asp Ala Ala Ile Leu Val Leu 725 730 735Leu Glu Pro Ile Glu Arg Lys
Ala Ile Pro Gln Arg Phe Cys Lys Leu 740 745 750Arg Lys Ile Met Asn
Thr Lys Thr Tyr Leu Glu Trp Pro Leu Asp Glu 755 760 765Gly Gln Gln
Glu Val Phe Trp Val Asn Leu Arg Thr Ala Ile Lys Ser 770 775
780321PRTHomo sapiens 3Lys Glu Glu Ser Ser Asn Gln Ala Ser Leu Ser
Cys Asp Arg Asn Gly1 5 10 15Ile Cys Lys Gly Ser20413PRTHomo sapiens
4Cys Ser Cys Glu Phe Leu Ser Phe Thr Gln Glu Gln Gln1 5
105586PRTHomo sapiens 5Met Pro His Thr Leu Trp Met Val Trp Val Leu
Gly Val Ile Ile Ser1 5 10 15Leu Ser Lys Glu Glu Ser Ser Asn Gln Ala
Ser Leu Ser Cys Asp Arg 20 25 30Asn Gly Ile Cys Lys Gly Ser Ser Gly
Ser Leu Asn Ser Ile Pro Ser 35 40 45Gly Leu Thr Glu Ala Val Lys Ser
Leu Asp Leu Ser Asn Asn Arg Ile 50 55 60Thr Tyr Ile Ser Asn Ser Asp
Leu Gln Arg Cys Val Asn Leu Gln Ala65 70 75 80Leu Val Leu Thr Ser
Asn Gly Ile Asn Thr Ile Glu Glu Asp Ser Phe 85 90 95Ser Ser Leu Gly
Ser Leu Glu His Leu Asp Leu Ser Tyr Asn Tyr Leu 100 105 110Ser Asn
Leu Ser Ser Ser Trp Phe Lys Pro Leu Ser Ser Leu Thr Phe 115 120
125Leu Asn Leu Leu Gly Asn Pro Tyr Lys Thr Leu Gly Glu Thr Ser Leu
130 135 140Phe Ser His Leu Thr Lys Leu Gln Ile Leu Arg Val Gly Asn
Met Asp145 150 155 160Thr Phe Thr Lys Ile Gln Arg Lys Asp Phe Ala
Gly Leu Thr Phe Leu 165 170 175Glu Glu Leu Glu Ile Asp Ala Ser Asp
Leu Gln Ser Tyr Glu Pro Lys 180 185 190Ser Leu Lys Ser Ile Gln Asn
Val Ser His Leu Ile Leu His Met Lys 195 200 205Gln His Ile Leu Leu
Leu Glu Ile Phe Val Asp Val Thr Ser Ser Val 210 215 220Glu Cys Leu
Glu Leu Arg Asp Thr Asp Leu Asp Thr Phe His Phe Ser225 230 235
240Glu Leu Ser Thr Gly Glu Thr Asn Ser Leu Ile Lys Lys Phe Thr Phe
245 250 255Arg Asn Val Lys Ile Thr Asp Glu Ser Leu Phe Gln Val Met
Lys Leu 260 265 270Leu Asn Gln Ile Ser Gly Leu Leu Glu Leu Glu Phe
Asp Asp Cys Thr 275 280 285Leu Asn Gly Val Gly Asn Phe Arg Ala Ser
Asp Asn Asp Arg Val Ile 290 295 300Asp Pro Gly Lys Val Glu Thr Leu
Thr Ile Arg Arg Leu His Ile Pro305 310 315 320Arg Phe Tyr Leu Phe
Tyr Asp Leu Ser Thr Leu Tyr Ser Leu Thr Glu 325 330 335Arg Val Lys
Arg Ile Thr Val Glu Asn Ser Lys Val Phe Leu Val Pro 340 345 350Cys
Leu Leu Ser Gln His Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser 355 360
365Glu Asn Leu Met Val Glu Glu Tyr Leu Lys Asn Ser Ala Cys Glu Asp
370 375 380Ala Trp Pro Ser Leu Gln Thr Leu Ile Leu Arg Gln Asn His
Leu Ala385 390 395 400Ser Leu Glu Lys Thr Gly Glu Thr Leu Leu Thr
Leu Lys Asn Leu Thr 405 410 415Asn Ile Asp Ile Ser Lys Asn Ser Phe
His Ser Met Pro Glu Thr Cys 420 425 430Gln Trp Pro Glu Lys Met Lys
Tyr Leu Asn Leu Ser Ser Thr Arg Ile 435 440 445His Ser Val Thr Gly
Cys Ile Pro Lys Thr Leu Glu Ile Leu Asp Val 450 455 460Ser Asn Asn
Asn Leu Asn Leu Phe Ser Leu Asn Leu Pro Gln Leu Lys465 470 475
480Glu Leu Tyr Ile Ser Arg Asn Lys Leu Met Thr Leu Pro Asp Ala Ser
485 490 495Leu Leu Pro Met Leu Leu Val Leu Lys Ile Ser Arg Asn Ala
Ile Thr 500 505 510Thr Phe Ser Lys Glu Gln Leu Asp Ser Phe His Thr
Leu Lys Thr Leu 515 520 525Glu Ala Gly Gly Asn Asn Phe Ile Cys Ser
Cys Glu Phe Leu Ser Phe 530 535 540Thr Gln Glu Gln Gln Ala Leu Ala
Lys Val Leu Ile Asp Trp Pro Ala545 550 555 560Asn Tyr Leu Cys Asp
Ser Pro Ser His Val Arg Gly Gln Gln Val Gln 565 570 575Asp Val Arg
Leu Ser Val Ser Glu Cys His 580 585
* * * * *
References