U.S. patent application number 16/131491 was filed with the patent office on 2019-01-03 for semaphorin 3a for treatment and prognosis of systemic lupus erythematosus.
This patent application is currently assigned to Medical Research & Development Fund for Health Services Bnai Zion Medical Center. The applicant listed for this patent is Medical Research & Development Fund for Health Services Bnai Zion Medical Center. Invention is credited to Elias Toubi, Zahava Vadasz.
Application Number | 20190000921 16/131491 |
Document ID | / |
Family ID | 52021734 |
Filed Date | 2019-01-03 |
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United States Patent
Application |
20190000921 |
Kind Code |
A1 |
Vadasz; Zahava ; et
al. |
January 3, 2019 |
SEMAPHORIN 3A FOR TREATMENT AND PROGNOSIS OF SYSTEMIC LUPUS
ERYTHEMATOSUS
Abstract
The subject matter relates to Semaphorin 3A (Sema3A) and its use
in treatment and prognosis of Systemic Lupus Erythematosus (SLE).
Provided are, inter-alia, methods of treating a subject afflicted
with SLE, comprising administering to the subject a pharmaceutical
composition comprising isolated Sema3A. Further provided are
methods for prognosis of SLE, comprising measuring Sema3A serum
concentration in a subject in need thereof.
Inventors: |
Vadasz; Zahava; (Haifa,
IL) ; Toubi; Elias; (Haifa, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medical Research & Development Fund for Health Services Bnai
Zion Medical Center |
Haifa |
|
IL |
|
|
Assignee: |
Medical Research & Development
Fund for Health Services Bnai Zion Medical Center
Haifa
IL
|
Family ID: |
52021734 |
Appl. No.: |
16/131491 |
Filed: |
September 14, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14897061 |
Dec 9, 2015 |
10105413 |
|
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PCT/IL2013/050504 |
Jun 13, 2013 |
|
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16131491 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/4716 20130101;
A61K 45/06 20130101; G01N 2800/104 20130101; G01N 33/564 20130101;
G01N 2800/52 20130101; A61K 38/1774 20130101; A61K 38/17 20130101;
G01N 2333/4703 20130101; G01N 33/68 20130101; A61K 38/1709
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; G01N 33/68 20060101 G01N033/68; A61K 45/06 20060101
A61K045/06; G01N 33/564 20060101 G01N033/564 |
Claims
1. A method for determining efficacy of a treatment for Systemic
Lupus Erythematosus in a subject in need thereof, the method
comprising: making a first measurement of serum Semaphorin 3A
concentration in a subject in need thereof prior to administration
of a treatment for Systemic Lupus Erythematosus to the subject;
making a second measurement of serum Semaphorin 3A concentration in
the subject following said treatment; and comparing said first
measurement and said second measurement, wherein an increase in
serum Semaphorin 3A concentration from the first to the second
measurement is indicative of said treatment being efficacious.
2. The method of claim 1, wherein said efficacy is further
indicated by at least one clinical outcome selected from the group
consisting of: an improvement in renal function, a decrease in
anti-dsDNA antibody concentration in the serum, a decrease in
anti-Cardiolipin antibody concentration in the serum, an increase
in serum concentration of complement factor C3 and an increase in
serum concentration of complement factor C4.
3. The method of claim 1, wherein said efficacy is further
indicated by a decrease in the Systemic Lupus Erythematosus Disease
Activity Index (SLEDAI) value of said subject.
4. The method of claim 1, wherein said treatment for Systemic Lupus
Erythematosus is selected from the group consisting of: a
corticosteroid, a cytotoxic drug, a non-steroidal anti-inflammatory
drug, an anti-malarial drug, a disease-modifying anti-rheumatic
drug, an immunosuppressive drug, an analgesic, intravenous
immunoglobulins and a combination thereof.
5. The method of claim 1, wherein said increase in serum Semaphorin
3A concentration is an increase of at least 10%.
6. The method of claim 1, wherein said Semaphorin 3A has a sequence
as set forth by SEQ ID NO: 1.
7. A method for prognosis of change in Systemic Lupus Erythematosus
disease activity in a subject in need thereof, the method
comprising: making a first measurement of serum Semaphorin 3A
concentration in a subject in need thereof; making a second
measurement of serum Semaphorin 3A concentration in the subject at
a later time point than said first measurement; and comparing said
first measurement and said second measurement; wherein an increase
in serum Semaphorin 3A concentration from the first to the second
measurement is indicative of a decrease in disease activity; and
wherein a decrease in serum Semaphorin 3A concentration from the
first to the second measurement is indicative of an increase in
disease activity.
8. The method of claim 7, wherein said Semaphorin 3A has a sequence
as set forth by SEQ ID NO: 1.
9. The method of claim 7, wherein said decrease in disease activity
is characterized by at least one clinical effect selected from the
group consisting of: an improvement in renal function, a decrease
in anti-dsDNA antibody concentration in the serum, a decrease in
anti-Cardiolipin antibody concentration in the serum, an increase
in serum concentration of complement factor C3 and an increase in
serum concentration of complement factor C4.
10. The method of claim 7, wherein said increase in disease
activity is characterized by at least one clinical effect selected
from the group consisting of: deterioration in renal function, an
increase in anti-dsDNA antibody concentration in the serum, an
increase in anti-Cardiolipin antibody concentration in the serum, a
decrease in serum concentration of complement factor C3 and a
decrease in serum concentration of complement factor C4.
11. The method of claim 7, wherein said decrease in disease
activity is evidenced by a decrease in the value of the Systemic
Lupus Erythematosus Disease Activity Index of said subject.
12. The method of claim 7, wherein said increase in disease
activity is evidenced by an increase in the value of the Systemic
Lupus Erythematosus Disease Activity Index of said subject.
13. The method of claim 7, wherein a measurement of up to 50 ng/ml
is indicative of Lupus Nephritis.
Description
SEQUENCE LISTING
[0001] The Sequence Listing submitted in text format (.txt) filed
on Sep. 14, 2018, named "SequenceListing.txt", created on Sep. 14,
2018 (16.2 KB), is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to Semaphorin 3A (Sema3A) and
its use in treatment and prognosis of Systemic Lupus Erythematosus
(SLE). The present invention provides methods of treating a subject
afflicted with SLE, comprising administering to the subject a
pharmaceutical composition comprising Sema3A, and methods for
prognosis of SLE, comprising measuring Sema3A serum concentration
in a subject in need thereof.
BACKGROUND OF THE INVENTION
Systemic Lupus Erythematosus (SLE)
[0003] Autoimmune diseases arise following an autoimmune response
of the body against substances and tissues normally present in the
body, resulting in continuous production of autoantibodies.
Autoantibodies are able to activate the complement system and are
deposited in many sites, thus inducing multi-organ inflammatory
reactions and possibly damage to organs such as the kidneys. More
than 5% of the world's population suffers from at least one of many
autoimmune diseases, such as lupus, rheumatoid arthritis,
scleroderma and others.
[0004] Systemic Lupus Erythematosus (SLE) is an autoimmune disease
manifested as a chronic syndrome with a multi-factorial etiology
and multiple symptoms affecting many organs such as the skin, the
kidneys and the brain. The multifactorial complexity of SLE
includes an overproduction of B cell activating factor (BAFF), an
escape of auto-reactive B cells from apoptosis and a dis-balanced
production of various inflammatory and protective cytokines. Severe
SLE involves glomerulonephritis, complications in the central
nervous system, and recurrent thrombosis.
[0005] Several symptoms that are associated with SLE include: an
increase in anti-cardiolipin auto-antibodies directed against
cardiolipin present in the mitochondrial inner-membrane, an
increase in anti-double-stranded DNA (anti-dsDNA) antibodies, a
decrease in complement-system component concentration and Lupus
Nephritis (LN)--an inflammation of the kidney leading to defects in
renal function and possibly renal failure. Lupus Nephritis is often
characterized by glomerulonephritis--an inflammation of the kidney
glomeruli.
[0006] The disease course of SLE is unpredictable, comprising
periods of illness (called flares) alternating with remissions and
displaying symptoms that vary widely. In order to evaluate SLE
disease activity, a scoring index is commonly used. The Systemic
Lupus Erythematosus Disease Activity Index (SLEDAI) is the most
commonly used scoring system to evaluate SLE disease activity
(Bombardier C. et al., 1992, Arthritis and Rheumatism,
35(6):630-640). In order to arrive at a SLEDAI score, each patient
is examined for the presence of 24 clinical characteristics in the
last 10 days. Each clinical characteristic is assigned a value and
the sum of these values is the patient's SLEDAI score.
[0007] There is no cure for SLE, thus treatment is focused on
reducing the severity of symptoms and/or on prevention of symptoms.
For many years, standard therapy for SLE included anti-malarials,
steroids and immunosuppressive drugs. Though efficient in improving
quality of life, survival and well-being, these treatments still
induce many undesired side effects.
[0008] Publication EP2123280 discloses an agent for prevention
and/or treatment of systemic lupus erythematosus, which comprises,
in combination,
2-benzyl-5-(4-chlorophenyl)-6-[4-(methylthio)phenyl]-2H-pyrida
zine-3-one or a solvate thereof and a corticosteroid.
[0009] Publication WO2004/093647 discloses a method for identifying
or monitoring SLE in an individual, including quantitating
complement component C4d on the surfaces of platelets and comparing
the amounts of C4d to reference levels of C4d on platelets of
individuals without SLE and/or on platelets of the individual
obtained at a different time.
Semaphorins and Semaphorin 3A
[0010] Semaphorins are a family of membrane bound and soluble
proteins classified into eight sub-classes based on their
structural domains. Semaphorins mainly regulate focal adhesion
assembly/disassembly and induce cytoskeletal remodeling, thus
affecting cell shape, cell attachment to the extracellular matrix,
cell motility, and cell migration. Although Semaphorins were
originally identified as affecting axon guidance during development
of the nervous system, they are now thought to fulfill diverse
physiological roles including organogenesis, vascularization,
angiogenesis, neuronal apoptosis, and neoplastic transformation.
Additionally, recent studies pointed to the involvement of
Neuropilin-1 and certain Semaphorins in the regulation of the
immune system, and thus these Semaphorins are denoted "immune
Semaphorins" (Kikutani H. et al., 2007, Advances in Immunology,
93:121-143).
[0011] The seven class-3 Semaphorins (Sema3s), designated by the
letters A-G, are the only vertebrate secreted Semaphorins.
Neuropilins (Nrps) and the type A/D family Plexins (Plexin-A1, -A2,
and -A3, and Plexin-D1) act as receptors for Sema3s. Each Sema3
family member shows distinct binding preference for Nrps. Each
Sema3-Nrp complex associates with specific plexins to mediate
downstream signaling. Most membrane-bound vertebrate Semaphorins
directly bind plexins, while class-3 Semaphorins require
Neuropilins as obligate co-receptors.
[0012] Semaphorin 3A (Sema3A), a class-3 secreted member of the
Semaphorin family, has been established as an axonal guidance
factor during development. Interestingly, several lines of evidence
suggest that Sema3A also affects immune cell functions. Sema3A has
been shown to be expressed by activated T cells and inhibit T cell
proliferation and cytokine secretion (Catalano, A et al, 2006,
Blood 107: 3321-3329; Lepelletier, Y. et al., 2006, Eur. J.
Immunol. 36: 1782-1793). Moreover, the expression of Sema3A,
Neuropilin 1 (NP-1), Neuropilin 2 (NP-2), and Plexins was found to
be increased on differentiating macrophages and on activated T
cells (Ji JD et al., 2009, Human Immunol., 70(4): 211-7).
Additionally, Neuropilin-1 expression on regulatory T cells has
been shown to enhance interactions with immature dendritic cells
(DCs) during antigen recognition, resulting in higher sensitivity
to limiting amounts of antigen (Sarris, M. et al., 2008, Immunity,
28: 402-13).
[0013] A recent study has shown that overexpression of Sema3A in a
mouse model of collagen-induced arthritis resulted in reduced
incidence, disease severity, and articular inflammation. Moreover,
in line with results in arthritic mice, the study showed a
defective Sema3A expression in CD4.sup.+ T cells derived from
patients with rheumatoid arthritis (Catalano A. et al., 2010, J.
Immunol., 185: 6373-83).
[0014] In another study, kidney biopsies from lupus
glomerulonephritis (LGN) patients showed stronger staining with
anti-NP-1, anti-Semaphorin 3A and anti-Semaphorin 4A antibodies as
compared with either normal biopsies or biopsies from patients with
primary nephropathy and proteinuria (Vadasz Z. et al., 2011, Lupus,
20:1466-1473). A subsequent study has shown that Sema 3A serum
levels in SLE patients are significantly lower than in healthy
individuals (Vadasz Z. et al, 2012, Arthritis Research &
Therapy, 14:R146).
[0015] U.S. Application Publication No. 2012/0251539 discloses a
method of treating an immune-related disorder in a subject,
comprising administering to the subject an effective amount of a
Sema3A inhibitor, resulting in reduced Sema3A activity in the
subject.
[0016] There is still an unmet need, however, for a safe and
effective treatment for Systemic Lupus Erythematosus.
SUMMARY OF THE INVENTION
[0017] The present invention provides methods of treatment for
Systemic Lupus Erythematosus (SLE), comprising administration of a
pharmaceutical composition comprising isolated Semaphorin 3A to a
subject in need thereof. The present invention further provides
methods for determining the efficiency of a treatment for Systemic
Lupus Erythematosus and methods for prognosis of change in Systemic
Lupus Erythematosus disease activity.
[0018] The present invention is based in part on the unexpected
discovery that serum concentration of Semaphorin 3A is inversely
correlated to Systemic Lupus Erythematosus disease activity. The
present invention if further based on the unexpected discovery that
incubation of B cells derived from SLE patients with Semaphorin 3A
containing media resulted in a decrease in Toll-Like Receptor 9
(TLR-9) expression on the cells, as exemplified herein below. As
known in the art, TLR-9 expression on B-cells is associated with
production of IL-10 and IL-6 cytokines and production of anti-dsDNA
antibodies in SLE patients.
[0019] According to one aspect, the present invention provides a
method for treating Systemic Lupus Erythematosus, the method
comprising administering to a subject in need thereof a
pharmaceutical composition comprising a therapeutically effective
amount of isolated Semaphorin 3A.
According to some embodiments, Semaphorin 3A has a sequence having
at least 90% homology to human Semaphorin 3A as set forth by SEQ ID
NO: 1. According to some embodiments, Semaphorin 3A has a sequence
as set forth by SEQ ID NO: 1. According to some embodiments, a
subject in need thereof is a subject afflicted by Systemic Lupus
Erythematosus.
[0020] According to some embodiments, treating a subject using a
pharmaceutical composition comprising Semaphorin 3A results in at
least one clinical outcome selected from the group consisting of:
an improvement in renal function, a decrease in anti-dsDNA antibody
concentration in the serum, a decrease in anti-Cardiolipin antibody
concentration in the serum, an increase in serum concentration of
complement factor C3 and an increase in serum concentration of
complement factor C4. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
treating a subject using a pharmaceutical composition comprising
isolated Semaphorin 3A results in a decrease in the Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI) value of the
subject.
[0021] According to some embodiments, administering to a subject in
need thereof is by a route selected from the group consisting of:
intravenous, intraarterial, subcutaneous and via direct injection
into a tissue or an organ. Each possibility represents a separate
embodiment of the present invention.
[0022] According to some embodiments, treating a subject using a
pharmaceutical composition comprising isolated Semaphorin 3A
further comprises administering an additional Systemic Lupus
Erythematosus treatment to the subject. According to some
embodiments, the Systemic Lupus Erythematosus treatment is selected
from the group consisting of: a corticosteroid, a cytotoxic drug, a
non-steroidal anti-inflammatory drug, an anti-malarial drug, a
disease-modifying anti-rheumatic drug, an immunosuppressive drug,
an analgesic, intravenous immunoglobulins and a combination
thereof. Each possibility represents a separate embodiment of the
present invention.
[0023] According to another aspect, the present invention provides
a method for determining efficacy of a treatment for Systemic Lupus
Erythematosus in a subject in need thereof, the method comprising:
making a first measurement of serum Semaphorin 3A concentration in
a subject in need thereof prior to administration of a treatment
for Systemic Lupus Erythematosus to the subject; making a second
measurement of serum Semaphorin 3A concentration in the subject
following the treatment; and comparing the first measurement and
the second measurement, wherein an increase in serum Semaphorin 3A
concentration from the first to the second measurement is
indicative of the treatment being efficacious.
[0024] According to another aspect, the present invention provides
a method for prognosis of change in Systemic Lupus Erythematosus
disease activity in a subject in need thereof, the method
comprising: making a first measurement of serum Semaphorin 3A
concentration in a subject in need thereof; making a second
measurement of serum Semaphorin 3A concentration in the subject at
a later time point than said first measurement; and comparing said
first measurement and said second measurement; wherein an increase
in serum Semaphorin 3A concentration from the first to the second
measurement is indicative of a decrease in disease activity; and
wherein a decrease in serum Semaphorin 3A concentration from the
first to the second measurement is indicative of an increase in
disease activity.
[0025] According to some embodiments, the efficacy of a treatment
for Systemic Lupus Erythematosus is indicated by at least one
clinical outcome selected from the group consisting of: an
improvement in renal function, a decrease in anti-dsDNA antibody
concentration in the serum, a decrease in anti-Cardiolipin antibody
concentration in the serum, an increase in serum concentration of
complement factor C3 and an increase in serum concentration of
complement factor C4. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
the efficacy of a treatment for Systemic Lupus Erythematosus is
indicated by a decrease in the Systemic Lupus Erythematosus Disease
Activity Index (SLEDAI) value of the treated subject. According to
some embodiments, the increase in serum Semaphorin 3A concentration
following an efficacious treatment for SLE is an increase of at
least 10%. According to some embodiments, the increase in serum
Semaphorin 3A concentration following an efficacious treatment for
SLE is an increase to a level of at least 50 ng/ml.
[0026] According to some embodiments, a decrease in SLE disease
activity is characterized by at least one clinical effect selected
from the group consisting of: an improvement in renal function, a
decrease in anti-dsDNA antibody concentration in the serum, a
decrease in anti-Cardiolipin antibody concentration in the serum,
an increase in serum concentration of complement factor C3 and an
increase in serum concentration of complement factor C4. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, a decrease in SLE disease
activity is evidenced by a decrease in the value of the Systemic
Lupus Erythematosus Disease Activity Index of the subject.
[0027] According to some embodiments, an increase in SLE disease
activity is characterized by at least one clinical effect selected
from the group consisting of: deterioration in renal function, an
increase in anti-dsDNA antibody concentration in the serum, an
increase in anti-Cardiolipin antibody concentration in the serum, a
decrease in serum concentration of complement factor C3 and a
decrease in serum concentration of complement factor C4. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, an increase in SLE
disease activity is evidenced by an increase in the value of the
Systemic Lupus Erythematosus Disease Activity Index of the
subject.
[0028] According to some embodiments, serum Semaphorin 3A
concentration measurement of up to 50 ng/ml is indicative of Lupus
Nephritis.
[0029] Further embodiments, features, advantages and the full scope
of applicability of the present invention will become apparent from
the detailed description and drawings given hereinafter. However,
it should be understood that the detailed description, while
indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 is a dot plot comparing Sema3A serum concentration in
Systemic Lupus Erythematosus patients (SLE), Rheumatoid Arthritis
patients (RA), as a disease control group, and healthy subjects
(Control).
[0031] FIGS. 2(A-C) depicts (A) a dot plot demonstrating the
negative correlation between Sema3A serum concentration and the
SLEDAI value in SLE patients, (B) bar graphs comparing the
percentages of SLE patients having kidney involvement (LN+) or not
having kidney involvement (LN-) amongst patients having less than
50 ng/ml serum Sema3A (Semaphorin 3A <50%) and patients having
more than 50 ng/ml serum Sema3A (Semaphorin 3A >50%), and (C)
bar graphs comparing the percentages of SLE patients having
anti-cardiolipin antibodies (anti-cardiolipin +) or not having
anti-cardiolipin antibodies (anti-cardiolipin -) amongst patients
having less than 50 ng/ml serum Sema3A (Semaphorin 3A <50%) and
patients having more than 50 ng/ml serum Sema3A (Semaphorin 3A
>50%).
[0032] FIG. 3(A-B) are dot plots demonstrating the positive
correlation between serum Sema3A concentration (ng/ml) in SLE
patients and complement C3 (A) or C4 (B) levels (mg/ml).
[0033] FIG. 4 depicts a bar graph comparing Sema3A expression on
CD19.sup.+CD25.sup.high B-Cells obtained from SLE patients and
healthy controls, as evaluated by Mean Fluorescent Intensity
(MFI).
[0034] FIG. 5 shows a dot plot comparing the percentage of
Toll-Like-Receptor 9 (TLR-9) expressing B cells out of B cells that
were isolated from SLE patients and treated either with Sema3A
containing medium (denoted as "After Sema3A") or cell culture
medium (denoted as "Baseline").
[0035] FIG. 6(A-E) show flow cytometry scatter micrographs plotting
CD19 positive B-cells vs. CD25 positive B-Cells (A,B) and
micrographs representing Sema3A expression on (C)
CD19.sup.-CD25.sup.low B-cells, (D) CD19.sup.+CD25.sup.low B cells
and (E) CD19.sup.+CD25.sup.high B cells.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention provides, according to one aspect, a
method for treating Systemic Lupus Erythematosus (SLE), the method
comprising administering to a subject in need thereof a
pharmaceutical composition comprising a therapeutically effective
amount of isolated Semaphorin 3A.
[0037] According to some embodiments, the present invention
provides a pharmaceutical composition comprising isolated
Semaphorin 3A for use in treating Systemic Lupus Erythematosus
(SLE). According to some embodiments, the present invention
provides a pharmaceutical composition comprising isolated
Semaphorin 3A for use in treating a subject afflicted with Systemic
Lupus Erythematosus (SLE).
[0038] As used herein, the terms "Systemic Lupus Erythematosus" and
"SLE" are used interchangeably. According to some embodiments,
treating SLE refers to ameliorating and/or preventing at least one
clinical symptom of SLE. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
treating SLE refers to ameliorating preventing at least one
clinical symptom of SLE. According to other embodiments, treating
SLE refers to ameliorating and/or preventing symptoms other than
symptoms of Lupus Nephritis. Each possibility represents a separate
embodiment of the present invention. As used herein, the term
"symptom" and "clinical symptom" are used interchangeably.
[0039] According to some embodiments, treating SLE refers to
inducing a decrease in disease activity. According to some
embodiments, treating SLE refers to inducing a decrease in disease
activity as evidenced by a decrease in Systemic Lupus Erythematosus
Disease Activity Index (SLEDAI) value in the treated subject. It is
to be noted that a lower SLEDAI value is indicative of lower SLE
disease activity.
[0040] According to some embodiments, treating SLE refers to
shortening a flare period present at the time of treatment.
According to some embodiments, treating SLE refers to shortening
flare periods. As used herein, the term "flare period" refers to a
time period in which at least one SLE symptom is manifested.
According to some embodiments, treating SLE refers to increasing
serum concentration of Semaphorin 3A. According to some
embodiments, treating SLE refers to increasing Semaphorin 3A serum
concentration to about 50 ng/ml or higher. Each possibility
represents a separate embodiment of the present invention. As used
herein, the term "about" refers to +/-10%, preferably +/-5%, most
preferably +/-1%. Each possibility represents a separate embodiment
of the present invention. According to some embodiments, treating
SLE refers to increasing serum concentration of Semaphorin 3A to at
least 50 ng/ml in a subject in need thereof. According to some
embodiments, treating SLE refers to increasing serum concentration
of Semaphorin 3A by at least 10%, preferably by at least 20%. Each
possibility represents a separate embodiment of the present
invention. Without wishing to be bound by theory or mechanism, the
increase in Semaphorin 3A serum concentration following treating
according to the present invention is dependent on the specific
physiological parameters of each subject. Therefore, an increase in
Semaphorin 3A of less than 10% may be accounted as treating
according to certain embodiments.
[0041] According to some embodiments, a subject in need thereof is
a subject afflicted with SLE. According to other embodiments, a
subject in need thereof is other than a subject afflicted with
Lupus Nephritis.
[0042] Symptoms of SLE may include, but are not limited to,
symptoms selected from the group consisting of: systemic symptoms,
skin and mucosal symptoms, muscular and articular symptoms, renal
symptoms, neurological and/or neuropsychiatric symptoms,
cardiovascular symptoms, pulmonary symptoms, gastrointestinal
symptoms, hematopoietic symptoms and a combination thereof. Each
possibility represents a separate embodiment of the present
invention.
[0043] According to some embodiments, treating SLE refers to
ameliorating and/or preventing at least one symptom caused by SLE
selected from the group consisting of: systemic symptoms, skin and
mucosal symptoms, muscular and articular symptoms, renal symptoms,
neurological and/or neuropsychiatric symptoms, cardiovascular
symptoms, pulmonary symptoms, gastrointestinal symptoms,
hematopoietic symptoms and a combination thereof. Each possibility
represents a separate embodiment of the present invention.
[0044] According to some embodiments, systemic symptoms comprise
symptoms selected from the group consisting of: systemic malaise,
fatigue, fever and a combination thereof. Each possibility
represents a separate embodiment of the present invention.
[0045] According to some embodiments, skin and mucosal symptoms
comprise symptoms selected from the group consisting of: malar rash
(butterfly erythema), discoid rash, chilblains, alopecia, mouth
ulcers and a combination thereof. Each possibility represents a
separate embodiment of the present invention.
[0046] According to some embodiments, muscular and articular
symptoms comprise symptoms selected from the group consisting of:
muscular pain, articular pain, arthritis and a combination thereof.
Each possibility represents a separate embodiment of the present
invention.
[0047] According to some embodiments, renal symptoms comprise
symptoms selected from the group consisting of: glomerulonephritis,
proteinuria, hematuria, renal failure, end-stage renal failure and
a combination thereof. Each possibility represents a separate
embodiment of the present invention.
[0048] As used herein, the terms "Lupus Nephritis" and "LN" are
used interchangeably and refer to SLE in which renal inflammation
is manifested. According to some embodiments, Lupus Nephritis
refers to SLE in which glomerulonephritis is manifested. As used
herein, the term "glomerulonephritis" refers to inflammation of
glomeruli in the kidney. According to some embodiments, SLE
patients with Lupus Nephritis develop renal failure. According to
some embodiments, Lupus Nephritis patients manifest at least one
symptom selected from the group consisting of: glomerulonephritis,
proteinuria, hematuria, elevated blood pressure, renal failure,
end-stage renal failure and a combination thereof. Each possibility
represents a separate embodiment of the present invention.
According to some embodiments, treating Lupus Nephritis results in
amelioration and/or prevention of at least one symptom selected
from the group consisting of: glomerulonephritis, proteinuria,
hematuria, renal failure, end-stage renal failure and a combination
thereof. Each possibility represents a separate embodiment of the
present invention.
[0049] As used herein, the terms "patient" and "subject" are used
interchangeably.
[0050] According to some embodiments, neurological and/or
neuropsychiatric symptoms comprise symptoms selected from the group
consisting of: headaches, cognitive dysfunction, mood disorder,
cerebrovascular disease, seizures, polyneuropathy, anxiety
disorder, psychosis, intracranial hypertension syndrome, acute
confusional state, Guillain-Barre syndrome, aseptic meningitis,
autonomic disorder, demyelinating syndrome, mononeuropathy,
movement disorder, myasthenia gravis-like symptoms, myelopathy,
cranial neuropathy, plexopathy, blood-brain barrier damage,
depression and a combination thereof. Each possibility represents a
separate embodiment of the present invention.
[0051] According to some embodiments, cardiovascular symptoms
comprise symptoms selected from the group consisting of:
pericarditis, myocarditis, endocarditis, atherosclerosis,
tachycardia, arrhythmia, aortic valve insufficiency, mitral valve
insufficiency, thrombophlebitis and a combination thereof. Each
possibility represents a separate embodiment of the present
invention.
[0052] According to some embodiments, pulmonary symptoms comprise
symptoms selected from the group consisting of: pleuritis, pleural
effusion, lupus pneumonitis, chronic diffuse interstitial lung
disease, pulmonary hypertension, pulmonary emboli, pulmonary
hemorrhage, shrinking lung syndrome and a combination thereof. Each
possibility represents a separate embodiment of the present
invention.
[0053] According to some embodiments, gastrointestinal symptoms
comprise symptoms selected from the group consisting of: mesenteric
vasculitis, lupus peritonitis, abdominal pain and a combination
thereof. Each possibility represents a separate embodiment of the
present invention.
[0054] According to some embodiments, hematopoietic symptoms
comprise symptoms selected from the group consisting of: anemia,
low platelet and/or white blood cell count, anti-phospholipid
antibody syndrome, presence of anti-cardiolipin antibodies,
presence of lupus-anticoagulant, presence of anti-dsDNA antibodies
and a combination thereof. Each possibility represents a separate
embodiment of the present invention.
[0055] According to some embodiments, symptoms of SLE include
reduced expression of Semaphorin 3A on CD19.sup.+CD25.sup.high B
cells, reduced expression of Neuropilin-1 on
CD19.sup.+CD25.sup.high B cells and a combination thereof. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, healthy subjects and SLE
patients show substantially similar expression level of Semaphorin
3A on T cells.
[0056] According to some embodiments, treating of SLE using the
composition of the invention results in at least one clinical
outcome selected from the group consisting of: an improvement in
renal function, a decrease in anti-dsDNA antibody concentration in
the serum, a decrease in anti-Cardiolipin antibody concentration in
the serum, an increase in serum concentration of complement factor
C3 and an increase in serum concentration of complement factor C4.
Each possibility represents a separate embodiment of the present
invention. Without wishing to be bound by any theory or mechanism,
administration of the composition of the invention to a subject
afflicted with SLE may result in a decrease in expression of
Toll-Like Receptor 9 and/or inflammatory cytokines, such as, but
not limited to, IL-1, IL-6, IL-17 and interferon-gamma, in
CD19.sup.+/CD27.sup.+ B cells of the subject. According to some
embodiments, the present invention provides a method for decreasing
Toll-Like Receptor 9 expression on CD19.sup.+/CD27.sup.+ B cells of
a subject afflicted with SLE, the method comprising administration
of a composition comprising therapeutically effective amount of
isolated Semaphorin 3A.
[0057] According to some embodiments, a therapeutically effective
amount refers to an amount sufficient to induce a decrease in SLE
disease activity. According to some embodiments, a therapeutically
effective amount refers to an amount sufficient to increase
Semaphorin 3A serum concentration by at least 10% in a subject in
need thereof. According to some embodiments, a therapeutically
effective amount refers to an amount sufficient to increase
Semaphorin 3A serum concentration to at least 50 ng/ml in a subject
in need thereof. According to some embodiments, a therapeutically
effective amount refers to an amount sufficient to ameliorate
and/or prevent at least one clinical symptom of SLE. Each
possibility represents a separate embodiment of the present
invention.
[0058] As used herein, the term "pharmaceutical composition",
"composition", "the composition of the invention" and "the
composition" are used interchangeably and refer to a composition
comprising isolated Semaphorin 3A. According to some embodiments,
Semaphorin3A is mammalian Semaphorin 3A. According to some
embodiments, Semaphorin3A is human Semaphorin 3A. According to some
embodiments, Semaphorin 3A is recombinant Semaphorin3A.
[0059] According to some embodiments, Semaphorin 3A refers to a
polypeptide having at least 90% homology to Semaphorin 3A.
According to some embodiments, Semaphorin 3A has a sequence having
at least 90% homology to human Semaphorin 3A as set forth by SEQ ID
NO: 1. According to some embodiments, a polypeptide having at least
90% homology to Semaphorin3A is about as functional as
Semaphorin3A. According to preferred embodiments, Semaphorin 3A as
used herein is human Semaphorin 3A having an amino-acid sequence as
set forth in SEQ ID NO: 1. The polynucleotide sequence as set forth
in SEQ ID NO: 2 corresponds to the cDNA encoding human Semaphorin
3A as set forth in SEQ ID NO: 1.
[0060] According to some embodiments, Semaphorin 3A according to
the present invention further comprises a protein tag. According to
some embodiments, Semaphorin3A comprises a protein tag upon
production but the tag is cleaved and/or removed from Semaphorin3A
prior to incorporation into the composition of the invention. Each
possibility represents a separate embodiment of the present
invention. Cleavage and/or removal of a tag may be performed by any
methods known in the art, such as, but not limited to, enzymatic
and/or chemical cleaving, so as long as Semaphorin 3A remains
functional. According to some embodiments, functional Semaphorin3A
refers to Semaphorin 3A which is able to reduce SLE disease
activity and/or ameliorate at least one SLE symptom. Each
possibility represents a separate embodiment of the present
invention.
[0061] As used herein, the term "protein tag" refers to a peptide
sequence bound to the N-terminus or C-terminus of a protein.
According to some embodiments, protein tags may comprise
glycoproteins. According to some embodiments, protein tags may be
used for separation and/or purification of the bound proteins. Each
possibility represents a separate embodiment of the present
invention. Non-limiting examples of protein tags are: Myc, Human
influenza hemaglutinin (HA), Flag, His, Gluthathione-S-Transferase
(GST) and a combination thereof. Each possibility represents a
separate embodiment of the present invention.
[0062] As used herein, the term "isolated" means either: 1)
separated from at least some of the components with which it is
usually associated in nature; 2) prepared or purified by a process
that involves the hand of man; and/or 3) not occurring in
nature.
[0063] According to some embodiments, isolated Semaphorin 3A as
disclosed herein may be produced by recombinant or chemical
synthetic methods. According to some embodiments, Semaphorin 3A as
disclosed herein may be produced by recombinant methods from
genetically-modified host cells. Any host cell known in the art for
the production of recombinant proteins may be used for the present
invention. In some embodiments, the host cell is a prokaryotic
cell. Representative, non-limiting examples of appropriate
prokaryotic hosts include bacterial cells, such as cells of
Escherichia coli and Bacillus subtilis. In other embodiments, the
host cell is a eukaryotic cell. In some exemplary embodiments, the
host cell is a fungal cell, such as yeast. Representative,
non-limiting examples of appropriate yeast cells include
Saccharomyces cerevisiae and Pichia pastoris. In additional
exemplary embodiments, the host cell is a plant cell.
[0064] Following are non-limiting examples of recombinant and
chemical synthetic methods suitable for production of Semaphorin
3A, according to the present invention.
Recombinant Expression
[0065] As used herein, the term "gene" has its meaning as
understood in the art. In general, a gene is taken to include gene
regulatory sequences (e.g. promoters, enhancers, etc.) and/or
intron sequences, in addition to coding sequences (open reading
frames).
[0066] As used herein, the terms "polypeptide" and "protein" are
used interchangeably and refer to a polymer of amino acid
residues.
[0067] As used herein, the term "DNA construct" refers to an
artificially assembled or isolated nucleic acid molecule which
comprises a gene of interest or a coding region of interest.
According to some embodiments, a gene of interest is a gene
encoding human Semaphorin 3A. According to some embodiments, a
coding region of interest is a coding region encoding Semaphorin
3A. According to some embodiments, a coding region of interest is a
coding region encoding for human Semaphorin 3A as set forth in SEQ
ID NO:2.
[0068] As used herein, the term "vector" refers to any recombinant
polynucleotide construct (such as a DNA construct) that may be used
for the purpose of transformation, i.e. the introduction of
heterologous DNA into a host cell. One exemplary type of vector is
a "plasmid" which refers to a circular double stranded DNA loop
into which additional DNA segments can be ligated. Another
exemplary type of vector is a viral vector, wherein additional DNA
segments can be ligated into the viral genome. Certain vectors are
capable of autonomous replication in a host cell into which they
are introduced.
[0069] As used herein, a "primer" defines an oligonucleotide which
is capable of annealing to (hybridizing with) a target nucleotide
sequence, thereby creating a double stranded region which can serve
as an initiation point for DNA synthesis under suitable
conditions.
[0070] As used herein, the terms "transformation" refers to the
introduction of foreign DNA into cells. The terms "transformants"
or "transformed cells" include the primary transformed cell and
cultures derived from that cell regardless to the number of
transfers. All progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same functionality as screened for in the originally
transformed cell are included in the definition of
transformants.
[0071] Semaphorin 3A may be synthesized by expressing a
polynucleotide molecule encoding Semaphorin 3A in a host cell, for
example, a microorganism cell transformed with the nucleic acid
molecule.
[0072] DNA sequences encoding wild type polypeptides, such as
Semaphorin 3A, may be isolated from any cell producing them, using
various methods well known in the art (see for example, Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold
Spring Harbor, N.Y., (2001)). For example, a DNA encoding the
wild-type polypeptide may be amplified from genomic DNA by
polymerase chain reaction (PCR) using specific primers, constructed
on the basis of the nucleotide sequence of the known wild type
sequence. Suitable techniques are well known in the art, described
for example in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159 and
4,965,188.
[0073] The genomic DNA may be extracted from the cell prior to the
amplification using various methods known in the art, see for
example, Marek P. M et al., "Cloning and expression in Escherichia
coli of Clostridium thermocellum DNA encoding p-glucosidase
activity", Enzyme and Microbial Technology Volume 9, Issue 8,
August 1987, Pages 474-478.
[0074] The isolated polynucleotide encoding the wild type
polypeptide may be cloned into a vector, such as, but not limited
to, the pET28a plasmid.
[0075] Upon isolation and cloning of the polynucleotide encoding
the wild type polypeptide, desired mutation(s) may be introduced by
modification at one or more base pairs, using methods known in the
art, such as for example, site-specific mutagenesis, cassette
mutagenesis, recursive ensemble mutagenesis and gene site
saturation mutagenesis. Methods are also well known for introducing
multiple mutations into a polynucleotide. For example, introduction
of two and/or three mutations can be performed using commercially
available kits, such as the QuickChange site-directed mutagenesis
kit (Stratagene).
[0076] An alternative method to producing a polynucleotide with a
desired sequence is the use of a synthetic gene. A polynucleotide
encoding a desired polypeptide may be prepared synthetically, for
example using the phosphoroamidite method (see, Beaucage et al.,
Curr Protoc Nucleic Acid Chem. 2001 May; Chapter 3: Unit 3.3;
Caruthers et al., Methods Enzymol 1987, 154:287-313).
[0077] The polynucleotide thus produced may then be subjected to
further manipulations, including one or more of purification,
annealing, ligation, amplification, digestion by restriction
endonucleases and cloning into appropriate vectors. The
polynucleotide may be ligated either initially into a cloning
vector, or directly into an expression vector that is appropriate
for its expression in a particular host cell type.
[0078] In the case of a fusion protein, or a protein fused with a
protein tag, different polynucleotides may be ligated to form one
polynucleotide. For example, different polynucleotides may be
ligated into linearized pET21a.
[0079] The polynucleotide encoding the polypeptide of the
invention, such as, but not limited to the polynucleotide encoding
human Semaphorin 3A (SEQ ID NO:2), may be incorporated into a wide
variety of expression vectors, which may be transformed into in a
wide variety of host cells.
[0080] Introduction of a polynucleotide into the host cell can be
effected by well-known methods, such as chemical transformation
(e.g. calcium chloride treatment), electroporation, conjugation,
transduction, calcium phosphate transfection, DEAE-dextran mediated
transfection, transvection, microinjection, cationic lipid-mediated
transfection, scrape loading, ballistic introduction and
infection.
[0081] Representative, non-limiting examples of appropriate hosts
include bacterial cells, such as cells of E. coli and Bacillus
subtilis.
[0082] The polypeptides may be expressed in any vector suitable for
expression. The appropriate vector is determined according to the
selected host cell. Vectors for expressing proteins in E. coli, for
example, include, but are not limited to, pET, pK233, pT7 and
lambda pSKF. Other expression vector systems are based on
betagalactosidase (pEX); maltose binding protein (pMAL); and
glutathione S-transferase (pGST).
[0083] The polypeptides may be designed to include a protein tag,
for example, a His-Tag (six consecutive histidine residues), which
can be isolated and purified by conventional methods.
[0084] Selection of a host cell transformed with the desired vector
may be accomplished using standard selection protocols involving
growth in a selection medium which is toxic to non-transformed
cells. For example, in the case of E. coli, it may be grown in a
medium containing an antibiotic selection agent; cells transformed
with the expression vector which further provides an antibiotic
resistance gene, will grow in the selection medium.
[0085] Upon transformation of a suitable host cell, and propagation
under conditions appropriate for protein expression, the
polypeptide may be identified in cell extracts of the transformed
cells. Transformed hosts expressing the polypeptide may be
identified by analyzing the proteins expressed by the host, for
example, using SDS-PAGE and comparing the gel to an SDS-PAGE gel
obtained from the host which was transformed with the same vector
but not containing a nucleic acid sequence encoding the desired
polypeptide.
[0086] The desired polypeptides which have been identified in cell
extracts may be isolated and purified by conventional methods,
including ammonium sulfate or ethanol precipitation, acid
extraction, salt fractionation, ion exchange chromatography,
hydrophobic interaction chromatography, gel permeation
chromatography, affinity chromatography, and combinations thereof.
The polypeptides of the invention may be produced as fusion
proteins, attached to an affinity purification protein tag, such as
a His-tag, in order to facilitate their rapid purification.
[0087] The isolated polypeptide may be analyzed for its various
properties, for example specific activity, using methods known in
the art. In a non-limiting example, isolated Semaphorin 3A may be
analyzed for its ability to reduce expression of Toll Like Receptor
9 (TLR-9) on memory CD19.sup.+/CD27.sup.+ B cells isolated from SLE
patients.
[0088] Conditions for carrying out the aforementioned procedures as
well as other useful methods are readily determined by those of
ordinary skill in the art (see for example, Current Protocols in
Protein Science, 1995 John Wiley & Sons).
[0089] A non-limiting example of recombinant production of
Semaphorin3A is disclosed by Kigel et al. (Kigel B. et al., 2008,
PLoS ONE, 3(9): e3287).
Synthetic Production:
[0090] Semaphorin 3A according to the present invention may also be
produced by synthetic means using well known techniques, such as
solid phase synthesis. Synthetic polypeptides may be produced using
commercially available laboratory peptide design and synthesis
kits. In addition, a number of available FMOC peptide synthesis
systems are available. Assembly of a polypeptide or fragment can be
carried out on a solid support using for example, an Applied
Biosystems, Inc. Model 431A automated peptide synthesizer. The
polypeptides may be made by either direct synthesis or by synthesis
of a series of fragments that can be coupled using other known
techniques.
[0091] Any suitable route of administration to a subject may be
used for the composition of the present invention, including but
not limited to, topical and systemic routes. According to some
embodiments, administering is administering systematically.
According to some embodiments, the composition is formulated for
systemic administration.
[0092] According to another embodiment, administration systemically
is through a parenteral route. According to some embodiments,
preparations of the composition of the invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions, each representing a separate embodiment
of the present invention. Non-limiting examples of non-aqueous
solvents or vehicles are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil and corn oil, gelatin, and
injectable organic esters such as ethyl oleate.
[0093] According to some embodiments, parenteral administration is
administration intravenously, intra-arterially, intramuscularly,
intraperitoneally, intradermally, intravitreally, or
subcutaneously. Each of the abovementioned administration routes
represents a separate embodiment of the present invention.
According to another embodiment, parenteral administration is
performed by bolus injection. According to another embodiment,
parenteral administration is performed by continuous infusion.
[0094] According to another embodiment, parenteral administration
is transmucosal administration. According to another embodiment,
transmucosal administration is transnasal administration. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art. The preferred mode of administration
will depend upon the particular indication being treated and will
be apparent to one of skill in the art.
[0095] According to another embodiment, systemic administration of
the composition is through injection. For administration through
injection, the composition may be formulated in an aqueous
solution, for example in a physiologically compatible buffer
including but not limited to Hank's solution, Ringer's solution, or
physiological salt buffer. Formulations for injection may be
presented in unit dosage forms, for example, in ampoules, or in
multi-dose containers with, optionally, an added preservative.
[0096] Aqueous injection suspensions may contain substances that
increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents that
increase the solubility of the active ingredients, to allow for the
preparation of highly concentrated solutions.
[0097] According to another embodiment, compositions formulated for
injection may be in the form of solutions, suspensions, dispersions
or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing, and/or
dispersing agents. Non-limiting examples of suitable lipophilic
solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acid esters such as ethyl oleate or
triglycerides.
[0098] According to another embodiment, the composition is
administered intravenously, and is thus formulated in a form
suitable for intravenous administration. According to another
embodiment, the composition is administered intra-arterially, and
is thus formulated in a form suitable for intra-arterial
administration. According to another embodiment, the composition is
administered intramuscularly, and is thus formulated in a form
suitable for intramuscular administration.
[0099] According to another embodiment, administration systemically
is through an enteral route. According to another embodiment,
administration through an enteral route is buccal administration.
According to another embodiment, administration through an enteral
route is oral administration. According to some embodiments, the
composition is formulated for oral administration.
[0100] According to some embodiments, oral administration is in the
form of hard or soft gelatin capsules, pills, capsules, tablets,
including coated tablets, dragees, elixirs, suspensions, liquids,
gels, slurries, syrups or inhalations and controlled release forms
thereof.
[0101] Suitable carriers for oral administration are well known in
the art. Compositions for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries as desired, to obtain tablets or dragee cores.
Non-limiting examples of suitable excipients include fillers such
as sugars, including lactose, sucrose, mannitol, or sorbitol,
cellulose preparations such as, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose,
and/or physiologically acceptable polymers such as
polyvinylpyrrolidone (PVP).
[0102] If desired, disintegrating agents, such as cross-linked
polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof,
such as sodium alginate, may be added. Capsules and cartridges of,
for example, gelatin, for use in a dispenser may be formulated
containing a powder mix of the composition of the invention and a
suitable powder base, such as lactose or starch.
[0103] Solid dosage forms for oral administration include capsules,
tablets, pill, powders, and granules. In such solid dosage forms,
the composition of the invention is admixed with at least one inert
pharmaceutically acceptable carrier such as sucrose, lactose, or
starch. Such dosage forms can also comprise, as it normal practice,
additional substances other than inert diluents, e.g., lubricating,
agents such as magnesium stearate. In the case of capsules, tablets
and pills, the dosage forms may also comprise buffering, agents.
Tablets and pills can additionally be prepared with enteric
coatings.
[0104] Liquid dosage forms for oral administration may further
contain adjuvants, such as wetting agents, emulsifying and
suspending agents, and sweetening, flavoring and perfuming agents.
According to some embodiments, enteral coating of the composition
is further used for oral or buccal administration. The term
"enteral coating", as used herein, refers to a coating which
controls the location of composition absorption within the
digestive system. Non-limiting examples for materials used for
enteral coating are fatty acids, waxes, plant fibers or
plastics.
[0105] According to some embodiments, administering is
administering topically. According to some embodiments, the
composition is formulated for topical administration. The term
"topical administration", as used herein, refers to administration
to body surfaces. Non-limiting examples of formulations for topical
use include cream, ointment, lotion, gel, foam, suspension, aqueous
or cosolvent solutions, salve and sprayable liquid form. Other
suitable topical product forms for the compositions of the present
invention include, for example, emulsion, mousse, lotion, solution
and serum.
[0106] According to some embodiments, administration of the
composition of the invention to a subject in need thereof is by a
route selected from the group consisting of: intravenous,
intraarterial, subcutaneous and via direct injection into a tissue
or an organ. Each possibility represents a separate embodiment of
the present invention. According to certain embodiments,
administration may be orally.
[0107] According to some embodiments, the method of treating SLE
according to the present invention further comprises administering
to the subject an additional treatment for SLE other than
administration of a pharmaceutical composition comprising isolated
Semaphorin 3A. According to some embodiments, treating SLE
according to the present invention comprises administration of a
pharmaceutical composition comprising isolated Semaphorin 3A and
administration of an additional treatment for SLE other than a
pharmaceutical composition comprising isolated Semaphorin 3A.
According to some embodiments, a treatment for SLE is selected from
the group consisting of: a corticosteroid, a cytotoxic drug, a
non-steroidal anti-inflammatory drug (NSAID), a disease-modifying
anti-rheumatic drug (DMARD), an anti-malarial drug, an
immunosuppressive drug, an analgesic, intravenous immunoglobulins
and a combination thereof. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
an additional treatment for SLE other than a pharmaceutical
composition comprising isolated Semaphorin 3A is selected from the
group consisting of: a corticosteroid, a cytotoxic drug, a
non-steroidal anti-inflammatory drug (NSAID), a disease-modifying
anti-rheumatic drug (DMARD), an anti-malarial drug, an
immunosuppressive drug, an analgesic, intravenous immunoglobulins
and a combination thereof. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
the composition of the invention further comprises a drug selected
from the group consisting of: a corticosteroid, a cytotoxic drug, a
non-steroidal anti-inflammatory drug, a disease-modifying
anti-rheumatic drug, an anti-malarial drug, an immunosuppressive
drug, an analgesic, immunoglobulins and a combination thereof. Each
possibility represents a separate embodiment of the present
invention. According to other embodiments, a treatment for SLE is a
treatment other than for Lupus Nephritis.
[0108] Non-limiting examples of disease-modifying anti-rheumatic
drugs (DMARDs) include, but are not limited to: abatacept,
adalimumab, azathioprine, chloroquine, hydroxychloroquine,
Cyclosporin A, etanercept, golimumab, infliximab, leflunomide,
methotrexate, minocycline, rituximab, sulfasalazine, Belimumab and
a combination thereof. Each possibility represents a separate
embodiment of the present invention.
[0109] According to some embodiments, the disease-modifying
anti-rheumatic drug (DMARD) is an antimalarial. Non-limiting
examples of antimalarial drugs include, but are not limited to:
Plaquenil, Quinine, Chloroquine, Amodiaquine, Pyrimethamine,
Proguanil, Mefloquine, Atovaquone, Primaquine, Artemisinin,
Halofantrine and a combination thereof. Each possibility represents
a separate embodiment of the present invention.
[0110] Non-limiting examples of cytotoxic drugs include, but are
not limited to: cyclophosphamide, mycophenolate and a combination
thereof. Each possibility represents a separate embodiment of the
present invention.
[0111] According to some embodiments, a non-steroidal
anti-inflammatory drug (NSAID) is selected from the group
consisting of: salicylates, propionic acid derivatives, acetic acid
derivatives, enolic acid derivatives (Oxicam), fenamic acid
derivatives (Fenamates), selective COX-2 inhibitors (Coxibs),
sulphonanilides and a combination thereof. Each possibility
represents a separate embodiment of the present invention.
According to some embodiments, the non-steroidal anti-inflammatory
drug (NSAID) is a selective COX-2 inhibitor (Coxibs), such as, but
not limited to: Celecoxib, Rofecoxib, Valdecoxib, Parecoxib,
Lumiracoxib, Etoricoxib and a combination thereof. Each possibility
represents a separate embodiment of the present invention.
[0112] According to another aspect, the present invention provides
a method for determining efficacy of a treatment for Systemic Lupus
Erythematosus in a subject in need thereof, the method comprising:
making a first measurement of serum Semaphorin 3A concentration in
a subject in need thereof; administering a treatment for Systemic
Lupus Erythematosus to the subject; making a second measurement of
serum Semaphorin 3A concentration in the subject following the
treatment; and comparing the first measurement and the second
measurement, wherein an increase in serum Semaphorin 3A
concentration from the first to the second measurement is
indicative of the treatment being efficacious.
[0113] According to some embodiments, the present invention
provides a method for determining efficacy of a treatment for Lupus
Nephritis in a subject in need thereof, the method comprising:
making a first measurement of serum Semaphorin 3A concentration in
a subject in need thereof; administering a treatment for Systemic
Lupus Erythematosus and/or Lupus Nephritis to the subject; making a
second measurement of serum Semaphorin 3A concentration in the
subject following the treatment; and comparing the first
measurement and the second measurement, wherein an increase in
serum Semaphorin 3A concentration from the first to the second
measurement is indicative of the treatment being efficacious. Each
possibility represents a separate embodiment of the present
invention.
[0114] According to another aspect, the present invention provides
a method for determining efficacy of a treatment for Systemic Lupus
Erythematosus in a subject in need thereof, the method comprising:
making a first measurement of serum Semaphorin 3A concentration in
a subject in need thereof prior to administration of a treatment
for Systemic Lupus Erythematosus to the subject; making a second
measurement of serum Semaphorin 3A concentration in the subject
following the treatment; and comparing the first measurement and
the second measurement, wherein an increase in serum Semaphorin 3A
concentration from the first to the second measurement is
indicative of the treatment being efficacious.
[0115] According to some embodiments, the present invention
provides a method for determining efficacy of a treatment for Lupus
Nephritis in a subject in need thereof, the method comprising:
making a first measurement of serum Semaphorin 3A concentration in
a subject in need thereof prior to administration of a treatment
for Systemic Lupus Erythematosus and/or Lupus Nephritis to the
subject; making a second measurement of serum Semaphorin 3A
concentration in the subject following the treatment; and comparing
the first measurement and the second measurement, wherein an
increase in serum Semaphorin 3A concentration from the first to the
second measurement is indicative of the treatment being
efficacious. Each possibility represents a separate embodiment of
the present invention.
[0116] The methods for determining efficacy of a treatment
according to the present invention are based in part on the
unexpected discovery that Semaphorin 3A serum concentration in a
subject is inversely correlated to SLE disease activity as measured
by SLEDAI value. According to some embodiments, a treatment for SLE
leading to an increase in Semaphorin 3A serum concentration in a
subject in need thereof, is an efficacious treatment. According to
some embodiments, a treatment for SLE and/or Lupus Nephritis
leading to an increase in Semaphorin 3A serum concentration in a
subject in need thereof, is an efficacious treatment. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, a treatment for SLE
leading to an increase in Semaphorin 3A serum concentration in a
subject in need thereof to at least 50 ng/ml, is an efficacious
treatment. According to some embodiments, a treatment for SLE
and/or Lupus Nephritis leading to an increase in Semaphorin 3A
serum concentration in a subject in need thereof to at least 50
ng/ml, is an efficacious treatment. Each possibility represents a
separate embodiment of the present invention.
[0117] According to some embodiments, the efficacy of a treatment
for SLE is indicated by its ability to induce an increase in
Semaphorin 3A serum concentration in a subject in need thereof.
According to some embodiments, the efficacy of a treatment for SLE
and/or Lupus Nephritis is indicated by its ability to induce an
increase in Semaphorin 3A serum concentration in a subject in need
thereof. Each possibility represents a separate embodiment of the
present invention. According to some embodiments, the efficacy of a
treatment for SLE and/or Lupus Nephritis is indicated by its
ability to induce an increase in Semaphorin 3A serum concentration
to at least 50 ng/ml in a subject in need thereof. Each possibility
represents a separate embodiment of the present invention.
According to some embodiments, the efficacy of a treatment for SLE
and/or Lupus Nephritis is indicated by its ability to induce an
increase of at least 10%, preferably 20%, in Semaphorin 3A serum
concentration in a subject in need thereof. Each possibility
represents a separate embodiment of the present invention.
According to some embodiments, the efficacy of a treatment for SLE
and/or Lupus Nephritis is indicated by its ability to induce an
increase of at least 10% in Semaphorin 3A serum concentration in a
subject in need thereof. Each possibility represents a separate
embodiment of the present invention. In certain embodiments, the
efficacy of a treatment for SLE and/or Lupus Nephritis is indicated
by its ability to induce an increase of more than 20% in Semaphorin
3A serum concentration in a subject in need thereof. Each
possibility represents a separate embodiment of the present
invention. Without wishing to be bound by theory or mechanism, the
increase in Semaphorin 3A serum concentration which is indicative
of the efficacy of a treatment for SLE and/or Lupus Nephritis is
dependent on the specific physiological parameters of each subject.
Therefore, Semaphorin 3A serum increase which is indicative of an
efficient treatment may be less than 10% or more than 20% according
to certain embodiments. Each possibility represents a separate
embodiment of the present invention.
[0118] According to some embodiments, the efficacy of a treatment
for SLE is further indicated by at least one clinical outcome
selected from the group consisting of: an improvement in renal
function, a decrease in anti-dsDNA antibody concentration in the
serum, a decrease in anti-Cardiolipin antibody concentration in the
serum, an increase in serum concentration of complement factor C3
and an increase in serum concentration of complement factor C4.
Each possibility represents a separate embodiment of the present
invention. According to some embodiments, the efficacy of a
treatment for SLE is further indicated by a decrease in the
Systemic Lupus Erythematosus Disease Activity Index value of the
treated subject.
[0119] According to some embodiments, the efficacy of a treatment
for SLE and/or Lupus Nephritis is further indicated by at least one
clinical outcome selected from the group consisting of: an
improvement in renal function, a decrease in anti-dsDNA antibody
concentration in the serum, a decrease in anti-Cardiolipin antibody
concentration in the serum, an increase in serum concentration of
complement factor C3 and an increase in serum concentration of
complement factor C4. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
the efficacy of a treatment for SLE and/or Lupus Nephritis is
further indicated by a decrease in the Systemic Lupus Erythematosus
Disease Activity Index value of the treated subject. Each
possibility represents a separate embodiment of the present
invention.
[0120] According to some embodiments, an efficacious treatment for
SLE and/or Lupus Nephritis is a treatment which ameliorates or
prevents at least one symptom of SLE and/or Lupus Nephritis,
respectively. Each possibility represents a separate embodiment of
the present invention. According to some embodiments, an
efficacious treatment for SLE and/or Lupus Nephritis is a treatment
that shortens at least one flare period. Each possibility
represents a separate embodiment of the present invention.
[0121] According to some embodiments, measurement of serum
Semaphorin 3A concentration may be performed by any method known in
the art. According to some embodiments, measurement of serum
Semaphorin 3A is performed on a sample obtained from a subject in
need thereof. According to some embodiments, measurement of serum
Semaphorin 3A is performed on a serum sample obtained from a
subject in need thereof. According to some embodiments, measurement
of serum Semaphorin 3A is performed on a blood sample obtained from
a subject in need thereof. According to some embodiments,
Semaphorin 3A can be detected and quantified by any of a number of
methods well known to those of skill in the art for polypeptide
detection. These may include, but are not limited to, analytic
biochemical methods such as electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography,
mass spectroscopy and the like, or various immunological methods
such as, but not limited to, fluid or gel precipitin reactions,
immunodiffusion (single or double), immunohistochemistry, affinity
chromatography, immunoelectrophoresis, radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay,
Western blotting, and the like. According to some embodiments,
measurement of serum Semaphorin 3A concentration is performed using
an enzyme-linked immunosorbent assay (ELISA).
[0122] According to some embodiments, the first measurement is
taken as close as possible prior to the beginning of the SLE/Lupus
Nephritis treatment, preferably within a day of the beginning of
treatment. Each possibility represents a separate embodiment of the
present invention. According to some embodiments, the first
measurement is taken at the time of diagnosing a subject with SLE
or as close as possible to the time of said diagnosis. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, the second measurement is
taken following termination of the SLE/Lupus Nephritis treatment.
Each possibility represents a separate embodiment of the present
invention. According to some embodiments, the second measurement is
taken in the course of treatment with the SLE/Lupus Nephritis
treatment. Each possibility represents a separate embodiment of the
present invention. According to some embodiments, the second
measurement is taken at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 30, 60,
90 days after the beginning of treatment with the SLE/Lupus
Nephritis treatment. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
the second measurement is taken at least a week after the beginning
of treatment with the SLE/Lupus Nephritis treatment. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, the second measurement is
taken at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 30, 60, 90 days
following termination of treatment with the SLE/Lupus Nephritis
treatment. Each possibility represents a separate embodiment of the
present invention. According to some embodiments, the second
measurement is taken at least a week following termination of
treatment with the SLE/Lupus Nephritis treatment. Each possibility
represents a separate embodiment of the present invention.
[0123] According to another aspect, the present invention provides
a method for prognosis of change in Systemic Lupus Erythematosus
disease activity in a subject in need thereof, the method
comprising: making a first measurement of serum Semaphorin 3A
concentration in a subject in need thereof; making a second
measurement of serum Semaphorin 3A concentration in the subject at
a later time point than said first measurement; and comparing said
first measurement and said second measurement; wherein an increase
in serum Semaphorin 3A concentration from the first to the second
measurement is indicative of a decrease in disease activity; and
wherein a decrease in serum Semaphorin 3A concentration from the
first to the second measurement is indicative of an increase in
disease activity.
[0124] According to some embodiments, a decrease in SLE disease
activity is characterized by amelioration of at least one symptom
of SLE. According to some embodiments, a decrease in SLE disease
activity is characterized by at least one clinical effect selected
from the group consisting of: an improvement in renal function, a
decrease in anti-dsDNA antibody concentration in the serum, a
decrease in anti-Cardiolipin antibody concentration in the serum,
an increase in serum concentration of complement factor C3 and an
increase in serum concentration of complement factor C4. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, a decrease in SLE disease
activity is evidenced by a decrease in the value of the Systemic
Lupus Erythematosus Disease Activity Index of the subject.
According to some embodiments, a decrease in disease activity, as
indicated by an increase in Semaphorin 3A serum concentration, is
indicative of the ending and/or clinical improvement of a flare
period. Each possibility represents a separate embodiment of the
present invention.
[0125] According to some embodiments, an increase in SLE disease
activity is characterized by worsening of at least one symptom of
SLE. According to some embodiments, an increase in SLE disease
activity is characterized by at least one clinical effect selected
from the group consisting of: deterioration in renal function, an
increase in anti-dsDNA antibody concentration in the serum, an
increase in anti-Cardiolipin antibody concentration in the serum, a
decrease in serum concentration of complement factor C3 and a
decrease in serum concentration of complement factor C4. Each
possibility represents a separate embodiment of the present
invention. According to some embodiments, an increase in SLE
disease activity is evidenced by an increase in the value of the
Systemic Lupus Erythematosus Disease Activity Index of the subject.
According to some embodiments, an increase in disease activity, as
indicated by a decrease in Semaphorin 3A serum concentration, is
indicative of the beginning and/or worsening of a flare period.
Each possibility represents a separate embodiment of the present
invention.
[0126] According to some embodiments, serum Semaphorin 3A
concentration is inversely correlated to SLE disease activity.
According to some embodiments, serum Semaphorin 3A concentration is
inversely correlated to SLEDAI value. According to some
embodiments, the present invention provides a method for prognosis
of lupus nephritis in a subject afflicted with SLE, the method
comprising measuring serum Semaphorin 3A concentration in the
subject, wherein a concentration up to 50 ng/ml is indicative of
Lupus Nephritis in the subject. According to some embodiments, a
measurement of serum Semaphorin 3A of up to 50 ng/ml is indicative
of Lupus Nephritis. According to some embodiments, a measurement of
serum Semaphorin 3A of up to 50 ng/ml is indicative of the presence
of serum anti-cardiolipin antibodies.
[0127] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a
variety of alternative forms without departing from the
invention.
EXAMPLES
Example 1
Correlation Between Presence and Severity of SLE and Sema3A Serum
Concentration
[0128] Serum samples from 32 SLE patients, 24 rheumatoid arthritis
(RA) patients (disease control) and 40 healthy controls, were all
analyzed for Sema3A serum level. SLE patients were evaluated for
disease activity by using the Systemic Lupus Erythematosus Disease
Activity Index (SLEDAI). All SLE patients were scored mild to
severe with a SLEDAI value ranging between 4 and 24. Of relevance,
blood was drawn before any steroid pulse or increase of cytotoxic
therapy was initiated when severity of disease activity required
it.
[0129] Table 1 summarizes the clinical and laboratory
characteristics of all SLE patients included in this study: SLEDAI
value, Sema 3A concentration (ng/ml), presence of anti-cardiolipin
antibodies (a.Cal), Lupus Nephritis (LN), presence of anti-dsDNA
antibodies (a. dsDNA), low (L) or normal (N) levels of complement
C3 and C4, and treatment with various drugs. Seven patients with
mild disease were treated only with hydroxychloroquine (HCQ),
another eight patients were treated with add-on 2.5 to 5 mg of
prednisone, and 17 patients, considered having moderate to severe
disease, received low to moderate daily doses of cytotoxic therapy.
Nine patients suffered from renal involvement, 19 patients were
anti-ds DNA antibody positive and 17 had anti-cardiolipin
antibodies of moderate to high titers.
[0130] The measurement of Sema3A serum level was conducted using a
commercial ELISA kit (USCNK Life Science, Wuhan, P. R. China)
according to the manufacturer instructions. The serum samples were
stored at -20.degree. until ELISA evaluation. Results of the SLE
patients' serum Sema3A levels were correlated with their SLEDAI
score, renal involvement and laboratory serologic studies including
anti-dsDNA anti-cardiolipin antibodies and C3-C4 serum levels.
[0131] As can be seen in FIG. 1, Sema 3A serum concentration in SLE
patients (55.04.+-.16.30 ng/ml) was significantly lower than that
of control subjects (74.41.+-.17.60 ng/ml, P<0.0001). As can be
seen in FIG. 2A, Sema 3A serum concentration was found to be
inversely correlated with disease activity of SLE patients as
represented by SLEDAI values (R=-0.89, P<0.0001). Moreover, as
can be seen in FIG. 2B, 73% of SLE patients which had a Sema3A
serum concentration below 50 ng/ml showed kidney involvement (Lupus
Nephritis) as compared to only 5% of patients having Sema3A serum
concentration above 50 ng/ml. FIG. 2C further demonstrates that 64%
of SLE patients which had a Sema3A serum concentration below 50
ng/ml were positive for anti-cardiolipin antibodies as compared to
only 33% of patients having Sema3A serum concentration above 50
ng/ml. In addition, a positive correlation was found between the
serum level of Sema3A and both C3 values (r=0.078, P=0.0008) and C4
values (r=0.523, P=0.005) (FIGS. 3A and 3B, respectively).
Example 2
Expression of Sema3A and NP-1 on B-Cells from Healthy Subjects and
SLE Patients
[0132] Flow cytometry was used in order to compare Sema3A or NP-1
expression in CD19.sup.+CD25.sup.high B cells of healthy subjects
vs. SLE patients. Briefly, whole blood samples were stained with
human anti-CD19 FITC/P antibody and human anti-CD25 PC5 antibody
(Immunotech, Beckman-Coulter, Marsellie, France). The blood samples
were further stained with either human anti-sema3A AlexaFluor 488
antibody or human anti-NP1 PE antibody (R&D, Minneapolis,
Minn., USA). The stained cells were then evaluated using a Flow
Cytometer (FC500 and CXP software, Beckman Coulter, Brea, Calif.,
USA).
[0133] Significantly less Sema3A had been expressed on
CD19.sup.+CD25.sup.high B cells derived from SLE patients than on
cells derived from healthy controls (52.2.+-.5.8% versus
82.6.+-.6.4% respectively, P<0.0001). FIG. 4 presents Sema 3A
levels as Mean Fluorescent Intensity (MFI) values of samples from
SLE patients vs. healthy subjects (2.68.+-.0.09 versus 4.27.+-.0.47
respectively, P=0.019).
[0134] NP-1 expression was also found to be significantly lower on
examined cells from SLE patients when compared to cells from
healthy individuals (10.8.+-.3.6% versus 15.4.+-.1.4% respectively,
P=0.03).
TABLE-US-00001 TABLE 1 Clinical characteristics of SLE patients
Sema3A a. a. Complement Patient SLEDAI (ng/ml) Cal LN dsDNA C3 C4
Treatment 1 16 44 + + L L HCQ; P 2 14 32 + + + L L HCQ; P 3 3 82 N
N HCQ 4 4 63 N L P 5 4 69 + N N HCQ; P; AZA 6 4 51 + N N HCQ;
Atabrin 7 5 69 + N L HCQ; P 8 8 53 N L HCQ; P; AZA 9 6 79 + + L L
HCQ; P; AZA 10 12 44 N L HCQ; P; AZA 11 6 65 N N HCQ; P 12 9 48 + N
L HCQ; P; AZA 13 6 65 + + L L HCQ; P; AZA 14 3 76 N L HCQ; P 15 12
42 + + N L HCQ; P; AZA 16 8 64 + + L L HCQ; P; 17 18 38 + + + L L
HCQ; P; AZA 18 18 33 + + + L L HCQ; P; MTX; IVIg 19 4 68 + N L HCQ;
P; AZA 20 6 55 N N HCQ; P 21 6 60 + + N L HCQ; P 22 24 20 + + + L L
HCQ; P; MTX; IVIg 23 5 70 + N N HCQ; P 24 10 50 + + + L L HCQ; P;
AZA 25 13 36 + + + L L HCQ; P; 26 4 65 + N N HCQ; 27 26 26 + + + L
L HCQ; P; CYC 28 6 61 N N HCQ; P; AZA 29 4 63 N N HCQ; P; AZA 30 4
59 N L HCQ 31 5 58 + + N N HCQ; P; AZA 32 20 34 + + + L L HCQ; P;
CYC a. Cal, anti-cardiolipin; a. dsDNA, anti-double-stranded DNA;
AZA, azathioprine; CYC, cyclophosphamide; F, female; HCQ,
hydroxychloroquine; IVIg, intravenous immunoglobulin; L, low; LN,
lupus nephritis; M, male; MTX, Methotrexate; N, normal; P,
prednisone; Sema3A, semaphorin 3A.
Example 3
Expression of Toll-Like Receptor 9 on B-Cells from SLE Patients
Incubated with Sema3A Conditioned Media
[0135] In order to examine the effect of treatment with Sema3A on
the expression of Toll-Like Receptor 9 (TLR-9) on B cells of SLE
patients, B cells were purified from peripheral blood of SLE
patients. Briefly, peripheral blood mononuclear cells (PBMCs) were
isolated on Lymphoprep (Axis-Shield, Oslo, Norway), and B
lymphocytes were isolated by positive selection using the CD22
microbeads (20 .mu.l/10.sup.7 cells; Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the manufacturer's instructions,
achieving >98% purity. Next, Purified B cells, gating on memory
CD19+/CD27+ B cells (Immunotech, Beckman-Coulter, Marseile, France)
from SLE patients were analyzed for TLR-9 expression:
[0136] Purified B cells from each patient, activated by
cytosine-phosphodiester-guanine oligodeoxynucleotides (ODN-CpG),
were incubated for 60 hours with conditioned media from
HEK293-cells infected by NSPI-CMV-FLAG lentivirus with or without
sema3A cDNA, as previously described (Bombardier C. et al., 1992,
Arthritis Rheum., 35:630-640). Accordingly, conditioned medium from
HEK293 cells infected by NSPI-CMV-FLAG lentivirus with Sema3A cDNA
contained FLAG-tagged Sema3A, as set forth in SEQ ID NO:3.
[0137] Following incubation, cells were fixed and permeabilized
using a commercial kit according to the manufacturer's instructions
(`Fix and Perm`, Invitrogen, Carlsbad, Calif., USA) and analyzed
for TLR-9 expression. Staining was performed using a specific
monoclonal anti-human TLR-9-PE antibody (Imgenex, San Diego,
Calif., USA) and evaluated using a FC500 flow cytometer.
[0138] Next, the percentage of cells expressing TLR-9 following
incubation with the Sema3A-containing media or the control culture
media was compared. FIG. 5 demonstrates that TLR-9 expression on
memory B cells was lower following incubation of the cells with
Sema3A ("After Sema3A") by almost 50% (P=0.001) as compared to
expression on cells incubated with medium alone ("Baseline"). Each
line in FIG. 5 represents the difference in TLR-9 expression
between cells from a single subject that were either incubated with
control or Sema 3A containing medium.
Example 4
Examining the Effect of Semaphorin 3A Administration in NZB/NZW Ft
Mice
[0139] In order to assess how Semaphorin 3A affects SLE disease
progression in NZB/NZW F1 mice (serving as a model system for SLE),
mice are divided into 4 groups:
[0140] Prevention group: In this group, 5 mice are injected with
recombinant Sema3A on a daily basis and 5 mice are injected with
PBS, as a control group. Mice are injected from the age of 6 weeks
for 90 days. During this period, both groups are assessed for the
development of auto-antibodies (e.g. anti-dsDNA and
anti-cardiolipin), kidney function tests (creatinine and BUN),
complete blood count on weekly basis and detection of early
proteinuria. In addition clinical status of the mice is evaluated
by assessing their weight. After this period, the mice are
sacrificed and a histological evaluation of their kidneys is
performed.
[0141] Treatment group: In this group, 5 mice are injected with
recombinant Sema3A on a daily basis and 5 mice are injected with
PBS, as a control group. Mice are injected from the onset of
clinical and laboratory signs of SLE (at four month of age with
early proteinuria) and continue for 90 days. During this period,
both groups are assessed for the development of auto-antibodies
(e.g. anti-dsDNA and anti-cardiolipin), kidney function tests
(creatinine and BUN), complete blood count on weekly basis and
detection of early proteinuria. In addition clinical status of the
mice is evaluated by assessing their weight. After this period, the
mice are sacrificed and a histological evaluation of their kidneys
is performed.
Example 5
Comparison of Semaphorin 3A Expression on CD19.sup.+CD25.sup.high
B-Cells and CD19.sup.+CD25.sup.low B-Cells
[0142] Flow cytometry was used in order to compare Sema3A
expression between CD19.sup.+CD25.sup.high and
CD19.sup.+CD25.sup.low B cells purified from peripheral blood of
SLE patients. Peripheral blood mononuclear cells (PBMCs) were
isolated on Lymphoprep (Axis-Shield, Oslo, Norway) from peripheral
blood of SLE patients, and B lymphocytes were isolated by positive
selection using the CD22 microbeads (20 .mu.l/10.sup.7 cells;
Miltenyi Biotec, Bergisch Gladbach, Germany) according to the
manufacturer's instructions. The purified B cells were stained with
monoclonal antibodies: human anti-CD19 FITC/PE and CD25 PC5
(Immunotech, Beckman-Coulter, Marsellie, France), and human
anti-sema3A AlexaFluor 488 (R&D, Minneapolis, Minn., USA), and
evaluated in Flow cytometry software (FC500 and CXP software,
Beckman Coulter, Brea, Calif., USA).
[0143] FIGS. 6A and 6B show the same flow cytometry scatter,
plotting CD19 positive cells vs. CD25 positive cells. FIG. 6A shows
the cell range used to examine Sema3A expression on
CD19.sup.-CD25.sup.low cells and CD19.sup.+CD25.sup.low B cells.
FIG. 6B shows the cell range used to examine Sema 3A expression on
CD19.sup.+CD25.sup.high B cells. As can be seen in FIGS. 6C-E,
CD19.sup.-CD25.sup.low cells showed 2.1% Semaphorin 3A expression
(marked Sema3A on CD19-non CD25 cells in FIG. 6C),
CD19.sup.+CD25.sup.low B cells showed 17% Semaphorin3A expression
(marked Sema3A on CD19-CD25 cells in FIG. 6D) and
CD19.sup.+CD25.sup.high B cells showed 35% Semaphorin 3A expression
(marked Sema3A on CD19-CD25 high cells in FIG. 6E).
Example 6
Comparison Between Semaphorin 3A Expression on CD4/CD25.sup.high T
Regulatory Cells of SLE Patients and Healthy Subjects
[0144] In order to examine Sema3A expression on CD4/CD25.sup.high T
regulatory cells, whole blood samples from healthy subjects and SLE
patients were stained using the following monoclonal antibodies:
human anti-CD4 PE and CD25 PC5 (Immunotech, Beckman-Coulter,
Marsellie, France), and human anti-sema3A AlexaFluor 488 (R&D,
Minneapolis, Minn., USA). The cells were evaluated using a flow
cytometry software (FC500 and CXP software, Beckman Coulter, Brea,
Calif., USA). Similar Sema3A expression was observed on
CD4/CD25.sup.high T regulatory cells from SLE patients and healthy
subjects.
[0145] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a
variety of alternative forms without departing from the invention.
It is to be understood that further trials are being conducted to
establish clinical effects.
Sequence CWU 1
1
31771PRTHomo sapiens 1Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe
Trp Gly Val Leu Leu 1 5 10 15 Thr Ala Arg Ala Asn Tyr Gln Asn Gly
Lys Asn Asn Val Pro Arg Leu 20 25 30 Lys Leu Ser Tyr Lys Glu Met
Leu Glu Ser Asn Asn Val Ile Thr Phe 35 40 45 Asn Gly Leu Ala Asn
Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu 50 55 60 Glu Arg Ser
Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe 65 70 75 80 Asp
Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser 85 90
95 Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys
100 105 110 Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln
Thr His 115 120 125 Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile
Cys Thr Tyr Ile 130 135 140 Glu Ile Gly His His Pro Glu Asp Asn Ile
Phe Lys Leu Glu Asn Ser 145 150 155 160 His Phe Glu Asn Gly Arg Gly
Lys Ser Pro Tyr Asp Pro Lys Leu Leu 165 170 175 Thr Ala Ser Leu Leu
Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala 180 185 190 Asp Phe Met
Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His 195 200 205 His
Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro 210 215
220 Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp
225 230 235 240 Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp
Gly Glu His 245 250 255 Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln
Ile Cys Lys Asn Asp 260 265 270 Phe Gly Gly His Arg Ser Leu Val Asn
Lys Trp Thr Thr Phe Leu Lys 275 280 285 Ala Arg Leu Ile Cys Ser Val
Pro Gly Pro Asn Gly Ile Asp Thr His 290 295 300 Phe Asp Glu Leu Gln
Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys 305 310 315 320 Asn Pro
Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys 325 330 335
Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe 340
345 350 Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val
Pro 355 360 365 Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys
Pro Ser Lys 370 375 380 Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu
Pro Asp Asp Val Ile 385 390 395 400 Thr Phe Ala Arg Ser His Pro Ala
Met Tyr Asn Pro Val Phe Pro Met 405 410 415 Asn Asn Arg Pro Ile Val
Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr 420 425 430 Gln Ile Val Val
Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val 435 440 445 Met Phe
Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile 450 455 460
Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met 465
470 475 480 Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu
Ser Thr 485 490 495 Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly
Val Ala Gln Leu 500 505 510 Pro Leu His Arg Cys Asp Ile Tyr Gly Lys
Ala Cys Ala Glu Cys Cys 515 520 525 Leu Ala Arg Asp Pro Tyr Cys Ala
Trp Asp Gly Ser Ala Cys Ser Arg 530 535 540 Tyr Phe Pro Thr Ala Lys
Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn 545 550 555 560 Gly Asp Pro
Leu Thr His Cys Ser Asp Leu His His Asp Asn His His 565 570 575 Gly
His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser 580 585
590 Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp
595 600 605 Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg
Val Asp 610 615 620 Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu
Arg Ser Leu Gln 625 630 635 640 Gln Lys Asp Ser Gly Asn Tyr Leu Cys
His Ala Val Glu His Gly Phe 645 650 655 Ile Gln Thr Leu Leu Lys Val
Thr Leu Glu Val Ile Asp Thr Glu His 660 665 670 Leu Glu Glu Leu Leu
His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr 675 680 685 Lys Glu Met
Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg 690 695 700 Asp
Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu 705 710
715 720 Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln
Arg 725 730 735 Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His
Leu Gln Glu 740 745 750 Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu
Phe Glu Arg Ala Pro 755 760 765 Arg Ser Val 770 22316DNAArtificial
SequencecDNA 2atgggctggt taactaggat tgtctgtctt ttctggggag
tattacttac agcaagagca 60aactatcaga atgggaagaa caatgtgcca aggctgaaat
tatcctacaa agaaatgttg 120gaatccaaca atgtgatcac tttcaatggc
ttggccaaca gctccagtta tcataccttc 180cttttggatg aggaacggag
taggctgtat gttggagcaa aggatcacat attttcattc 240gacctggtta
atatcaagga ttttcaaaag attgtgtggc cagtatctta caccagaaga
300gatgaatgca agtgggctgg aaaagacatc ctgaaagaat gtgctaattt
catcaaggta 360cttaaggcat ataatcagac tcacttgtac gcctgtggaa
cgggggcttt tcatccaatt 420tgcacctaca ttgaaattgg acatcatcct
gaggacaata tttttaagct ggagaactca 480cattttgaaa acggccgtgg
gaagagtcca tatgacccta agctgctgac agcatccctt 540ttaatagatg
gagaattata ctctggaact gcagctgatt ttatggggcg agactttgct
600atcttccgaa ctcttgggca ccaccaccca atcaggacag agcagcatga
ttccaggtgg 660ctcaatgatc caaagttcat tagtgcccac ctcatctcag
agagtgacaa tcctgaagat 720gacaaagtat actttttctt ccgtgaaaat
gcaatagatg gagaacactc tggaaaagct 780actcacgcta gaataggtca
gatatgcaag aatgactttg gagggcacag aagtctggtg 840aataaatgga
caacattcct caaagctcgt ctgatttgct cagtgccagg tccaaatggc
900attgacactc attttgatga actgcaggat gtattcctaa tgaactttaa
agatcctaaa 960aatccagttg tatatggagt gtttacgact tccagtaaca
ttttcaaggg atcagccgtg 1020tgtatgtata gcatgagtga tgtgagaagg
gtgttccttg gtccatatgc ccacagggat 1080ggacccaact atcaatgggt
gccttatcaa ggaagagtcc cctatccacg gccaggaact 1140tgtcccagca
aaacatttgg tggttttgac tctacaaagg accttcctga tgatgttata
1200acctttgcaa gaagtcatcc agccatgtac aatccagtgt ttcctatgaa
caatcgccca 1260atagtgatca aaacggatgt aaattatcaa tttacacaaa
ttgtcgtaga ccgagtggat 1320gcagaagatg gacagtatga tgttatgttt
atcggaacag atgttgggac cgttcttaaa 1380gtagtttcaa ttcctaagga
gacttggtat gatttagaag aggttctgct ggaagaaatg 1440acagtttttc
gggaaccgac tgctatttca gcaatggagc tttccactaa gcagcaacaa
1500ctatatattg gttcaacggc tggggttgcc cagctccctt tacaccggtg
tgatatttac 1560gggaaagcgt gtgctgagtg ttgcctcgcc cgagaccctt
actgtgcttg ggatggttct 1620gcatgttctc gctattttcc cactgcaaag
agacgcacaa gacgacaaga tataagaaat 1680ggagacccac tgactcactg
ttcagactta caccatgata atcaccatgg ccacagccct 1740gaagagagaa
tcatctatgg tgtagagaat agtagcacat ttttggaatg cagtccgaag
1800tcgcagagag cgctggtcta ttggcaattc cagaggcgaa atgaagagcg
aaaagaagag 1860atcagagtgg atgatcatat catcaggaca gatcaaggcc
ttctgctacg tagtctacaa 1920cagaaggatt caggcaatta cctctgccat
gcggtggaac atgggttcat acaaactctt 1980cttaaggtaa ccctggaagt
cattgacaca gagcatttgg aagaacttct tcataaagat 2040gatgatggag
atggctctaa gaccaaagaa atgtccaata gcatgacacc tagccagaag
2100gtctggtaca gagacttcat gcagctcatc aaccacccca atctcaacac
aatggatgag 2160ttctgtgaac aagtttggaa aagggaccga aaacaacgtc
ggcaaaggcc aggacatacc 2220ccagggaaca gtaacaaatg gaagcactta
caagaaaata agaaaggtag aaacaggagg 2280acccacgaat ttgagagggc
acccaggagt gtctga 23163779PRTArtificial SequenceRecombinant
polypeptide 3Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly
Val Leu Leu 1 5 10 15 Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn
Asn Val Pro Arg Leu 20 25 30 Lys Leu Ser Tyr Lys Glu Met Leu Glu
Ser Asn Asn Val Ile Thr Phe 35 40 45 Asn Gly Leu Ala Asn Ser Ser
Ser Tyr His Thr Phe Leu Leu Asp Glu 50 55 60 Glu Arg Ser Arg Leu
Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe 65 70 75 80 Asp Leu Val
Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser 85 90 95 Tyr
Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys 100 105
110 Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His
115 120 125 Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr
Tyr Ile 130 135 140 Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys
Leu Glu Asn Ser 145 150 155 160 His Phe Glu Asn Gly Arg Gly Lys Ser
Pro Tyr Asp Pro Lys Leu Leu 165 170 175 Thr Ala Ser Leu Leu Ile Asp
Gly Glu Leu Tyr Ser Gly Thr Ala Ala 180 185 190 Asp Phe Met Gly Arg
Asp Phe Ala Ile Phe Arg Thr Leu Gly His His 195 200 205 His Pro Ile
Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro 210 215 220 Lys
Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp 225 230
235 240 Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu
His 245 250 255 Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys
Lys Asn Asp 260 265 270 Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp
Thr Thr Phe Leu Lys 275 280 285 Ala Arg Leu Ile Cys Ser Val Pro Gly
Pro Asn Gly Ile Asp Thr His 290 295 300 Phe Asp Glu Leu Gln Asp Val
Phe Leu Met Asn Phe Lys Asp Pro Lys 305 310 315 320 Asn Pro Val Val
Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys 325 330 335 Gly Ser
Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe 340 345 350
Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro 355
360 365 Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser
Lys 370 375 380 Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp
Asp Val Ile 385 390 395 400 Thr Phe Ala Arg Ser His Pro Ala Met Tyr
Asn Pro Val Phe Pro Met 405 410 415 Asn Asn Arg Pro Ile Val Ile Lys
Thr Asp Val Asn Tyr Gln Phe Thr 420 425 430 Gln Ile Val Val Asp Arg
Val Asp Ala Glu Asp Gly Gln Tyr Asp Val 435 440 445 Met Phe Ile Gly
Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile 450 455 460 Pro Lys
Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met 465 470 475
480 Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr
485 490 495 Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala
Gln Leu 500 505 510 Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys
Ala Glu Cys Cys 515 520 525 Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp
Gly Ser Ala Cys Ser Arg 530 535 540 Tyr Phe Pro Thr Ala Lys Arg Arg
Thr Arg Arg Gln Asp Ile Arg Asn 545 550 555 560 Gly Asp Pro Leu Thr
His Cys Ser Asp Leu His His Asp Asn His His 565 570 575 Gly His Ser
Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser 580 585 590 Thr
Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp 595 600
605 Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp
610 615 620 Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser
Leu Gln 625 630 635 640 Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala
Val Glu His Gly Phe 645 650 655 Ile Gln Thr Leu Leu Lys Val Thr Leu
Glu Val Ile Asp Thr Glu His 660 665 670 Leu Glu Glu Leu Leu His Lys
Asp Asp Asp Gly Asp Gly Ser Lys Thr 675 680 685 Lys Glu Met Ser Asn
Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg 690 695 700 Asp Phe Met
Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu 705 710 715 720
Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg 725
730 735 Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln
Glu 740 745 750 Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu
Arg Ala Pro 755 760 765 Arg Ser Val Asp Tyr Lys Asp Asp Asp Asp Lys
770 775
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