U.S. patent application number 15/562602 was filed with the patent office on 2018-04-26 for inhibitors of neuroligin 4 - neurexin 1-beta protein-protein interaction for treatment of liver disorders.
This patent application is currently assigned to HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD.. The applicant listed for this patent is HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD.. Invention is credited to Rifaat SAFADI.
Application Number | 20180111989 15/562602 |
Document ID | / |
Family ID | 57004841 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180111989 |
Kind Code |
A1 |
SAFADI; Rifaat |
April 26, 2018 |
INHIBITORS OF NEUROLIGIN 4 - NEUREXIN 1-BETA PROTEIN-PROTEIN
INTERACTION FOR TREATMENT OF LIVER DISORDERS
Abstract
Provided is a method for treating, attenuating and/or preventing
progression of a liver disorder in a subject, the method including
administering a therapeutically effective amount of an agent
capable of interfering with, inhibiting and/or preventing
neuroligin 4 (NLGn4)-Neurexin 1-beta (Nrx1b) protein-protein
interaction. Also provided are compositions including the
agent.
Inventors: |
SAFADI; Rifaat; (Nazareth
Elit, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD. |
Jerusalem |
|
IL |
|
|
Assignee: |
HADASIT MEDICAL RESEARCH SERVICES
AND DEVELOPMENT LTD.
Jerusalem
IL
|
Family ID: |
57004841 |
Appl. No.: |
15/562602 |
Filed: |
March 31, 2016 |
PCT Filed: |
March 31, 2016 |
PCT NO: |
PCT/IL2016/050351 |
371 Date: |
September 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62141299 |
Apr 1, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/76 20130101; A61K 38/16 20130101; C07K 2319/30 20130101;
A61P 1/16 20180101; C07K 2317/52 20130101; C07K 16/28 20130101;
A61K 38/1709 20130101; A61K 38/177 20130101; C07K 2317/34
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 1/16 20060101 A61P001/16; A61K 38/17 20060101
A61K038/17 |
Claims
1.-21. (canceled)
22. A composition comprising a therapeutically effective amount of
an agent capable of interfering with, inhibiting and/or preventing
neuroligin 4 (NLGn4)-Neurexin 1.beta. (Nrx1b) protein-protein
interaction and a pharmaceutically acceptable carrier wherein the
therapeutic agent comprises an anti-NLGn4 antibody, or a fragment,
derivative or analog thereof, comprising at least the antigen
binding portion thereof.
23. The composition of claim 22, wherein the anti-NLGn4 antibody is
capable of binding specifically to amino acids 359-364 of the human
NLGN4 protein.
24. The composition of claim 22, wherein the anti-NLGn4 antibody is
capable of binding to an epitope within a sequence selected from
the group of: residues 261-270 of the human NLGn4, as set forth in
SEQ ID NO: 3 (SLLTLSHYSE); residues 265-275, as set forth in SEQ ID
NO: 5 (LSHYSEGLFQK), of the human NLGn4; and residues 461-470 of
the human NLGn4, as set forth in SEQ ID NO: 4 (AQYGSPTYFY).
25. A method of treating, attenuating and/or preventing progression
of a liver disorder, comprising administering a therapeutically
effective amount of an agent capable of interfering with,
inhibiting and/or preventing neuroligin 4 (NLGn4)-Neurexin 1.beta.
(Nrx1b) protein-protein interaction and a pharmaceutically
acceptable carrier, thereby treating, attenuating and/or preventing
progression of a liver disorder.
26. The method of claim 25, wherein the agent comprises recombinant
soluble NLGn4 (rsNLGn4); wherein the rsNLGn4 comprises the
extracellular domain of NLGn4 or a fragment, derivative or analog
thereof.
27. The method of claim 26, wherein the extracellular domain of
NLGn4 consists of SEQ ID NO: 7; or wherein the rsNLGn4 consists of
SEQ ID NO: 7, or a fragment, derivative or analog thereof; or
wherein the rsNLGn4 competes with endogenous NLGn4 for binding to
Nrx1b; or wherein the rsNLGn4 is devoid of the intracellular domain
and/or the transmembrane domain of NLGn4.
28. The method of claim 25, wherein the agent comprises recombinant
soluble Nrx1b (rsNrx1b); wherein the rsNrx1b comprises the
extracellular domain of Nrx1b or a fragment, derivative or analog
thereof capable of binding the NLGn4.
29. The method of claim 28, wherein the extracellular domain of
Nrx1b consists of SEQ ID NO: 10; wherein the rsNrx1b consists of
SEQ ID NO: 10, or a fragment, derivative or analog thereof; wherein
the rsNrx1b competes with endogenous Nrx1b for binding to NLGn4; or
wherein the rsNrx1b is devoid of the intracellular domain and/or
the transmembrane domain of Nrx1b.
30. The method of claim 25, wherein NLGn4 is encoded by the
sequence set forth in SEQ ID NO: 1.
31. The method of claim 25, wherein Nrx1b is encoded by the
sequence set forth in SEQ ID NO: 6.
32. The method of claim 25, the agent comprises a fusion protein
comprising the Fc portion of an immunoglobulin molecule and SEQ ID
NO: 7 or a fragment thereof.
33. The method of claim 25, wherein the agent comprises a fusion
protein comprising the Fc portion of an immunoglobulin molecule and
SEQ ID NO: 10 or a fragment thereof.
34. The method of claim 25, wherein the therapeutic agent comprises
an anti-NLGn4 antibody, or a fragment, derivative or analog
thereof, comprising at least the antigen-binding portion thereof;
wherein the anti-NLGn4 antibody is capable of binding specifically
to an interaction domain of human NLGn4.
35. The method of claim 34, wherein the anti-NLGn4 antibody is
capable of binding specifically to amino acids 359-364 of the human
NLGN4 protein.
36. The method of claim 34, wherein the anti-NLGn4 antibody is
capable of binding to an epitope within a sequence selected from
the group of: residues 261-270 of the human NLGn4, as set forth in
SEQ ID NO: 3 (SLLTLSHYSE); residues 265-275, as set forth in SEQ ID
NO: 5 (LSHYSEGLFQK), of the human NLGn4; and residues 461-470 of
the human NLGn4, as set forth in SEQ ID NO: 4 (AQYGSPTYFY).
37. The method of claim 25, wherein the liver disorder is selected
from the group consisting of: non-alcoholic fatty liver disease
(NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis,
hepatitis, liver adenoma, insulin hypersensitivity, liver cancer
and any combination thereof.
38. The method of claim 37, wherein the liver disorder is
NAFLD.
39. The composition of claim 37, wherein the liver disorder is
hepatocellular carcinoma.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to antibodies and recombinant
proteins capable of interfering with, inhibiting and/or preventing
neuroligin-4 (NLGn4)-neurexin 113 (Nrx1b) protein-protein
interaction, and to the use thereof for the treatment and/or
attenuation of liver disorders.
BACKGROUND OF THE INVENTION
[0002] The normal liver is composed of hepatocytes and
non-parenchymal cells, which include Kupffer cells, sinusoidal
endothelial cells, and myofibroblasts known as Hepatic Stellate
Cells (HSCs). HSCs are considered involved in the pathogenesis of
liver fibrosis from any etiology, including NASH-related hepatic
fibrosis and hepatocellular carcinoma. In normal liver, HSCs are in
a quiescent state and serve to store retinoids (vitamin A).
Quiescent stellate cells represent 5-8% of the total number of
liver cells. When the liver is damaged, HSCs change into an
activated state characterized by contractions, loss of lipid
droplets, enhanced proliferation, cell migration and cellular
adhesion. HSCs are also unequivocally the main cells involved in
the production of excessive ECM seen in liver fibrosis. Since
activated HSCs themselves secrete inflammatory chemokines, a
vicious cycle is formed, whereby fibrogenic and inflammatory cells
stimulate each other and perpetuate a process of liver damage and
repair.
[0003] Natural killer (NK) cells are a key component of the innate
immune system, and play a critical role in the early stages of the
immune response against tumor cells, as well as those infected by
viral and microbial pathogens.
[0004] In humans, two NK-cell subsets have been characterized
according to the cell-surface density of CD56 and expression of
CD16. CD56.sup.dimCD16.sup.bright NK cells (hereinafter
CD56.sup.dim) compose approximately 90% of circulating NK cells;
CD56.sup.brightCD16.sup.dim NK cells (hereinafter CD56.sup.bright)
constitute approximately 10%. CD56.sup.bright NK cells proliferate
and produce interferon in response to stimulation with
interleukin-12 (IL-12), whereas CD56.sup.dim NK cells are more
cytolytic and produce significant amounts of cytokine when their
activating receptors are engaged.
[0005] In a paper on which the inventor is a principle
investigator, it was found that, as opposed to CD8 immune cells, NK
cells have anti-fibrotic activity through HSC killing (Melhem et
al., J. Hepatology; 2006; 45: 60-71). It has also been reported
that the function of NK cells decreases when the liver disease
progresses into cirrhosis, suggesting that attenuating NK function
is a prerequisite for the progression of the disease (Seki et al.;
Clin Dev Immunol.; 2011; Article ID 868345).
[0006] Human neuroligin-4 (NLG4, NLGn4, NLGn4X) is a family member
of neuronal cell surface proteins called the Neuroligins. FIG. 1
illustrates the neuroligins and their interactions. Members of this
family are membrane-anchored proteins acting as ligands for
beta-neurexins and are thought to be involved in the formation and
remodeling of central nervous system synapses. The encoded protein
interacts with discs, large (Drosophila) homolog 4 (DLG4).
Mutations in this gene have been associated with autism and
Asperger syndrome. High levels of NLGn4 has been reported heart
tissue and lower levels in liver, skeletal muscle and pancreas.
[0007] Neurexin (NRXN) is a presynaptic protein that helps to glue
together neurons at the synapse. Neurexins are located mostly on
the presynaptic membrane and contain a single transmembrane domain.
The extracellular domain interacts with proteins in the synaptic
cleft, most notably neuroligin, while the intracellular cytoplasmic
portion interacts with proteins associated with exocytosis.
[0008] Non-alcoholic fatty-liver disease (NAFLD) is one of the most
prevalent liver diseases in western countries. The full
pathophysiology of NAFLD is still unknown. Both obesity and insulin
resistance are considered to play a strong role in the disease
process. Indeed, the rising rates of obesity and diabetes mellitus
correlate with the increasing incidence of NAFLD, which is the
hepatic and early manifestation of metabolic syndrome. Estimates
suggest that about 20% to 30% of adults in developed countries have
excess fat accumulation in the liver, 50% among people with
diabetes, and about 80% in the obese and morbidly obese
individuals.
[0009] Non-alcoholic steatohepatitis (NASH) is the most severe form
of NAFLD, and can progress to more severe forms of liver disease,
including fibrosis, cirrhosis, and hepatocellular carcinoma.
[0010] The disease begins with the aberrant accumulation of
triglycerides in the liver, resulting in simple steatosis; most
patients who develop steatosis are stable and further disease does
not develop. However, some individuals progress to NASH, the severe
form of NAFLD. In NASH, up to 20% of patients' progress into
cirrhosis. The clinical implications of NAFLD are derived mostly
from its potential to progress to cirrhosis and liver failure.
[0011] There is an unmet medical need for compositions and methods
for treating NAFLD and preventing the progression to cirrhosis and
hepatocellular carcinoma. Nowhere in the art has it been suggested
that treating, attenuating and/or preventing progression of liver
disorders can be achieved by administering an agent capable of
interfering with, inhibiting and/or preventing NLGn4-Nrx1b
protein-protein interaction.
SUMMARY OF THE INVENTION
[0012] The present invention stems, in part, from the surprising
finding that liver disorders can be treated and/or attenuated by
administering to a subject in need thereof an agent capable of
interfering, inhibiting and/or preventing NLGn4-Nrx1b
protein-protein interaction. The agent may be an anti-NLGn4
antibody, an anti-Nrx1b antibody, recombinant NLGn4, recombinant
Nrx1b or any fragment, analog or derivative thereof capable of
interfering, inhibiting and/or preventing the NLGn4-Nrx1b
interaction.
[0013] According to some embodiments, the invention provides a
method of treating, attenuating and/or preventing progression of a
liver disorder in a subject, the method comprising administering to
the subject in need thereof a therapeutically effective amount of
an agent capable of interfering, inhibiting and/or preventing with
neuroligin 4 (NLGn4)-Neurexin 10 (Nrx1b) protein interaction;
thereby treating, attenuating and/or preventing progression of the
liver disorder.
[0014] According to some embodiments, the invention provides a
composition for use in the treatment, attenuation and/or prevention
of progression of a liver disorder, the composition comprising a
therapeutically effective amount of an agent capable of
interfering, inhibiting and/or preventing with neuroligin 4
(NLGn4)-Neurexin 1.beta. (Nrx1b) protein interaction. According to
some embodiments, the composition is suitable for administration to
a subject suffering from a liver disorder. According to some
embodiments, the composition is suitable for administration to an
immune cell population of the subject.
[0015] According to some embodiments, the agent comprises
recombinant NLGn4.
[0016] According to some embodiments, the recombinant NLGn4 is
soluble (rsNLGn4); wherein the rsNLGn4 comprises the extracellular
domain of NLGn4 as set forth in SEQ ID NO: 7 or a fragment,
derivative or analog thereof. According to some embodiments, the
rsNLGn4 consists essentially of SEQ ID NO: 7 or a fragment,
derivative or analog thereof. According to some embodiments, the
recombinant NLGn4 comprises Gln42-Ser676 of NP_065793. According to
some embodiments, the recombinant NLGn4 consists essentially of
Gln42-Ser676 of NP_065793.
[0017] According to some embodiments, the rsNLGn4 competes with
endogenous NLGn4 for binding to Nrx1b.
[0018] According to some embodiments, the rsNLGn4 is devoid of the
intracellular domain and/or the transmembrane domain of NLGn4.
[0019] According to some embodiments, the agent comprises a fusion
protein comprising rsNLGn4 and an immunoglobulin molecule.
According to some embodiments, the agent comprises a fusion protein
comprising rsNLGn4 and an Fc domain of an immunoglobulin molecule,
also referred to herein as rsNGLn4-Fc fusion protein and rsNLGn4-Fc
chimera. According to some embodiments, the Fc domain may include
part or the entire CH1 domain, CH2 domain, CH3 domain and hinge
region of IgG1, IgG2, IgG3 and IgG4. According to some embodiments,
the Fc domain may be devoid of the CH1 domain. According to some
embodiments, the Fc domain comprises Pro100-Lys330 of Human IgG1.
According to some embodiments, the Fc domain consists essentially
of Pro100-Lys330 of Human IgG1. According to some embodiments, the
Fc domain may be linked to rsNLGn4 through a linker. According to
some embodiments, the linker may include the amino acid sequence
set forth in SEQ ID NO: 13 (IEGRMD).
[0020] According to some embodiments, the agent is a chimeric
protein formed from NLGn4 polypeptides or fragments fused with a
second polypeptide to form a soluble NLGn4. According to some
embodiments, the agent comprises a DNA sequences, which combine two
partial DNA sequences, one sequence encoding soluble fragments of
NLGn4 (i.e. the DNA sequence encoding the extracellular domain of
NLGn4) and the other partial sequence encoding all domains except
the first domain of the constant region of the heavy chain of human
immunoglobulin IgG, IgA, IgM, or IgE. These DNA sequences may
subsequently be expressed in target cells using expression vectors
as known in the art, thereby obtaining endogenous expression of
recombinant proteins having the extracellular domain of NLGn4
joined to the Fc fragment of an immunoglobulin molecule.
[0021] According to some embodiments, the agent comprises
recombinant Nrx1b. According to some embodiments, the recombinant
Nrx1b is soluble (rsNrx1b); wherein the rsNrx1b comprises the
extracellular domain of Nrx1b as set forth in SEQ ID NO: 10 or a
fragment, derivative or analog thereof. According to some
embodiments, the recombinant Nrx1b comprises Ala51-Ser363 of
NP_620072. According to some embodiments, the recombinant Nrx1b
consists essentially of Ala51-Ser363 of NP_620072. According to
some embodiments, the rsNrx1b consists essentially of SEQ ID NO: 10
or a fragment, derivative or analog thereof.
[0022] According to some embodiments, the rsNrx1b competes with
endogenous Nrx1b for binding to NLGn4.
[0023] According to some embodiments, the rsNrx1b is devoid of the
intracellular domain and/or the transmembrane domain of Nrx1b.
[0024] According to some embodiments, NLGn4 is encoded by the
sequence set forth in SEQ ID NO: 1.
[0025] According to some embodiments, the agent comprises a fusion
protein comprising rsNrx1b and an immunoglobulin molecule.
According to some embodiments, the agent comprises a fusion protein
comprising rsNrx1b and an Fc domain of an immunoglobulin molecule,
also referred to herein as rsNrx1b-Fc fusion protein and rsNrx1b-Fc
chimera. According to some embodiments, the Fc domain may include
part or all of the CH1 domain, the CH2 domain, the CH3 domain and
hinge region of IgG1, IgG2, IgG3 and IgG4. According to some
embodiments, the Fc domain may be devoid of the CH1 domain.
According to some embodiments, the Fc domain may include all
domains of the Fc domain apart from CH1. According to some
embodiments, the Fc domain comprises Pro100-Lys330 of Human IgG1.
According to some embodiments, the Fc domain consists essentially
of Pro100-Lys330 of Human IgG1. According to some embodiments, the
Fc domain may be linked to rsNrx1b through a linker. According to
some embodiments, the linker may have the amino acid sequence set
forth in SEQ ID NO: 13 (IEGRMD).
[0026] According to some embodiments, the agent is a chimeric
protein formed from Nrx1b polypeptides or fragments fused with a
second polypeptide to form a soluble Nrx1. According to some
embodiments, the agent comprises a DNA sequences, which combine two
partial DNA sequences, one sequence encoding soluble fragments of
Nrx1b (i.e. the DNA sequence encoding the extracellular domain of
Nrx1b) and the other partial sequence encoding all domains except
the first domain of the constant region of the heavy chain of human
immunoglobulin IgG, IgA, IgM, or IgE. These DNA sequences may
subsequently be expressed in target cells using expression vectors
as known in the art, thereby obtaining endogenous expression of
recombinant proteins having the extracellular domain of Nrx1b
joined to the Fc fragment of an immunoglobulin molecule.
[0027] According to some embodiments, Nrx1b is encoded by the
sequence set forth in SEQ ID NO: 6.
[0028] According to some embodiments, the agent is an antibody to
human NLGn4 and/or its receptor Nrx1b.
[0029] According to some embodiments, the antibody is a monoclonal
antibody, according to additional embodiments the antibody is a
humanized monoclonal antibody.
[0030] According to some embodiments, the humanized monoclonal
antibody is an antibody directed to human NLGn4.
[0031] According to some embodiments, the antibody interferes with
the binding of NLGn4 to NrX1b. According to some embodiments, the
antibody is capable of reducing NLGn4 protein levels.
[0032] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to the
interaction domain of NLGn4, the interaction domain mediating the
interaction between NLGn4 to NrX1b.
[0033] According to some embodiments, the anti-NLGn4 antibody or a
fragment, derivative or analog thereof is capable of binding to
amino acids 359-364, set forth in SEQ ID NO: 2 (QGEFLN), of the
human NLGN4 protein.
[0034] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid E361 of human NLGn4.
[0035] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid L363 of human NLGn4.
[0036] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope within residues 261-270, set forth in SEQ ID NO: 3
(SLLTLSHYSE), of human NLGn4.
[0037] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid H267 of human NLGn4.
[0038] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope within residues 461-470, set forth in SEQ ID NO: 4
(AQYGSPTYFY), of human NLGn4.
[0039] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid Y463 of human NLGn4.
[0040] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope within residues 265-275, set forth in SEQ ID NO: 5
(LSHYSEGLFQK), of human NLGn4.
[0041] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid E270 of human NLGn4.
[0042] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising an amino acid selected from the group consisting
of E361, L363, H267, Y463, E270 or any combination thereof. Each
possibility is a separate embodiment of the invention.
[0043] According to some embodiments, antibody is an antibody to
human Nrx1b.
[0044] According to some embodiments, the Nrx1b monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to the interaction domain of Nrx1b, the interaction domain
mediating the interaction between NLGn4 to NrX1b.
[0045] According to some embodiments, the anti-Nrx1b antibody or a
fragment, derivative or analog thereof is capable of binding to
amino acids 103-109, set forth in SEQ ID NO: 7 (LLADTPV) and/or
amino acids 234-239 as set forth in SEQ ID NO: 7 (CSVVDD), of the
human Nrxb1 protein. Each possibility is a separate embodiment of
the invention.
[0046] According to some embodiments, the liver disorder is
selected from the group consisting of: non-alcoholic fatty liver
disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis,
hepatitis, liver adenoma, insulin hypersensitivity, liver cancer
and any combination thereof. Each possibility is a separate
embodiment of the invention.
[0047] According to some embodiments, the liver disorder is
characterized by NLGn4 overexpression. According to some
embodiments, NLGn4 overexpression comprises a 2, 3, 4, 5-10 fold or
more increase in NLGn4 expression relative to the expression level
obtained in normal subjects. Each possibility is a separate
embodiment of the invention. According to some embodiments, the
overexpression attenuates NK cell activity, inhibits the expression
of NLGn4 and modulates and/or activates the function of the NK
cell.
[0048] According to some embodiments, the liver disorder is NAFLD
and/or hepatocellular carcinoma. According to some embodiments, the
liver disorder is NAFLD. According to some embodiments, the liver
disorder is hepatocellular carcinoma.
[0049] According to some embodiments, administering to the subject
a therapeutically effective amount of a humanized, monoclonal
antibody to NLGn4 and/or its receptor Nrx1b comprises administering
the composition to an immune cell population of the subject.
[0050] According to some embodiments, administering a
therapeutically effective amount of a humanized, monoclonal
antibody to NLGn4 to a subject reduces the NLGn4 protein levels in
the subject's NK cells. According to some embodiments, reducing
NLGn4 protein levels in NK cells reduces the activity of hepatic
stellate cells (also referred to herein as HSCs). According to some
embodiments, reducing NLGn4 protein levels increases apoptosis of
the hepatic stellate cells.
[0051] According to some embodiments, there is provided a humanized
monoclonal antibody reactive with a human NLGN4. According to some
embodiments, the human NLGn4 protein is a polypeptide sequence
comprising SEQ ID NO: 1.
[0052] According to some embodiments, the anti-NLGn4 monoclonal
antibody or a fragment, derivative or analog thereof is capable of
binding to amino acids 359-364, set forth in SEQ ID NO: 2 (QGEFLN),
of the human NLGN4 protein.
[0053] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope comprising the amino acid E361 of human
NLGn4.
[0054] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope comprising the amino acid L363 of human
NLGn4.
[0055] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope within residues 261-270, set forth in SEQ ID
NO: 3 (SLLTLSHYSE), of human NLGn4.
[0056] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope comprising the amino acid H267 of human
NLGn4.
[0057] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope within residues 461-470, set forth in SEQ ID
NO: 4 (AQYGSPTYFY), of human NLGn4.
[0058] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope comprising the amino acid Y463 of human
NLGn4.
[0059] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an epitope within residues 265-275, set forth in SEQ ID
NO: 5 (LSHYSEGLFQK), of human NLGn4.
[0060] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to an amino acid selected from the group consisting of
E361, L363, H267, Y463, E270 or any combination thereof, of human
NLGn4.
[0061] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
reducing human NLGn4 protein levels.
[0062] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
interfering with, inhibiting and/or preventing binding of NLGn4 to
Nrx1b.
[0063] According to some embodiments, there is provided a
composition comprising a therapeutically effective amount of an
agent capable of interfering with, inhibiting and/or preventing
NLGn4-Nrx1b protein-protein interaction and a pharmaceutically
acceptable carrier. According to some embodiments, the composition
is for the use of treating a liver disorder. According to some
embodiments, the liver disorder is selected from the group
consisting of: non-alcoholic fatty liver disease (NAFLD),
non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, liver
adenoma, insulin hypersensitivity, liver cancer and any combination
thereof. Each possibility is a separate embodiment of the
invention. According to some embodiments, the liver disorder is
NAFLD and/or hepatocellular carcinoma. According to some
embodiments, the liver disorder is NAFLD. According to some
embodiments, the liver disorder is hepatocellular carcinoma.
[0064] According to some embodiments, the agent comprises
recombinant NLGn4. According to some embodiments, the recombinant
NLGn4 is soluble (rsNLGn4); wherein the rsNLGn4 comprises the
extracellular domain of NLGn4 or a fragment, derivative or analog
thereof.
[0065] According to some embodiments, the extracellular domain of
NLGn4 consists of SEQ ID NO: 7. According to some embodiments, the
rsNLGn4 consists of SEQ ID NO: 7, or a fragment, derivative or
analog thereof.
[0066] According to some embodiments, the recombinant NLGn4
comprises Gln42-Ser676 of NP_065793. According to some embodiments,
the recombinant NLGn4 consists essentially of Gln42-Ser676 of
NP_065793.
[0067] According to some embodiments, the rsNLGn4 competes with
endogenous NLGn4 for binding to Nrx1b.
[0068] According to some embodiments, the rsNLGn4 is devoid of the
intracellular domain and/or the transmembrane domain of NLGn4.
[0069] According to some embodiments, the agent comprises a fusion
protein comprising rsNLGn4 and an immunoglobulin molecule.
According to some embodiments, the agent comprises a fusion protein
comprising rsNLGn4 and an Fc domain of an immunoglobulin molecule.
According to some embodiments, the Fc domain may include part or
the entire CH1 domain, CH2 domain, CH3 domain and hinge region of
IgG1, IgG2, IgG3 and IgG4. According to some embodiments, the Fc
domain may be devoid of the CH1 domain. According to some
embodiments, the Fc domain comprises Pro100-Lys330 of Human IgG1.
According to some embodiments, the Fc domain consists essentially
of Pro100-Lys330 of Human IgG1. According to some embodiments, the
Fc domain may be linked to rsNLGn4 through a linker. According to
some embodiments, the linker may include the amino acid sequence
set forth in SEQ ID NO: 13 (IEGRMD).
[0070] According to some embodiments, the agent is a chimeric
protein formed from NLGn4 polypeptides or fragments fused with a
second polypeptide to form a soluble NLGn4. According to some
embodiments, the agent comprises a DNA sequences, which combine two
partial DNA sequences, one sequence encoding soluble fragments of
NLGn4 (i.e. the DNA sequence encoding the extracellular domain of
NLGn4) and the other partial sequence encoding all domains except
the first domain of the constant region of the heavy chain of human
immunoglobulin IgG, IgA, IgM, or IgE. These DNA sequences may
subsequently be expressed in target cells using expression vectors
as known in the art, thereby obtaining endogenous expression of
recombinant proteins having the extracellular domain of NLGn4
joined to the Fc fragment of an immunoglobulin molecule.
[0071] According to some embodiments, NLGn4 is encoded by the
sequence set forth in SEQ ID NO: 1.
[0072] According to some embodiments, the agent comprises
recombinant Nrx1b. According to some embodiments, the recombinant
Nrx1b is soluble (rsNrx1b); wherein the rsNrx1b comprises the
extracellular domain of Nrx1b or a fragment, derivative or analog
thereof capable of binding NLGn4.
[0073] According to some embodiments, the recombinant Nrx1b
comprises Ala51-Ser363 of NP_620072. According to some embodiments,
the recombinant Nrx1b consists essentially of Ala51-Ser363 of
NP_620072.
[0074] According to some embodiments, the extracellular domain of
Nrx1b consists of SEQ ID NO: 10. According to some embodiments, the
rsNrx1b consists of SEQ ID NO: 10, or a fragment, derivative or
analog thereof.
[0075] According to some embodiments, the rsNrx1b competes with
endogenous Nrx1b for binding to NLGn4.
[0076] According to some embodiments, the rsNrx1b is devoid of the
intracellular domain and/or the transmembrane domain of Nrx1b.
[0077] According to some embodiments, Nrx1b is encoded by the
sequence set forth in SEQ ID NO: 6.
[0078] According to some embodiments, the agent comprises a fusion
protein comprising rsNrx1b and an immunoglobulin molecule.
According to some embodiments, the agent comprises a fusion protein
comprising rsNrx1b and an Fc domain of an immunoglobulin molecule.
According to some embodiments, the Fc domain may include part or
the entire CH1 domain, the CH2 domain, the CH3 domain and hinge
region of IgG1, IgG2, IgG3 and IgG4. According to some embodiments,
the Fc domain may be devoid of the CH1 domain. According to some
embodiments, the Fc domain may include all domains of the Fc domain
apart from CH1. According to some embodiments, the Fc domain
comprises Pro100-Lys330 of Human IgG1. According to some
embodiments, the Fc domain consists essentially of Pro100-Lys330 of
Human IgG1. According to some embodiments, the Fc domain may be
linked to rsNrx1b through a linker. According to some embodiments,
the linker may have the amino acid sequence set forth in SEQ ID NO:
13 (IEGRMD).
[0079] According to some embodiments, the agent is a chimeric
protein formed from Nrx1b polypeptides or fragments fused with a
second polypeptide to form a soluble Nrx1. According to some
embodiments, the agent comprises a DNA sequences, which combine two
partial DNA sequences, one sequence encoding soluble fragments of
Nrx1b (i.e. the DNA sequence encoding the extracellular domain of
Nrx1b) and the other partial sequence encoding all domains except
the first domain of the constant region of the heavy chain of human
immunoglobulin IgG, IgA, IgM, or IgE. These DNA sequences may
subsequently be expressed in target cells using expression vectors
as known in the art, thereby obtaining endogenous expression of
recombinant proteins having the extracellular domain of Nrx1b
joined to the Fc fragment of an immunoglobulin molecule.
[0080] According to some embodiments, the agent comprises
recombinant NLGn4 and recombinant Nrx1b. According to some
embodiments, the recombinant NlGn4 and Nrx1b are soluble (rsNLGN4
and rsNrx1b, respectively); wherein the rsNLGn4 comprises the
extracellular domain of NLGn4 or a fragment, derivative or analog
thereof capable of binding NLGn4; and wherein the rsNrx1b comprises
the extracellular domain of Nrx1b or a fragment, derivative or
analog thereof capable of binding NLGn4.
[0081] According to some embodiments, the agent is an antibody to
NLGn4. According to some embodiments, the antibody is a monoclonal
antibody. According to some embodiments, the agent is an antibody
to Nrxb1. According to some embodiments, the antibody is a
monoclonal antibody. According to some embodiments, the antibody is
a humanized antibody. According to some embodiments, there is
provided a composition comprising a therapeutically effective
amount of monoclonal antibodies to NLGn4 and to Nrxb1.
[0082] According to some embodiments, the composition is for the
use in treating, attenuating and/or preventing progression of a
liver disorder in a subject.
[0083] According to some embodiments, the composition further
comprises a GLUT4 antagonist. According to some embodiments, NLGn4
expression is regulated by a specific type of ionotropic glutamate
receptor N-methyl-D-aspartate (NMDA or GLUT4 receptor; NMDAR).
According to some embodiments, NLGn4 is linked to NMDR and both
localize and bind PSD-95; a post synaptic density protein (PSD)
According to some embodiments, the composition comprises an NMDAR
antagonist selected from the group consisting of: Ketamin,
Amantadine, Phencyclidine, Nitrous oxide, Dextromethorphan (and
dextrorphan), Memantine, Ethanol, Riluzole, Xenon, HU-211, Lead
(Pb2+), Conantokins, and Huperzine A.
[0084] According to an alternative embodiment, administering an
N-methyl D aspartate receptor (NMDAR) agonist can increase
NMDAR-mediated NLGn4 expression and as a result attenuate NK cell
activity. Non-limiting examples of NMDAR agonists are
Aminocyclopropanecarboxylic acid, D-Cycloserine,
cis-2,3-Piperidinedicarboxylic acid, L-aspartate, L-alanine,
Quinolinate, Homocysterate, D-serine, and ACPL.
[0085] According to some embodiments, the pharmaceutical
composition further comprises a pharmaceutically acceptable
carrier.
[0086] According to some embodiments, there is provided method of
treating, attenuating and/or preventing progression of a liver
disorder in a subject, the method comprising administering to the
subject in need thereof a therapeutically effective amount of an
agent capable of interfering with, inhibiting and/or preventing
neuroligin 4 (NLGn4)-Neurexin 1.beta. (Nrx1b) protein-protein
interaction; thereby treating, attenuating and/or preventing
progression of the liver disorder.
[0087] According to some embodiments, there is provided composition
for use in treating, attenuating and/or preventing progression of a
liver disorder in a subject, the composition comprising a
therapeutically effective amount of an agent capable of interfering
with, inhibiting and/or preventing neuroligin 4 (NLGn4)-Neurexin 10
(Nrx1b) protein-protein interaction.
[0088] According to some embodiments, there is provided composition
comprising a therapeutically effective amount of an agent capable
of interfering with, inhibiting and/or preventing neuroligin 4
(NLGn4)-Neurexin 1.beta. (Nrx1b) protein-protein interaction for
use in treating, attenuating and/or preventing progression of a
liver disorder.
[0089] According to some embodiments, there is provided an agent
capable of interfering with, inhibiting and/or preventing
neuroligin 4 (NLGn4)-Neurexin 10 (Nrx1b) protein-protein
interaction for use in preparation of a composition for treating,
attenuating and/or preventing progression of a liver disorder.
[0090] According to some embodiments, the agent comprises
recombinant soluble NLGn4 (rsNLGn4); wherein the rsNLGn4 comprises
the extracellular domain of NLGn4 or a fragment, derivative or
analog thereof. According to some embodiments, the extracellular
domain of NLGn4 consists of SEQ ID NO: 7 or a fragment, derivative
or analog thereof. According to some embodiments, the rsNLGn4
competes with endogenous NLGn4 for binding to Nrx1b. According to
some embodiments, the rsNLGn4 is devoid of the intracellular domain
and/or transmembrane domain of NLGn4. According to some
embodiments, NLGn4 is encoded by the sequence set forth in SEQ ID
NO: 1. According to some embodiments, the agent comprises a fusion
protein comprising the Fc portion of an immunoglobulin molecule and
SEQ ID NO: 7 or a fragment thereof.
[0091] According to some embodiments, the therapeutic agent
comprises recombinant soluble Nrx1b (rsNrx1b); wherein the rsNrx1b
comprises the extracellular domain of Nrx1b or a fragment,
derivative or analog thereof capable of binding the NLGn4.
According to some embodiments, the extracellular domain of Nrx1b
consists of SEQ ID NO: 10. According to some embodiments, the
rsNrx1b consists of SEQ ID NO: 10, or a fragment, derivative or
analog thereof. According to some embodiments, the rsNrx1b competes
with endogenous Nrx1b for binding to NLGn4. According to some
embodiments, the rsNrx1b is devoid of the intracellular domain
and/or the transmembrane domain of Nrx1b. According to some
embodiments, the Nrx1b is encoded by the sequence set forth in SEQ
ID NO: 6. According to some embodiments, the agent comprises a
fusion protein comprising the Fc portion of an immunoglobulin
molecule and SEQ ID NO: 10 or a fragment thereof.
[0092] According to some embodiments, the therapeutic agent
comprises an anti-NLGn4 antibody, or a fragment, derivative or
analog thereof, comprising at least the antigen-binding portion
thereof. According to some embodiments, the antibody is a humanized
monoclonal anti-NLGn4 antibody. According to some embodiments, the
anti-NLGn4 antibody is capable of binding specifically to an
interaction domain of human NLGn4. According to some embodiments,
the anti-NLGn4 antibody is capable of binding specifically to amino
acids 359-364 of the human NLGN4 protein. According to some
embodiments, the anti-NLGn4 antibody is capable of binding to an
epitope within residues 261-270 of the human NLGn4, as set forth in
SEQ ID NO: 3 (SLLTLSHYSE). According to some embodiments, the
anti-NLGn4 antibody is capable of binding to an epitope within
residues 265-275, as set forth in SEQ ID NO: 5 (LSHYSEGLFQK), of
the human NLGn4. According to some embodiments, the anti-NLGn4
antibody is capable of binding to an epitope within residues
461-470 of the human NLGn4, as set forth in SEQ ID NO: 4
(AQYGSPTYFY).
[0093] According to some embodiments, the liver disorder is
selected from the group consisting of: non-alcoholic fatty liver
disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis,
hepatitis, liver adenoma, insulin hypersensitivity, liver cancer
and any combination thereof. According to some embodiments, the
liver disorder is NAFLD.
[0094] According to some embodiments, the liver disorder is
hepatocellular carcinoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIG. 1 shows a schematic representation of the Neuroligins
and their interactions.
[0096] FIG. 2 shows NLGn4 expression in NK cells obtained from NASH
patients with hepatic fibrosis. Isolated NK cells from healthy and
NASH cirrhotic cases were assessed by DNA spotted Microarrays.
[0097] FIG. 3. Shows NLGn4 expression levels in a leptin-deficient
fatty liver disease mouse model as compared control mice.
[0098] FIG. 4A shows serum ALT levels in homozygous NLG4-/- mice as
compared to WT, upon induction of fibrosis.
[0099] FIG. 4B shows alpha-SMA levels in homozygous NLG4-/- mice as
compared to WT, upon induction of fibrosis.
[0100] FIG. 5A shows NLGn4 protein expression levels in NK cells
isolated from NAFLD patients and incubated with a hepatic stellate
cell line (LX2) in the absence or in the presence of increasing
concentrations of an anti-NLGn4 antibody.
[0101] FIG. 5B shows CD107a levels in NK cells isolated from NAFLD
patients and incubated with a hepatic stellate cell line in the
absence or in the presence of increasing concentrations of an
anti-NLGn4 antibody.
[0102] FIG. 5C shows alpha-SMA intensities (LX2 activity) in LX2
cells, incubated with NK cells isolated from NAFLD patients in the
absence or in the presence of increasing concentrations of an
anti-NLGn4 antibody.
[0103] FIG. 6A shows CD107a levels in NK cells isolated from mice
induced with carbon tetrachloride to cause acute hepatic fibrosis
(or left untreated) and either injected with an anti-NLGn4 antibody
or left untreated.
[0104] FIG. 6B shows serum ALT levels in WT mice induced with
carbon tetrachloride to cause acute hepatic fibrosis (or left
untreated) and either injected with an anti-NLGn4 antibody or left
untreated.
[0105] FIG. 6C shows alpha-SMA levels in WT mice induced with
carbon tetrachloride to cause acute hepatic fibrosis (or left
untreated) and either injected with an anti-NLGn4 antibody or left
untreated.
[0106] FIG. 7 shows .alpha.-feto-protein levels in mice induced for
hepatocellular carcinoma by injection of human hepatoma cells
(Hep3B) co-cultured with either wt or NLGn4-/- NK cells.
[0107] FIG. 8A shows CD107a levels in NK cells isolated from NAFLD
patients and incubated with a hepatic stellate cell line in the
absence or in the presence of recombinant Nrx1b (4 nM, 10 nM).
[0108] FIG. 8B shows alpha-SMA intensities (LX2 activity) in LX2
cells, incubated with NK cells isolated from NAFLD patients in the
absence or in the presence of recombinant Nrx1b (4 nM, 10 nM).
[0109] FIG. 9A shows serum ALT levels in WT mice induced with
carbon tetrachloride to cause acute hepatic fibrosis (or left
untreated) and either injected with a recombinant Nrx1b or left
untreated.
[0110] FIG. 9B shows alpha-SMA levels in WT mice induced with
carbon tetrachloride to cause acute hepatic fibrosis (or left
untreated) and either injected with recombinant Nrx1b or left
untreated.
DETAILED DESCRIPTION
[0111] In the following description, various aspects of the
invention will be described. For the purpose of explanation,
specific details are set forth in order to provide a thorough
understanding of the invention. However, it will also be apparent
to one skilled in the art that the invention may be practiced
without specific details being presented herein. Furthermore,
well-known features may be omitted or simplified in order not to
obscure the invention.
[0112] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0113] The following are terms which are used throughout the
description and which should be understood in accordance with the
various embodiments to mean as follows:
[0114] The terms "administration" and "administering" refer to
providing or giving a subject a therapeutic agent (e.g. a
recombinant polypeptide), by any effective route. Exemplary routes
of administration include, but are not limited to, injection or
infusion (such as subcutaneous, intramuscular, intradermal,
intraperitoneal, intrathecal, intravenous, intracerebroventricular,
intrastriatal, intracranial and into the spinal cord), oral,
intraductal, sublingual, rectal, transdermal, intranasal, vaginal
and inhalation routes. Each possibility is a separate
embodiment.
[0115] The terms "peptide", "polypeptide" and "protein" as used
herein are intended to encompass any amino acid sequence including
modified sequences, such as but not limited to modified NLGn4
and/or Nrx1b. The terms "peptide" and "polypeptide" are
specifically intended to cover naturally occurring proteins, as
well as those, which are recombinantly or synthetically produced.
"Peptides" of the invention also include modified peptides (with
amino acid substitutions, both conservative and non-conservative as
described below) that have the same or improved activity as a
wild-type or unmodified peptide. "Salts" of the peptides of the
invention contemplated by the invention are physiologically and
pharmaceutically acceptable organic and inorganic salts.
[0116] Isolated polynucleotide sequences comprising at least one
sequence encoding a peptide, peptide analog, conjugate or fusion
protein of NLGn4 or Nrx1b are also included in the scope of the
present invention. According to some embodiments, the
polynucleotide sequence encoding the peptide or peptide analog is
translationally linked to another polynucleotide sequence such as
an RNA or DNA molecule and is recombinantly expressed within target
cells. According to other embodiments, the polynucleotide sequence
is part of a recombinant viral or bacterial vector.
[0117] The term "analog" refers to a molecule, which has the amino
acid sequence according to the invention except for one or more
amino acid changes. Analogs according to the present invention may
include peptidomimetics. "Peptidomimetic" refers to a peptide
modified in such a way that it includes at least one non-coded
residue or non-peptidic bond. Such modifications include, e.g.,
alkylation and more specific methylation of one or more residues,
insertion of or replacement of natural amino acid by non-natural
amino acids, replacement of an amide bond with another covalent
bond. A peptidomimetic according to the present invention may
optionally comprise at least one bond, which is an
amide-replacement bond such as urea bond, carbamate bond,
sulfonamide bond, hydrazine bond, or any other covalent bond. The
design of appropriate "analogs" may be computer assisted. Analogs
are included in the invention as long as they remain
pharmaceutically acceptable.
[0118] The amino acids used in this invention are those, which are
available commercially or are available by routine synthetic
methods. Certain residues may require special methods for
incorporation into the peptide, and either sequential, divergent or
convergent synthetic approaches to the peptide sequence are useful
in this invention. Natural coded amino acids and their derivatives
are represented by three-letter codes according to IUPAC
conventions. When there is no indication, the L isomer was used.
The D isomers are indicated by "D" before the residue
abbreviation.
[0119] Conservative substitutions of amino acids as known to those
skilled in the art are within the scope of the present invention.
Conservative amino acid substitutions include replacement of one
amino acid with another having the same type of functional group or
side chain, e.g., aliphatic, aromatic, positively charged,
negatively charged. These substitutions may enhance oral
bioavailability, penetration into the islets, targeting to specific
beta cell populations, immunogenicity, and the like. One of skill
will recognize that individual substitutions, deletions or
additions to a peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" where the alteration results in the substitution
of an amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art.
[0120] The following six groups each contain amino acids that are
conservative substitutions for one another:
[0121] 1) Alanine (A), Serine (S), Threonine (T);
[0122] 2) Aspartic acid (D), Glutamic acid (E);
[0123] 3) Asparagine (N), Glutamine (Q);
[0124] 4) Arginine (R), Lysine (K);
[0125] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0126] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0127] The peptides of the present invention may be produced by any
method known in the art, including recombinant and synthetic
methods. Synthetic methods include exclusive solid phase synthesis,
partial solid phase synthesis, fragment condensation, or classical
solution synthesis. Solid phase peptide synthesis procedures are
well known to one skilled in the art and described, for example by
John Morrow Stewart and Janis Dillaha Young, Solid Phase
Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In
some embodiments, synthetic peptides are purified by preparative
high performance liquid chromatography (Creighton T. (1983)
Proteins, structures and molecular principles. WH Freeman and Co.
N.Y.) The peptide sequence may be confirmed by amino acid
sequencing using methods known to one skilled in the art.
[0128] In some embodiments, recombinant protein techniques are used
to generate the peptide of the present invention. In some
embodiments, recombinant protein techniques are used for generation
of relatively long polypeptides (typically longer than 18 amino
acids) or nucleic acid sequences or viral or bacterial vectors for
vaccine formulation. Recombinant techniques are described for
example by Bitter et al., (1987) Methods in Enzymol. 153:516-544,
Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al.
(1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J.
6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et
al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell.
Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for
Plant Molecular Biology, Academic Press, NY, Section VIII, pp
421-463.
[0129] The terms "recombinant" and "recombinant protein" refer,
according to some embodiments, to proteins having sequence that is
not naturally occurring. The term recombinant includes proteins
that have been altered by addition, substitution, or deletion of a
portion of a natural nucleic acid molecule or protein. According to
some embodiments, the term recombinant refers to proteins that have
been altered so as to generate soluble forms of NLGn4 and/or
NRx1b.
[0130] As used herein, the term "soluble" refers to a polypeptide
that is not bound to the cell membrane. Usually, a receptor is in
soluble form when its amino acid sequence lacks the transmembrane
domain. In this context, a form will be soluble if using
conventional assays known to one of skill in the art most of this
form can be detected in fractions that are not associated with the
membrane, e.g., in cellular supernatants or serum.
[0131] In the present invention, "Fc domain" means the constant
domain of an antibody. It is preferably the Fc domain of human
IgG1, but is not limited thereto. In addition to human IgG1, IgG2,
IgG3 and IgG4 may also be used, and the Fc domain may comprise.
Preferably, it may comprise the CH2 domain, the CH3 domain and the
hinge region, excluding the CH1 domain.
[0132] As used herein, the term "NLGn4-Fc fusion protein" refers to
a product resulting from linkage of the entire or a part of the
NLGn4 protein with immunoglobulin Fc region (or part thereof). As
used herein, the term "rsNLGn4-Fc fusion protein" refers to a
product resulting from linkage of the extracellular domain of NLGn4
protein with immunoglobulin Fc region (or part thereof). The
linkage may be by enzymatic action, or resulting from expression of
two polypeptides into a single polypeptide by gene manipulation. In
the NLGn4-Fc and rsNLGn4-Fc fusion proteins, the NLGn4 protein (or
part thereof) and the immunoglobulin Fc region may be directly
linked with each other, or linked via a peptide linker, but is not
limited thereto.
[0133] As used herein, the term "Nrx1b-Fc fusion protein" refers to
a product resulting from linkage of the entire or a part of the
Nrx1b protein with immunoglobulin Fc region (or part thereof). As
used herein, the term "rsNrx1b-Fc fusion protein" refers to a
product resulting from linkage of the extracellular domain of Nrx1b
protein with immunoglobulin Fc region (or part thereof). The
linkage may be by enzymatic action, or resulting from expression of
two polypeptides into a single polypeptide by gene manipulation. In
the Nrx1b-Fc and rsNrx1b fusion proteins, the Nrx1b protein (or
part thereof) and the immunoglobulin Fc region may be directly
linked with each other, or linked via a peptide linker, but is not
limited thereto.
[0134] As used herein, the term "immunoglobulin (Ig) Fc region"
refers to a part of immunoglobulin that contains the heavy-chain
constant region 2 (CH2), the heavy-chain constant region 3 (CH3),
and a hinge region, excluding the variable regions of the heavy and
light chains, the heavy-chain constant region 1 (CH1) and the
light-chain constant region 1 (CL1) of the immunoglobulin. The
immunoglobulin Fc region of the present invention includes a native
amino acid sequence, and a sequence derivative thereof. In
addition, the immunoglobulin Fc region may be an Fc region that is
derived from IgG, IgM, IgE, IgA or IgD, or that is made by
combinations thereof or hybrids thereof. Preferably, it is derived
from IgG, which is known to enhance the half-life of binding
proteins. More preferably, it is derived from IgG1, but is not
limited thereto.
[0135] The Ig combinations, as used herein, refer to polypeptides
encoding single-chain immunoglobulin Fc regions of the same origin
are linked to a single-chain polypeptide of a different origin to
form a dimer or multimer. That is, a dimer or multimer may be
formed from two or more fragments selected from the group
consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc
fragments.
[0136] Ig hybrids, as used herein, refer to sequences encoding two
or more immunoglobulin Fc regions of different origin are present
in a single-chain immunoglobulin Fc region. In the present
invention, various types of hybrids are possible. That is, domain
hybrids may be composed of one to four domains selected from the
group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA
Fc, IgE Fc and IgD Fc, and may include the hinge region. On the
other hand, IgG is also divided into IgG1, IgG2, IgG3 and IgG4
subclasses, and the present invention includes combinations and
hybrids thereof.
[0137] The term "sequence identity/similarity" refers to the degree
of similarity between two or more nucleic acid sequences, or two or
more amino acid sequences, and is measured as percentage identity;
the higher the percentage, the more identical the sequences are.
Sequence similarity can be measured in terms of percentage
similarity (which takes into account conservative amino acid
substitutions); the higher the percentage, the more similar the
sequences are. Homologs or orthologs of nucleic acid or amino acid
sequences possess a relatively high degree of sequence
identity/similarity when aligned using standard methods.
[0138] The terms "liver disorder", "liver disease" and "hepatic
disease" are used interchangeably and refer to diseases and
disorders that cause the liver to function improperly or to stop
functioning.
[0139] The terms "subject" and "patient" are interchangeable and as
used herein refer to any individual suffering from a liver
disorder.
[0140] The term "treatment" as used herein refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with the disorder
as well as those in which the disorder is to be prevented.
[0141] The terms "composition" and "pharmaceutical composition" are
used interchangeably and as used herein refer to any composition
comprising at least one chemical or biological agent, for example
an antibody.
[0142] The term "pharmaceutically acceptable carrier" refer to any
carrier conventional used in the production of pharmaceutical
compositions. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition, 1975, describes
compositions and formulations suitable for pharmaceutical delivery
of the compositions disclosed herein.
[0143] The terms "Neuroligin 4", "Neuroligin 4X", "NLGn4" and
"NLG4" are interchangeable and as used herein refer to the protein
product of the NLGn4 gene e.g., NP_001269075.1, NP_001269074.1,
NP_851849.1 and NP.sub..crclbar.065793.1.
[0144] The terms "anti-NLGn4 antibody", "an antibody which
recognizes NLGn4", "an antibody against NLGn4" and "an antibody to
NLGn4" are interchangeable, and as used herein refer to an antibody
that binds to the NLGn4 protein with sufficient affinity and
specificity. According to some embodiments, the term refer to an
antibody capable of interfering, inhibiting and/or preventing with
NLGn4 binding to Nrx1b.
[0145] The terms "neurexin-1-beta", "Nrx1b" and "Nrxn1" are
interchangeable and as used herein refer to the protein product of
the Nrx1b gene e.g., NP_001129131.1 NP_004792.1 NP_620072.1.
[0146] The terms "anti-Nrx1b antibody", "an antibody which
recognizes Nrx1b", "an antibody against Nrx1b" and "an antibody to
Nrx1b" are interchangeable, and as used herein refer to an antibody
that binds to the Nrx1b protein with sufficient affinity and
specificity. According to some embodiments, the term refer to an
antibody capable of interfering with, inhibiting and/or preventing
Nrx1b binding to NLGn4.
[0147] It should be understood that, according to the present
invention, anti-human-NLGn4 antibodies and/or anti-human-Nrx1b
antibodies may bind to NLGn4 and/or Nrx1b both in-vivo, i.e. bind
NLGn4/Nrx1b in a patient's NK cells, and/or bind NLGn4/Nrx1b in NK
cells in-vitro, i.e. bind NLGn4/Nrx1b in NK cells extracted from
the patient's body, the NK cells later administered to the
patient.
[0148] The term "antigen" as used herein refers to a molecule or a
portion of a molecule capable of eliciting antibody formation and
being bound by an antibody. An antigen may have one or more than
one epitope. The specific reaction referred to above is meant to
indicate that the antigen will react, in a highly selective manner,
with its corresponding antibody and not with the multitude of other
antibodies, which may be evoked by other antigens. According to
some embodiments, the antigen is an NLGn4 protein or a fragment
thereof. According to some embodiments, the antigen is an Nrx1b
protein or a fragment thereof.
[0149] The term "antigenic determinant" or "epitope" as used herein
refers to the region of an antigen molecule that specifically
reacts with a particular antibody. Peptide sequences derived from
an epitope can be used, alone or in conjunction with a carrier
moiety, applying methods known in the art, to immunize animals and
to produce polyclonal or monoclonal antibodies. Isolated peptides
derived from an epitope may be used in diagnostic methods to detect
antibodies and as therapeutic agents when inhibition of antibodies
is required.
[0150] Antibodies, or immunoglobulins, comprise two heavy chains
linked together by disulfide bonds and two light chains, each light
chain being linked to a respective heavy chain by disulfide bonds
in a "Y" shaped configuration. Proteolytic digestion of an antibody
yields Fv (Fragment variable) and Fc (Fragment crystalline)
domains. The antigen binding domains, Fab, include regions where
the polypeptide sequence varies. The term F(ab').sub.2 represents
two Fab' arms linked together by disulfide bonds. The central axis
of the antibody is termed the Fc fragment. Each heavy chain has at
one end a variable domain (V.sub.H) followed by a number of
constant domains (C.sub.H). Each light chain has a variable domain
(V.sub.L) at one end and a constant domain (C.sub.L) at its other
end, the light chain variable domain being aligned with the
variable domain of the heavy chain and the light chain constant
domain being aligned with the first constant domain of the heavy
chain (CH1). The variable domains of each pair of light and heavy
chains form the antigen-binding site. The domains on the light and
heavy chains have the same general structure and each domain
comprises four framework regions, whose sequences are relatively
conserved, joined by three hyper-variable domains known as
complementarity determining regions (CDRs 1-3). These domains
contribute specificity and affinity of the antigen-binding site.
The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu)
determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM,
respectively). The light chain is either of two isotypes (kappa,
.kappa. or lambda, .lamda.) found in all antibody classes.
[0151] The term "antibody" is used in the broadest sense and
includes monoclonal antibodies (including full length or intact
monoclonal antibodies), polyclonal antibodies, multivalent
antibodies, multi-specific antibodies (e.g., bi-specific
antibodies), and antibody fragments long enough to exhibit the
desired biological activity.
[0152] The antibody according to the present invention is a
molecule comprising at least the antigen-binding portion of an
antibody. Antibody or antibodies according to the invention include
intact antibodies, such as polyclonal antibodies or monoclonal
antibodies (mAbs), as well as proteolytic fragments thereof, such
as the Fab or F(ab')2 fragments. Single chain antibodies also fall
within the scope of the present invention.
[0153] "Antibody fragments" comprise only a portion of an intact
antibody, generally including an antigen-binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having VL, CL, VH and CH1 domains;
(ii) the Fab' fragment, which is a Fab fragment having one or more
cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd
fragment having VH and CH1 domains; (iv) the Fd' fragment having VH
and CH1 domains and one or more cysteine residues at the C-terminus
of the CH1 domain; (v) the Fv fragment having the VL and VH domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 1989, 341, 544-546) which consists of a VH domain; (vii)
isolated CDR regions; (viii) F(ab').sub.2 fragments, a bivalent
fragment including two Fab' fragments linked by a disulphide bridge
at the hinge region; (ix) single chain antibody molecules (e.g.
single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426;
and Huston et al., PNAS (USA) 1988, 85, 5879-5883); (x) "diabodies"
with two antigen binding sites, comprising a heavy chain variable
domain (VH) connected to a light chain variable domain (VL) in the
same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448);
(xi) "linear antibodies" comprising a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions (Zapata et al.
Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).
[0154] Single chain antibodies can be single chain composite
polypeptides having antigen binding capabilities and comprising
amino acid sequences homologous or analogous to the variable
regions of an immunoglobulin light and heavy chain i.e. linked
VH-VL or single chain Fv (scFv).
[0155] The term "neutralizing antibody" as used herein refers to a
molecule having an antigen-binding site to a specific receptor or
ligand target capable of reducing or inhibiting (blocking) activity
or signaling through a receptor, as determined by in-vivo or
in-vitro assays, as per the specification.
[0156] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. mAbs may be
obtained by methods known to those skilled in the art. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al., Nature 1975, 256, 495, or may be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature 1991,
352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597, for
example.
[0157] The mAbs of the present invention may be of any
immunoglobulin class including IgG, IgM, IgE, IgA. A hybridoma
producing a mAb may be cultivated in-vitro or in-vivo. High titers
of mAbs can be obtained by in-vivo production where cells from the
individual hybridomas are injected intra-peritoneally (i.p.) into
pristine-primed Balb/c mice to produce ascites fluid containing
high concentrations of the desired mAbs. mAbs of isotype IgM or IgG
may be purified from such ascites fluids, or from culture
supernatants, using column chromatography methods well known to
those of skill in the art.
[0158] The terms "human antibody" and "humanized antibody" are
interchangeable and as used herein refer to an antibody that
possesses an amino acid sequence, which corresponds to that of an
antibody produced by a human and/or has been made using any of the
techniques for making human antibodies as disclosed herein.
[0159] The terms "molecule having the antigen-binding portion of an
antibody" and "antigen-binding-fragments" as used herein is
intended to include not only intact immunoglobulin molecules of any
isotype and generated by any animal cell line or microorganism, but
also the antigen-binding reactive fraction thereof, including, but
not limited to, the Fab fragment, the Fab' fragment, the
F(ab').sub.2 fragment, the variable portion of the heavy and/or
light chains thereof, Fab mini-antibodies (see WO 93/15210, U.S.
patent application Ser. No. 08/256,790, WO 96/13583, U.S. patent
application Ser. No. 08/817,788, WO 96/37621, U.S. patent
application Ser. No. 08/999,554, the entire contents of which are
incorporated herein by reference), dimeric bispecific
mini-antibodies (see Muller et al., 1998) and single-chain
antibodies incorporating such reactive fraction, as well as any
other type of molecule in which such antibody reactive fraction has
been physically inserted. Such molecules may be provided by any
known technique, including, but not limited to, enzymatic cleavage,
peptide synthesis or recombinant techniques.
[0160] The invention also provides conservative amino acid variants
of the antibody molecules according to the invention. Variants
according to the invention also may be made that conserve the
overall molecular structure of the encoded proteins. Given the
properties of the individual amino acids comprising the disclosed
protein products, some rational substitutions will be recognized by
the skilled worker. Amino acid substitutions, i.e. "conservative
substitutions," may be made, for instance, on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved.
[0161] The term "antibody analog" as used herein refers to an
antibody derived from another antibody by one or more conservative
amino acid substitutions.
[0162] The term "antibody variant" as used herein refers to any
molecule comprising the antibody of the present invention. For
example, fusion proteins in which the antibody or an
antigen-binding-fragment thereof is linked to another chemical
entity is considered an antibody variant.
[0163] The phrase "capable of inhibiting or blocking the
interaction" as used herein refers to the capability of an agent,
for example an antibody, to interact with a at least one target,
for example a cell's membrane-bound receptor, in such a way that
the target becomes less accessible, preferably inaccessible, to
binding by at least one other agent, for example the receptor's
natural antigen.
[0164] Antibodies according to the invention can be obtained by
administering NLGn4 or Nrx1b peptides, analogs, or cells expressing
these, to an animal, preferably a nonhuman, using routine
protocols. For preparation of mAbs, any technique known in the art
that provides antibodies produced by continuous cell line cultures
can be used. Examples include various techniques, such as those in
Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et
al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in
Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc.
(1985).
[0165] Besides the conventional method of raising antibodies in
vivo, antibodies can be generated in vitro using phage display
technology. Such a production of recombinant antibodies is much
faster compared to conventional antibody production and they can be
generated against an enormous number of antigens. Moreover,
affinity maturation (i.e., increasing the affinity and specificity)
of recombinant antibodies is very simple and relatively fast.
Finally, large numbers of different antibodies against a specific
antigen can be generated in one selection procedure. To generate
recombinant mAbs one can use various methods all based on display
libraries to generate a large pool of antibodies with different
antigen recognition sites. Such a library can be made in several
ways: One can generate a synthetic repertoire by cloning synthetic
CDR regions in a pool of H chain germline genes and thus generating
a large antibody repertoire, from which recombinant antibody
fragments with various specificities can be selected. One can use
the lymphocyte pool of humans as starting material for the
construction of an antibody library. It is possible to construct
naive repertoires of human IgM antibodies and thus create a human
library of large diversity.
[0166] Non-human antibodies may be humanized by any methods known
in the art. In one method, the non-human CDRs are inserted into a
human antibody or consensus antibody FR sequence. Further changes
can then be introduced into the antibody framework to modulate
affinity or immunogenicity.
[0167] Anti-idiotype antibodies specifically immunoreactive with an
antibody of the invention are also comprehended.
[0168] Techniques for the production of single-chain antibodies can
be adapted to produce single-chain antibodies to polypeptides or
polynucleotides of this invention. Also, transgenic mice, or other
organisms such as other mammals, can be used to express humanized
antibodies specific to the polypeptides or polynucleotides of the
invention.
[0169] The invention also provides conservative amino acid variants
of the peptides and antibody molecules according to the invention.
Variants according to the invention also may be made that conserve
the overall molecular structure of the encoded proteins or
peptides. Given the properties of the individual amino acids
comprising the disclosed protein products, some rational
substitutions will be recognized by the skilled worker. Amino acid
substitutions, i.e. "conservative substitutions," may be made, for
instance, on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues involved.
[0170] A humanized antibody, typically has a human FR grafted with
non-human CDRs. Thus, a humanized antibody has one or more amino
acid sequence introduced into it from a source, which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies wherein substantially less than
an intact human V domain has been substituted by the corresponding
sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies.
[0171] The choice of human VH and VL domains to be used in making
the humanized antibodies is very important for reducing
immunogenicity. According to the so-called "best-fit" method, the
sequence of the V domain of a rodent antibody is screened against
the entire library of known human-domain sequences. The human
sequence, which is closest to that of the rodent, is then accepted
as the human FR for the humanized antibody (Sims et al., J.
Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901
(1987)). Another method uses a particular FR derived from the
consensus sequence of all human antibodies of a particular subgroup
of H or L chains. It is further important that antibodies be
humanized with retention of high specificity and affinity for the
antigen and other favorable biological properties. To achieve this
goal, according to a preferred method, humanized antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three-dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the CDR residues are directly and most substantially
involved in influencing antigen binding.
[0172] According to some aspects, the present invention provides
methods and compositions for treating liver disorders by activating
attenuated natural killer (NK) cells and thereby reducing Hepatic
stellate cell (HCSs) induced fibrosis and/or hepatocellular
carcinoma.
[0173] According to some aspects, the present invention provides a
composition for use in treating liver disorders, the composition
comprising an agent capable of activating attenuated natural killer
(NK) cells, thereby reducing Hepatic stellate cell (HCSs) induced
fibrosis and/or hepatocellular carcinoma.
[0174] According to some embodiments, the method includes
administering to the subject in need thereof a therapeutically
effective amount of an agent capable of interfering with,
inhibiting and/or preventing neuroligin 4 (NLGn4)-Neurexin 1.beta.
(Nrx1b) protein-protein interaction; thereby treating, attenuating
and/or preventing progression of the liver disorder. According to
some embodiments, the composition comprises a therapeutically
effective amount of an agent capable of interfering with,
inhibiting and/or preventing neuroligin 4 (NLGn4)-Neurexin 1.beta.
(Nrx1b) protein-protein interaction. According to some embodiments,
the composition is suitable for administration to a subject.
[0175] According to some embodiments, the agent comprises
recombinant soluble NLGn4 (rsNLGn4).
[0176] According to some embodiments, the recombinant soluble human
NLGn4 may function as a decoy competing with endogenous NLGn4 for
binding to Nrx1b.
[0177] According to some embodiments, the recombinant NLGn4
comprises Gln42-Ser676 of NP_065793. According to some embodiments,
the recombinant NLGn4 consists essentially of Gln42-Ser676 of
NP_065793.
[0178] According to some embodiments, the rsNLGn4 comprises the
extracellular domain of NLGn4, the extracellular domain of NLGn4
having the polypeptide sequence set forth in SEQ ID: 7.
TABLE-US-00001 SEQ ID NO: 7:
QAQYPVVNTNYGKIRGLRTPLPNEILGPVEQYLGVPYASPPTGERRFQPP
EPPSSWTGIRNTTQFAAVCPQHLDERSLLHDMLPIWFTANLDTLMTYVQD
QNEDCLYLNIYVPTEDDIHDQNSKKPVMVYIHGGSYMEGTGNMIDGSILA
SYGNVIVITINYRLGILGFLSTGDQAAKGNYGLLDQIQALRWIEENVGAF
GGDPKRVTIFGSGAGASCVSLLTLSHYSEGLFQKAIIQSGTALSSWAVNY
QPAKYTRILADKVGCNMLDTTDMVECLRNKNYKELIQQTITPATYHIAFG
PVIDGDVIPDDPQILMEQGEFLNYDIMLGVNQGEGLKFVDGIVDNEDGVT
PNDFDFSVSNFVDNLYGYPEGKDTLRETIKFMYTDWADKENPETRRKTLV
ALFTDHQWVAPAVATADLHAQYGSPTYFYAFYHHCQSEMKPSWADSAHGD
EVPYVFGIPMIGPTELFSCNFSKNDVMLSAVVMTYWTNFAKTGDPNQPVP
QDTKFIHTKPNRFEEVAWSKYNPKDQLYLHIGLKPRVRDHYRATKVAFWL
ELVPHLHNLNEIFQYVSTTTKVPPPDMTSFPYGTRRSPAKIWPTTKRPAI
TPANNPKHSKDPHKTGPEDTTVLIETKRDYSTELS
[0179] According to some embodiments, the rsNLGn4 consists of SEQ
ID NO: 7, or a fragment, derivative or analog thereof. According to
some embodiments, the rsNLGn4 has at least 70%, at least 80%, at
least 90%, at least 95% or at least 98% sequence similarity to SEQ
ID NO: 7. Each possibility is separate embodiment.
[0180] According to some embodiments, the rsNLGn4 is devoid of the
intracellular domain of NLGn4 set forth in SEQ ID NO: 8:
TABLE-US-00002 SEQ ID NO: 8:
YKKDKRRHETHRRPSPQRNTTNDIAHIQNEEIMSLQMKQLEHDHECESLQ
AHDTLRLTCPPDYTLTLRRSPDDIPLMTPNTITMIPNTLTGMQPLHTFNT
FSGGQNSTNLPHGHSTTRV
[0181] According to some embodiments, the rsNLGn4 is devoid of the
transmembrane domain of NLGn4 set forth in SEQ ID NO: 9:
TABLE-US-00003 SEQ ID NO: 9: VTIAVGASLLFLNILAFAALY
[0182] According to some embodiments, the agent comprises
recombinant soluble Nrx1b (rsNrx1b).
[0183] According to some embodiments, the recombinant Nrx1b
comprises Ala51-Ser363 of NP_620072. According to some embodiments,
the recombinant Nrx1b consists essentially of Ala51-Ser363 of
NP_620072.
[0184] According to some embodiments, recombinant soluble human
Nrx1b may function as a decoy competing with endogenous Nrx1b for
binding to NLGn4.
[0185] According to some embodiments, the rsNrx1b comprises the
extracellular domain of Nrx1b, the extracellular domain of Nrx1b
having the polypeptide sequence set forth in SEQ ID: 10.
TABLE-US-00004 SEQ ID NO: 10:
ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGGHAGTTYIFSKGGGQI
TYKWPPNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGLGDYLELHIHQG
KIGVKFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNATLQVDSWPVIE
RYPAGRQLTIFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLNMAAEND
ANIAIVGNVRLVGEVPSSMTTESTATAMQSEMSTSIMETTTTLATSTARR
GKPPTKEPISQTTDDILVASAECPSDDEDIDPCEPSSGGLANPTRAGGRE
PYPGSAEVIRESS
[0186] According to some embodiments, the rsNrx1b consists of SEQ
ID NO: 10, or a fragment, derivative or analog thereof. According
to some embodiments, the rsNrx1b has at least 70%, at least 80%, at
least 90%, at least 95% or at least 98% sequence similarity to SEQ
ID NO: 10. Each possibility is separate embodiment.
[0187] According to some embodiments, the rsNrx1b is devoid of the
intracellular domain of Nrx1b set forth in SEQ ID NO: 11:
TABLE-US-00005 SEQ ID NO: 11:
MYKYRNRDEGSYHVDESRNYISNSAQSNGAVVKEKQPSSAKSSNKNKKNK DKEYYV
[0188] According to some embodiments, the rsNLGn4 is devoid of the
transmembrane domain of NLGn4 set forth in SEQ ID NO: 12:
TABLE-US-00006 SEQ ID NO: 12: STTGMVVGIVAAAALCILILLYA
[0189] According to some embodiments, the agent comprises a
combination of recombinant soluble NLGn4 and recombinant soluble
Nrx1b (rsNrx1b).
[0190] According to some embodiments, the liver disorder is
selected from the group consisting of: non-alcoholic fatty liver
disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis,
hepatitis, liver adenoma, insulin hypersensitivity, liver cancer
and any combination thereof.
[0191] According to some embodiments, of the agent comprises an
anti-NLGn4 antibody.
[0192] According to some embodiments, administering a
therapeutically effective amount of the agent includes contacting
an immune cell, such as an NK cell and/or a CD56.sup.dim NK cell
subset, with the therapeutically effective amount of the agent.
[0193] According to some embodiments, the antibody is a monoclonal
antibody. According to some embodiments, the antibody is a
humanized antibody. According to some embodiments, the antibody
does not cross the blood-brain barrier (BBB).
[0194] According to some embodiments, the antibody is an anti-NLGn4
monoclonal antibody or a fragment, derivative or analog
thereof.
[0195] According to some embodiments, the polypeptide sequence of
the NLGn4 is set forth in SEQ ID NO: 1:
TABLE-US-00007 MSRPQGLLWLPLLFTPVCVMLNSNVLLWLTALAIKFTLIDSQAQYPVVNT
NYGKIRGLRTPLPNEILGPVEQYLGVPYASPPTGERRFQPPEPPSSWTGI
RNTTQFAAVCPQHLDERSLLHDMLPIWFTANLDTLMTYVQDQNEDCLYLN
IYVPTEDDIHDQNSKKPVMVYIHGGSYMEGTGNMIDGSILASYGNVIVIT
INYRLGILGFLSTGDQAAKGNYGLLDQIQALRWIEENVGAFGGDPKRVTI
FGSGAGASCVSLLTLSHYSEGLFQKAIIQSGTALSSWAVNYQPAKYTRIL
ADKVGCNMLDTTDMVECLRNKNYKELIQQTITPATYHIAFGPVIDGDVIP
DDPQILMEQGEFLNYDIMLGVNQGEGLKFVDGIVDNEDGVTPNDFDFSVS
NFVDNLYGYPEGKDTLRETIKFMYTDWADKENPETRRKTLVALFTDHQWV
APAVATADLHAQYGSPTYFYAFYHHCQSEMKPSWADSAHGDEVPYVFGIP
MIGPTELFSCNFSKNDVMLSAVVMTYWTNFAKTGDPNQPVPQDTKFIHTK
PNRFEEVAWSKYNPKDQLYLHIGLKPRVRDHYRATKVAFWLELVPHLHNL
NEIFQYVSTTTKVPPPDMTSFPYGTRRSPAKIWPTTKRPAITPANNPKHS
KDPHKTGPEDTTVLIETKRDYSTELSVTIAVGASLLFLNILAFAALYYKK
DKRRHETHRRPSPQRNTTNDIAHIQNEEIMSLQMKQLEHDHECESLQAHD
TLRLTCPPDYTLTLRRSPDDIPLMTPNTITMIPNTLTGMQPLHTFNTFSG
GQNSTNLPHGHSTIRV
[0196] According to some embodiments, the NLGn4 monoclonal
antibody, or a fragment, derivative or analog thereof is capable of
binding to the interaction domain of NLGn4, mediating the
interaction between NLGn4 and NrX1b.
[0197] According to some embodiments, the anti-NLGn4 monoclonal
antibody or a fragment, derivative or analog thereof is capable of
binding to amino acids 359-364 of the human NLGN4 protein as set
forth in SEQ ID NO: 2 (QGEFLN).
[0198] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid E361 of human NLGn4.
[0199] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid L363 of human NLGn4.
[0200] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope within residues 261-270, as set forth in SEQ ID NO: 3
(SLLTLSHYSE), of human NLGn4.
[0201] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid H267 of human NLGn4.
[0202] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope within residues 461-470, as set forth in SEQ ID NO: 4
(AQYGSPTYFY), of human NLGn4.
[0203] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid Y463 of human NLGn4.
[0204] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope within residues 265-275, as set forth in SEQ ID NO: 5
(LSHYSEGLFQK), of human NLGn4.
[0205] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising the amino acid E270 of human NLGn4.
[0206] According to some embodiments, the NLGn4 antibody, or a
fragment, derivative or analog thereof is capable of binding to an
epitope comprising an amino acid selected from the group consisting
of E361, L363, H267, Y463, E270 or any combination thereof. Each
possibility is a separate embodiment.
[0207] According to some embodiments, the antibody is an anti-Nrx1b
antibody or a fragment, derivative or analog thereof. According to
some embodiments, the Nrx1b antibody, or a fragment, derivative or
analog thereof is capable of binding to the interaction domain of
Nrx1b, mediating the interaction between NLGn4 to NrX1b.
[0208] According to some embodiments, the polypeptide sequence of
the Nrx1b is set forth in SEQ ID NO: 6:
TABLE-US-00008 MGTALLQRGGCFLLCLSLLLLGCWAELGSGLEFPGAEGQWTRFPKWNACC
ESEMSFQLKTRSARGLVLYFDDEGFCDFLELILTRGGRLQLSFSIFCAEP
ATLLADTPVNDGAWHSVRIRRQFRNTTLFIDQVEAKWVEVKSKRRDMTVF
SGLFVGGLPPELRAAALKLTLASVREREPFKGWIRDVRVNSSQVLPVDSG
EVKLDDEPPNSGGGSPCEAGEEGEGGVCLNGGVCSVVDDQAVCDCSRTGF
RGKDCSQEIKFGLQCVLPVLLHDNDQGKYCCINTAKPLTEKDNNVEGLAH
LMMGDQGKSKGKEEYIATFKGSEYFCYDLSQNPIQSSSDEITLSFKTLQR
NGLMLHTGKSADYVNLALKNGAVSLVINLGSGAFEALVEPVNGKFNDNAW
HDVKVTRNLRQHSGIGHAMVNKLHCSVTISVDGILTTTGYTQEDYTMLGS
DDFFYVGGSPSTADLPGSPVSNNFMGCLKEVVYKNNDVRLELSRLAKQGD
PKMKIHGVVAFKCENVATLDPITFETPESFISLPKWNAKKTGSISFDFRT
TEPNGLILFSHGKPRHQKDAKHPQMIKVDFFAIEMLDGHLYLLLDMGSGT
IKIKALLKKVNDGEWYHVDFQRDGRSGTISVNTLRTPYTAPGESEILDLD
DELYLGGLPENKAGLVFPTEVWTALLNYGYVGCIRDLFIDGQSKDIRQMA
EVQSTAGVKPSCSKETAKPCLSNPCKNNGMCRDGWNRYVCDCSGTGYLGR
SCEREATVLSYDGSMFMKIQLPVVMHTEAEDVSLRFRSQRAYGILMATTS
RDSADTLRLELDAGRVKLTVNLDCIRINCNSSKGPETLFAGYNLNDNEWH
TVRVVRRGKSLKLTVDDQQAMTGQMAGDHTRLEFHNIETGIITERRYLSS
VPSNFIGHLQSLTFNGMAYIDLCKNGDIDYCELNARFGFRNIIADPVTFK
TKSSYVALATLQAYTSMHLFFQFKTTSLDGLILYNSGDGNDFIVVELVKG
YLHYVFDLGNGANLIKGSSNKPLNDNQWHNVMISRDTSNLHTVKIDTKIT
TQITAGARNLDLKSDLYIGGVAKETYKSLPKLVHAKEGFQGCLASVDLNG
RLPDLISDALFCNGQIERGCEGPSTTCQEDSCSNQGVCLQQWDGFSCDCS
MTSFSGPLCNDPGTTYIFSKGGGQITYKWPPNDRPSTRADRLAIGFSTVQ
KEAVLVRVDSSSGLGDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDG
KYHVVRFTRSGGNATLQVDSWPVIERYPAGNNDNERLAIARQRIPYRLGR
VVDEWLLDKGRQLTIFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLNM
AAENDANIAIVGNVRLVGEVPSSMTTESTATAMQSEMSTSIMETTTTLAT
STARRGKPPTKEPISQTTDDILVASAECPSDDEDIDPCEPSSGGLANPTR
AGGREPYPGSAEVIRESSSTTGMVVGIVAAAALCILILLYAMYKYRNRDE
GSYHVDESRNYISNSAQSNGAVVKEKQPSSAKSSNKNKKNKDKEYYV
[0209] According to some embodiments, the anti-Nrx1b monoclonal
antibody or a fragment, derivative or analog thereof is capable of
binding to amino acids 103-109 as set forth in SEQ ID NO: 7
(LLADTPV) and/or amino acids 234-239 as set forth in SEQ ID NO: 8
(CSVVDD) of the human Nrx1b protein. Each possibility is a separate
embodiment of the invention.
[0210] According to some embodiments, the liver disorders is
Non-alcoholic fatty liver disease (NAFLD), and Non-alcoholic
steatohepatitis (NASH). Additionally or alternatively as the liver
disorder is cirrhosis, hepatitis, liver adenoma, insulin
resistance, liver cancer, or any NK related inflammatory or
neoplastic disorder well. The clinical implications of NAFLD are
derived mostly from its potential to progress to Non-alcoholic
steatohepatitis, cirrhosis and liver failure. In accordance, the
invention, addresses the long felt need to attenuate the
progression of NAFLD into cirrhosis and liver failure by
inhibiting, attenuating and/or preventing NLGn4-Nrx1b
protein-protein interaction and thereby modulating the cytotoxic
activity of NK cells. According to some embodiments, the invention
provides a method for modulating the activity of a natural killer
(NK) cell.
[0211] According to some embodiments, the NK cells are liver NK
cells, which are attenuated in patients having a liver disorder.
According to yet another embodiment, the liver disorder is
characterized by overexpression of NLGn4 protein. Such
overexpression can attenuate NK cell activity.
[0212] According to some embodiments and without being bound to any
theory, reducing NLGn4 protein levels and/or interfering with the
binding of NLGn4 to Nrx1b, modulates the function of the NK cell
for example by activating the NK cell and/or the CD56.sup.dim NK
cell subset. As a result of NK activation, the activity of hepatic
stellate cells (HSCs) and hence fibrosis is reduced. In addition,
and according to yet another embodiment, modulating and/or
activating the NK cells increases the apoptosis of the HSCs.
[0213] According to yet another embodiment there is provided a
method for modulating the activity of a natural killer (NK) cell
and/or treating, preventing and/or attenuating a liver disorder by
administering to a patient a composition comprising an anti-NLGn4
antibody and/or an anti-Nrx1b antibody.
[0214] According to some embodiments, the composition may further
include a GLUT4 antagonist. Such antagonist can according to the
present invention inhibit GLUT4 mediated NLGn4 expression. The
antagonist can be selected from the group comprising Ketamine,
Amantadine, Phencyclidine, Nitrous oxide, Dextromethorphan (and
dextrorphan), Memantine, Ethanol, Riluzole (used in ALS), Xenon,
HU-211 (also a cannabinoid), Lead (Pb2+), Conantokins, and
Huperzine A.
[0215] Without being bound to any theory, reducing NLGn4 protein
levels and/or inhibiting NLGn4-Nrx1b binding can, according to the
present invention, enhances the cytotoxicity of the NK cells and/or
specific NK cell subpopulations. According to certain embodiments,
and without being bound to any theory, enhancing the cytotoxicity
comprises enhancing the expression of CD107a (marker of NK cell
functional activity) on said NK cell.
[0216] In certain liver disorders, NK cell function can be
attenuated. According to the present invention and without being
bound by any theory, such attenuation can be a result of NLGn4
overexpression. In accordance, reducing NLGn4 protein levels
modulates and/or activates the function of attenuated NK cell. In
turn, activating the NK cell may reduce HSC activity and/or
increase their apoptosis.
[0217] As further demonstrated herein below, it has now
surprisingly been shown that administration of anti-NLGn4
antibodies, increases NK cell activity and attenuates HSC
activation, both in-vitro and in-vivo. These results show that
anti-NLGn4 antibodies can serve as therapeutically agents in the
treatment of liver disorders such as but not limited to NAFLD and
preventing its progression to cirrhosis.
[0218] According to some embodiments, there is provided a
composition comprising a therapeutically effective amount of an
agent capable of interfering with, inhibiting and/or preventing
neuroligin 4 (NLGn4)-Neurexin 1.beta. (Nrx1b) protein-protein
interaction and a pharmaceutically acceptable carrier.
[0219] According to some embodiments, the recombinant NLGn4
comprises Gln42-Ser676 of NP_065793. According to some embodiments,
the recombinant NLGn4 consists essentially of Gln42-Ser676 of
NP_065793.
[0220] According to some embodiments, the agent comprises
recombinant soluble NLGn4 (rsNLGn4); wherein the rsNLGn4 comprises
the extracellular domain of NLGn4 or a fragment, derivative or
analog thereof.
[0221] According to some embodiments, the extracellular domain of
NLGn4 consists of SEQ ID NO: 7. According to some embodiments, the
rsNLGn4 consists of SEQ ID NO: 7, or a fragment, derivative or
analog thereof.
[0222] According to some embodiments, the rsNLGn4 competes with
endogenous NLGn4 for binding to Nrx1b.
[0223] According to some embodiments, the rsNLGn4 is devoid of the
intracellular domain and/or the transmembrane domain of NLGn4.
[0224] According to some embodiments, the agent comprises a fusion
protein comprising rsNLGn4 and an immunoglobulin molecule.
According to some embodiments, the agent comprises a fusion protein
comprising rsNLGn4 and an Fc domain of an immunoglobulin molecule.
According to some embodiments, the Fc domain may include part or
the entire CH1 domain, CH2 domain, CH3 domain and hinge region of
IgG1, IgG2, IgG3 and IgG4. According to some embodiments, the Fc
domain may be devoid of the CH1 domain. According to some
embodiments, the Fc domain comprises Pro100-Lys330 of Human IgG1.
According to some embodiments, the Fc domain consists essentially
of Pro100-Lys330 of Human IgG1. According to some embodiments, the
Fc domain may be linked to rsNLGn4 through a linker. According to
some embodiments, the linker may have the amino acid sequence set
forth in SEQ ID NO: 13 (IEGRMD).
[0225] According to some embodiments, the agent is a chimeric
protein formed from NLGn4 polypeptides or fragments fused with a
second polypeptide to form a soluble NLGn4. According to some
embodiments, the agent comprises a DNA sequences, which combine two
partial DNA sequences, one sequence encoding soluble fragments of
NLGn4 (i.e. the DNA sequence encoding the extracellular domain of
NLGn4) and the other partial sequence encoding all domains except
the first domain of the constant region of the heavy chain of human
immunoglobulin IgG, IgA, IgM, or IgE. These DNA sequences may
subsequently be expressed in target cells using expression vectors
as known in the art, thereby obtaining endogenous expression of
recombinant proteins having the extracellular domain of NLGn4
joined to the Fc fragment of an immunoglobulin molecule.
[0226] According to some embodiments, the NLGn4 is encoded by the
sequence set forth in SEQ ID NO: 1.
[0227] According to some embodiments, the recombinant Nrx1b
comprises Ala51-Ser363 of NP_620072. According to some embodiments,
the recombinant Nrx1b consists essentially of Ala51-Ser363 of
NP_620072.
[0228] According to some embodiments, the agent comprises
recombinant soluble Nrx1b (rsNrx1b); wherein the rsNrx1b comprises
the extracellular domain of Nrx1b or a fragment, derivative or
analog thereof capable of binding NLGn4.
[0229] According to some embodiments, the extracellular domain of
Nrx1b consists of SEQ ID NO: 10. According to some embodiments, the
rsNrx1b consists of SEQ ID NO: 10, or a fragment, derivative or
analog thereof.
[0230] According to some embodiments, the rsNrx1b competes with
endogenous Nrx1b for binding to NLGn4.
[0231] According to some embodiments, the rsNrx1b is devoid of the
intracellular domain and/or the transmembrane domain of Nrx1b.
[0232] According to some embodiments, Nrx1b is encoded by the
sequence set forth in SEQ ID NO: 6.
[0233] According to some embodiments, the agent comprises a fusion
protein comprising rsNrx1b and an immunoglobulin molecule.
According to some embodiments, the agent comprises a fusion protein
comprising rsNrx1b and an Fc domain of an immunoglobulin molecule,
also referred to herein as rsNrx1b-Fc fusion protein. According to
some embodiments, the Fc domain may include part or the entire CH1
domain, the CH2 domain, the CH3 domain and hinge region of IgG1,
IgG2, IgG3 and IgG4. According to some embodiments, the Fc domain
may be devoid of the CH1 domain. According to some embodiments, the
Fc domain may include all domains of the Fc domain apart from CH1.
According to some embodiments, the Fc domain comprises
Pro100-Lys330 of Human IgG1. According to some embodiments, the Fc
domain consists essentially of Pro100-Lys330 of Human IgG1.
According to some embodiments, the Fc domain may be linked to
rsNrx1b through a linker. According to some embodiments, the linker
may have the amino acid sequence set forth in SEQ ID NO: 13
(IEGRMD).
[0234] According to some embodiments, the agent is a chimeric
protein formed from Nrx1b polypeptides or fragments fused with a
second polypeptide to form a soluble Nrx1. According to some
embodiments, the agent comprises a DNA sequences, which combine two
partial DNA sequences, one sequence encoding soluble fragments of
Nrx1b (i.e. the DNA sequence encoding the extracellular domain of
Nrx1b) and the other partial sequence encoding all domains except
the first domain of the constant region of the heavy chain of human
immunoglobulin IgG, IgA, IgM, or IgE. These DNA sequences may
subsequently be expressed in target cells using expression vectors
as known in the art, thereby obtaining endogenous expression of
recombinant proteins having the extracellular domain of Nrx1b
joined to the Fc fragment of an immunoglobulin molecule.
[0235] According to some embodiments, the agent comprises a
combination of recombinant soluble NLGn4 and recombinant soluble
Nrx1b (rsNrx1b).
[0236] The following examples are presented to provide a more
complete understanding of the invention. The specific techniques,
conditions, materials, proportions and reported data set forth to
illustrate the principles of the invention are exemplary and should
not be construed as limiting the scope of the invention.
EXAMPLES
Example 1: NLGn4 Gene is Over-Expressed in NASH Patients with
Hepatic Fibrosis
[0237] Heparinized-blood samples of healthy volunteers and NAFLD
patients and/or cirrhotic cases were obtained. Mononuclear cells
were isolated by centrifugation over ficoll-hypaque (Pharmacia).
After three washes in saline, cells were resuspended in medium of
Roswell Park Memorial Institute 1640 with 10% FBS. Human NK cells
were isolated from Peripheral blood lymphocytes (PBLs) using a
magnetic cell sorting kit (Miltenyi Biotec) according to
manufacturer's instructions. RNA was extracted, converted to cDNA
and hybridized onto a spotted microarray.
[0238] As seen in FIG. 2, a 4-fold increase in NLGn4 gene
expression on NASH NK cells was obtained (in the zone of down- and
up-expressed genes with a cutoff p-value of 0.015 showed),
suggesting a correlation between NLGn4 expression levels and
NAFLD/NASH in humans.
Example 2: Leptin-Deficient Mice (Ob/Ob) (Fatty Liver Disease
Model) Show Increased NLGn4 Expression on Liver NK Cells
[0239] RNA was extracted from Leptin deficient ob/ob mice (fatty
liver disease mouse model), converted into cDNA and the expression
level of NLGn4 analyzed by RT-PCR using NLGn4 specific primers. As
seen in FIG. 3, the ob/ob mice showed a 4-fold increase in mRNA
NLG4 expressions as compared to WT c57bl mice, serving as control.
This result shows a correlation between NLGn4 overexpression and
fatty liver disease in mice.
Example 3: Homozygous NLG4-/- (KO) Mice Show Decreased Inflammation
and Fibrosis
[0240] Naive Wild Type (WT) and homozygote NLG4-/- mice were
induced for fibrosis by i.p injections of carbon tetrachloride
2.times./week for 6 weeks and evaluated for serum levels of Alanine
transaminase (ALT--biomarker for liver health). As seen from FIG.
4A, induction of fibrosis, by carbon tetrachloride injection, was
significantly attenuated in the NLG4-/- KO mice as compared to WT.
Furthermore, as shown in FIG. 4B, alpha-SMA (marker of HSC
activation) levels were lower in the NLG4-/- mice compared to WT,
as evaluated by quantitative western blot analysis.
[0241] These results show the involvement of NLGn4 in HSC activity
and in the induction of fibrosis.
Example 4: Incubation of NK Cells with Anti-NLGn4 Antibodies
Increases NK Cell Activation and Reduces HSC Activation in an
In-Vitro Assay
[0242] NK cells were isolated form peripheral blood derived from
NAFLD patients with advanced fibrosis scoring (histology
documentation of F3, F4). Patients without medications and lacking
full metabolic syndrome were included. Isolated NK cells were
incubated with 4, 8, 10 or 12 .mu.M of anti-NLG4 (human NLGN4
Antibody, Antigen Affinity-purified Polyclonal Sheep IgG, R&D
Systems Catalog # AF5158) for 3 hours prior to incubation with the
myofibroblasts LX2-cell line (HSCs) o left untreated (0). Following
24 hours of NK-LX2 co-culture, harvested cells were analyzed by
flow-cytometry for NK NLG4 and CD107a expressions as well as LX2
alpha-SMA intensity.
[0243] Incubation of the NK cells with an anti-NLG4 antibody
significantly inhibited NLGn4 protein levels (FIG. 5A, P<0.03)
and increased CD107a expression on the NK cells (FIG. 5B,
P<0.01) in all tested concentrations. These results show that
incubation of NK cells with anti-NLGn4 antibodies increase NK cell
activity in-vitro.
[0244] The increased NK activity was accompanied with increased LX2
killing; as alpha-SMA mean fluorescence intensities decreased with
the increase in anti-NLG4 concentrations (FIG. 5C, P<0.01). This
shows that incubation of NK cells with anti-NLGn4 antibodies
attenuates HSC activity in-vitro.
Example 5: Incubation with Anti-NLGn4 Antibodies Increases NK Cell
Activation and Reduces HSC Activation in an In-Vivo Assay in
Mice
[0245] 25 WT naive C57/BL mice (12 weeks) mice were either induced
for acute hepatic fibrosis by i.p injections of carbon
tetrachloride (3.times./week) or left untreated (induced n=10,
control n=10). One day after the last injection, the mice were
either injected i.p. with NLGn4-antibodies (anti-human NLGN4
Antibody, Antigen Affinity-purified Polyclonal Sheep IgG, R&D
Systems Catalog # AF5158) at concentrations of 82 mg/mice or left
untreated (5 mice of each of the induced and control mice). 24
hours after injection with the NLGn4 antibodies, the mice were
sacrificed and sera were collected for detection of CD107a
expression (marker of NK activity), ALT levels biomarker for liver
health and livers were harvested for assessment of alpha-SMA
expression (marker of HSC activation) using an RT PCR assay.
[0246] As seen in FIG. 6A mice induced for acute liver fibrosis
that were exposed to the NLGn4 antibody had increased CD107a
expression (marker of NK activity), a lesser degree of liver
injuries and fibrosis as evidenced by reduced serum ALT levels
(P=0.03) (FIG. 6B) and alpha-SMC expression (FIG. 6C).
[0247] These results show that treatment with anti-NLGn4 antibodies
reduces liver injuries and fibrosis in-vivo as well as in-vitro and
show that anti-NLGn4 antibodies can serve as therapeutic agents in
the treatment of liver disorders such as but not limited to NAFLD
and for preventing progression to cirrhosis.
Example 6: Mice Induced for Hepatocellular Carcinoma by Injection
of Human Hepatoma Cells (Hep3B) Co-Cultured with NLGn4-/- NK Cells
Show Reduced Levels of the Cancer Marker .alpha.-Feto-Protein
[0248] Human hepatoma cells (Hep3B) co-cultured for 24 h with
either wt NK cells (n=4) or NLGn4-/- NK cells (n=4) were injected
subcutaneously (2.times.10.sup.6) into C57BI6 mice (13 week old).
The mice were subsequently irradiated (5Gry) to induce tumor
growth. Subsequently, plasma alpha-fetoprotein (AFP) levels were
evaluated. AFP is a biomarker of liver cancers and levels above 500
nanograms/milliliter of AFP in adults is indicative of
hepatocellular carcinoma, and metastatic cancers of the liver. As
seen from FIG. 7, AFP levels were significantly lowered in mice
injected with Hep3B cells incubated with NLG4-/- NK cells as
compared to mice injected with Hep3B cells incubated with wt NK
cells.
[0249] These results show the involvement of NLGn4 in NK cell
attenuation and hepatoma formation and demonstrate that reducing
NLGn4 protein levels inhibit tumor growth.
Example 7: Incubation of NK Cells with Recombinant Nrx1b Increases
NK Cell Activation and Reduces HSC Activation in an In-Vitro
Assay
[0250] NK cells were isolated form peripheral blood derived from
NAFLD patients with advanced fibrosis scoring (histology
documentation of F3, F4). Patients without medications and lacking
full metabolic syndrome were included. Isolated NK cells were
incubated with 4 nM or 10 nM recombinant Nrx1b-Fc Chimera
(Recombinant Human Neurexin 1.beta./NXRN1b Fc Chimera, R&D
Systems Catalog Number: 5268-NX) including amino acids Ala51-Ser363
of Nrx1b (NP_620072) and Pro100-Lys330 of Human IgG1 linked by
linker sequence IEGRMD, for 3 hours prior to the NK cells being
co-cultured with the hepatic stellate cell line LX2. Following 24
hours of co-culture, harvested cells were analyzed by
flow-cytometry for NK CD107a expression as well as LX2 alpha-SMA
intensity.
[0251] Incubation of the NK cells with recombinant Nrx1b
significantly increased CD107a expression on the NK cells (FIG. 8A)
at both concentrations. These results show that incubation of NK
cells with recombinant Nrx1b increases NK cell activity
in-vitro.
[0252] The increased NK activity was accompanied with increased LX2
killing; as alpha-SMA mean fluorescence intensities decreased with
the increase in Nrx1b concentrations (FIG. 8B). This shows that
co-culturing HSC with NK cells pre-incubated with recombinant Nrx1b
attenuate HSC activity in-vitro.
Example 8: Incubation with Recombinant Nrx1b Increases NK Cell
Activation and Reduces HSC Activation in an In-Vivo Assay
[0253] 25 WT naive C57/BL mice (12 weeks) mice were either induced
for acute hepatic fibrosis by i.p. injections of carbon
tetrachloride (CCl.sub.4--3.times./week) or left untreated (induced
n=12, control n=12). Either one day after the last injection (D9)
or the day after each injection (D1, D3 and D7), the mice were
injected i.p. with 0.2 .mu.g recombinant Nrx1b/g body weight or
left untreated (3 mice of each of the induced and control mice). 24
hours after the last injection with the recombinant Nrx1b-Fc
Chimera (Recombinant Human Neurexin 1.beta./NXRN1b Fc Chimera,
R&D Systems Catalog Number: 5268-NX) including amino acids
Ala51-Ser363 of Nrx1b (NP_620072) and Pro100-Lys330 of Human IgG1
linked by linker sequence IEGRMD, the mice were sacrificed and sera
were collected for detection of ALT levels and livers were
harvested for assessment of alpha-SMA expression using an RT PCR
assay.
[0254] As seen in FIGS. 9A and 9B mice induced for acute liver
fibrosis that were exposed to the recombinant Nrx1b had a lesser
degree of liver injuries and fibrosis as evidenced by reduced serum
ALT levels (FIG. 9A) and reduced alpha-SMC expression (FIG.
9B).
[0255] These results show that treatment with recombinant Nrx1b
reduces liver injuries and fibrosis in-vivo as well as in-vitro and
show that recombinant Nrx1b can serve as therapeutic agents in the
treatment of liver disorders such as but not limited to NAFLD and
for preventing progression to cirrhosis.
[0256] 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.
Sequence CWU 1
1
131816PRTHomo sapiens 1Met Ser Arg Pro Gln Gly Leu Leu Trp Leu Pro
Leu Leu Phe Thr Pro 1 5 10 15 Val Cys Val Met Leu Asn Ser Asn Val
Leu Leu Trp Leu Thr Ala Leu 20 25 30 Ala Ile Lys Phe Thr Leu Ile
Asp Ser Gln Ala Gln Tyr Pro Val Val 35 40 45 Asn Thr Asn Tyr Gly
Lys Ile Arg Gly Leu Arg Thr Pro Leu Pro Asn 50 55 60 Glu Ile Leu
Gly Pro Val Glu Gln Tyr Leu Gly Val Pro Tyr Ala Ser 65 70 75 80 Pro
Pro Thr Gly Glu Arg Arg Phe Gln Pro Pro Glu Pro Pro Ser Ser 85 90
95 Trp Thr Gly Ile Arg Asn Thr Thr Gln Phe Ala Ala Val Cys Pro Gln
100 105 110 His Leu Asp Glu Arg Ser Leu Leu His Asp Met Leu Pro Ile
Trp Phe 115 120 125 Thr Ala Asn Leu Asp Thr Leu Met Thr Tyr Val Gln
Asp Gln Asn Glu 130 135 140 Asp Cys Leu Tyr Leu Asn Ile Tyr Val Pro
Thr Glu Asp Asp Ile His 145 150 155 160 Asp Gln Asn Ser Lys Lys Pro
Val Met Val Tyr Ile His Gly Gly Ser 165 170 175 Tyr Met Glu Gly Thr
Gly Asn Met Ile Asp Gly Ser Ile Leu Ala Ser 180 185 190 Tyr Gly Asn
Val Ile Val Ile Thr Ile Asn Tyr Arg Leu Gly Ile Leu 195 200 205 Gly
Phe Leu Ser Thr Gly Asp Gln Ala Ala Lys Gly Asn Tyr Gly Leu 210 215
220 Leu Asp Gln Ile Gln Ala Leu Arg Trp Ile Glu Glu Asn Val Gly Ala
225 230 235 240 Phe Gly Gly Asp Pro Lys Arg Val Thr Ile Phe Gly Ser
Gly Ala Gly 245 250 255 Ala Ser Cys Val Ser Leu Leu Thr Leu Ser His
Tyr Ser Glu Gly Leu 260 265 270 Phe Gln Lys Ala Ile Ile Gln Ser Gly
Thr Ala Leu Ser Ser Trp Ala 275 280 285 Val Asn Tyr Gln Pro Ala Lys
Tyr Thr Arg Ile Leu Ala Asp Lys Val 290 295 300 Gly Cys Asn Met Leu
Asp Thr Thr Asp Met Val Glu Cys Leu Arg Asn 305 310 315 320 Lys Asn
Tyr Lys Glu Leu Ile Gln Gln Thr Ile Thr Pro Ala Thr Tyr 325 330 335
His Ile Ala Phe Gly Pro Val Ile Asp Gly Asp Val Ile Pro Asp Asp 340
345 350 Pro Gln Ile Leu Met Glu Gln Gly Glu Phe Leu Asn Tyr Asp Ile
Met 355 360 365 Leu Gly Val Asn Gln Gly Glu Gly Leu Lys Phe Val Asp
Gly Ile Val 370 375 380 Asp Asn Glu Asp Gly Val Thr Pro Asn Asp Phe
Asp Phe Ser Val Ser 385 390 395 400 Asn Phe Val Asp Asn Leu Tyr Gly
Tyr Pro Glu Gly Lys Asp Thr Leu 405 410 415 Arg Glu Thr Ile Lys Phe
Met Tyr Thr Asp Trp Ala Asp Lys Glu Asn 420 425 430 Pro Glu Thr Arg
Arg Lys Thr Leu Val Ala Leu Phe Thr Asp His Gln 435 440 445 Trp Val
Ala Pro Ala Val Ala Thr Ala Asp Leu His Ala Gln Tyr Gly 450 455 460
Ser Pro Thr Tyr Phe Tyr Ala Phe Tyr His His Cys Gln Ser Glu Met 465
470 475 480 Lys Pro Ser Trp Ala Asp Ser Ala His Gly Asp Glu Val Pro
Tyr Val 485 490 495 Phe Gly Ile Pro Met Ile Gly Pro Thr Glu Leu Phe
Ser Cys Asn Phe 500 505 510 Ser Lys Asn Asp Val Met Leu Ser Ala Val
Val Met Thr Tyr Trp Thr 515 520 525 Asn Phe Ala Lys Thr Gly Asp Pro
Asn Gln Pro Val Pro Gln Asp Thr 530 535 540 Lys Phe Ile His Thr Lys
Pro Asn Arg Phe Glu Glu Val Ala Trp Ser 545 550 555 560 Lys Tyr Asn
Pro Lys Asp Gln Leu Tyr Leu His Ile Gly Leu Lys Pro 565 570 575 Arg
Val Arg Asp His Tyr Arg Ala Thr Lys Val Ala Phe Trp Leu Glu 580 585
590 Leu Val Pro His Leu His Asn Leu Asn Glu Ile Phe Gln Tyr Val Ser
595 600 605 Thr Thr Thr Lys Val Pro Pro Pro Asp Met Thr Ser Phe Pro
Tyr Gly 610 615 620 Thr Arg Arg Ser Pro Ala Lys Ile Trp Pro Thr Thr
Lys Arg Pro Ala 625 630 635 640 Ile Thr Pro Ala Asn Asn Pro Lys His
Ser Lys Asp Pro His Lys Thr 645 650 655 Gly Pro Glu Asp Thr Thr Val
Leu Ile Glu Thr Lys Arg Asp Tyr Ser 660 665 670 Thr Glu Leu Ser Val
Thr Ile Ala Val Gly Ala Ser Leu Leu Phe Leu 675 680 685 Asn Ile Leu
Ala Phe Ala Ala Leu Tyr Tyr Lys Lys Asp Lys Arg Arg 690 695 700 His
Glu Thr His Arg Arg Pro Ser Pro Gln Arg Asn Thr Thr Asn Asp 705 710
715 720 Ile Ala His Ile Gln Asn Glu Glu Ile Met Ser Leu Gln Met Lys
Gln 725 730 735 Leu Glu His Asp His Glu Cys Glu Ser Leu Gln Ala His
Asp Thr Leu 740 745 750 Arg Leu Thr Cys Pro Pro Asp Tyr Thr Leu Thr
Leu Arg Arg Ser Pro 755 760 765 Asp Asp Ile Pro Leu Met Thr Pro Asn
Thr Ile Thr Met Ile Pro Asn 770 775 780 Thr Leu Thr Gly Met Gln Pro
Leu His Thr Phe Asn Thr Phe Ser Gly 785 790 795 800 Gly Gln Asn Ser
Thr Asn Leu Pro His Gly His Ser Thr Thr Arg Val 805 810 815
26PRTArtificial Sequencepeptide 2Gln Gly Glu Phe Leu Asn 1 5
310PRTArtificial Sequencepeptide 3Ser Leu Leu Thr Leu Ser His Tyr
Ser Glu 1 5 10 410PRTArtificial Sequencepeptide 4Ala Gln Tyr Gly
Ser Pro Thr Tyr Phe Tyr 1 5 10 511PRTArtificial Sequencepeptide
5Leu Ser His Tyr Ser Glu Gly Leu Phe Gln Lys 1 5 10 61547PRTHomo
sapiens 6Met Gly Thr Ala Leu Leu Gln Arg Gly Gly Cys Phe Leu Leu
Cys Leu 1 5 10 15 Ser Leu Leu Leu Leu Gly Cys Trp Ala Glu Leu Gly
Ser Gly Leu Glu 20 25 30 Phe Pro Gly Ala Glu Gly Gln Trp Thr Arg
Phe Pro Lys Trp Asn Ala 35 40 45 Cys Cys Glu Ser Glu Met Ser Phe
Gln Leu Lys Thr Arg Ser Ala Arg 50 55 60 Gly Leu Val Leu Tyr Phe
Asp Asp Glu Gly Phe Cys Asp Phe Leu Glu 65 70 75 80 Leu Ile Leu Thr
Arg Gly Gly Arg Leu Gln Leu Ser Phe Ser Ile Phe 85 90 95 Cys Ala
Glu Pro Ala Thr Leu Leu Ala Asp Thr Pro Val Asn Asp Gly 100 105 110
Ala Trp His Ser Val Arg Ile Arg Arg Gln Phe Arg Asn Thr Thr Leu 115
120 125 Phe Ile Asp Gln Val Glu Ala Lys Trp Val Glu Val Lys Ser Lys
Arg 130 135 140 Arg Asp Met Thr Val Phe Ser Gly Leu Phe Val Gly Gly
Leu Pro Pro 145 150 155 160 Glu Leu Arg Ala Ala Ala Leu Lys Leu Thr
Leu Ala Ser Val Arg Glu 165 170 175 Arg Glu Pro Phe Lys Gly Trp Ile
Arg Asp Val Arg Val Asn Ser Ser 180 185 190 Gln Val Leu Pro Val Asp
Ser Gly Glu Val Lys Leu Asp Asp Glu Pro 195 200 205 Pro Asn Ser Gly
Gly Gly Ser Pro Cys Glu Ala Gly Glu Glu Gly Glu 210 215 220 Gly Gly
Val Cys Leu Asn Gly Gly Val Cys Ser Val Val Asp Asp Gln 225 230 235
240 Ala Val Cys Asp Cys Ser Arg Thr Gly Phe Arg Gly Lys Asp Cys Ser
245 250 255 Gln Glu Ile Lys Phe Gly Leu Gln Cys Val Leu Pro Val Leu
Leu His 260 265 270 Asp Asn Asp Gln Gly Lys Tyr Cys Cys Ile Asn Thr
Ala Lys Pro Leu 275 280 285 Thr Glu Lys Asp Asn Asn Val Glu Gly Leu
Ala His Leu Met Met Gly 290 295 300 Asp Gln Gly Lys Ser Lys Gly Lys
Glu Glu Tyr Ile Ala Thr Phe Lys 305 310 315 320 Gly Ser Glu Tyr Phe
Cys Tyr Asp Leu Ser Gln Asn Pro Ile Gln Ser 325 330 335 Ser Ser Asp
Glu Ile Thr Leu Ser Phe Lys Thr Leu Gln Arg Asn Gly 340 345 350 Leu
Met Leu His Thr Gly Lys Ser Ala Asp Tyr Val Asn Leu Ala Leu 355 360
365 Lys Asn Gly Ala Val Ser Leu Val Ile Asn Leu Gly Ser Gly Ala Phe
370 375 380 Glu Ala Leu Val Glu Pro Val Asn Gly Lys Phe Asn Asp Asn
Ala Trp 385 390 395 400 His Asp Val Lys Val Thr Arg Asn Leu Arg Gln
His Ser Gly Ile Gly 405 410 415 His Ala Met Val Asn Lys Leu His Cys
Ser Val Thr Ile Ser Val Asp 420 425 430 Gly Ile Leu Thr Thr Thr Gly
Tyr Thr Gln Glu Asp Tyr Thr Met Leu 435 440 445 Gly Ser Asp Asp Phe
Phe Tyr Val Gly Gly Ser Pro Ser Thr Ala Asp 450 455 460 Leu Pro Gly
Ser Pro Val Ser Asn Asn Phe Met Gly Cys Leu Lys Glu 465 470 475 480
Val Val Tyr Lys Asn Asn Asp Val Arg Leu Glu Leu Ser Arg Leu Ala 485
490 495 Lys Gln Gly Asp Pro Lys Met Lys Ile His Gly Val Val Ala Phe
Lys 500 505 510 Cys Glu Asn Val Ala Thr Leu Asp Pro Ile Thr Phe Glu
Thr Pro Glu 515 520 525 Ser Phe Ile Ser Leu Pro Lys Trp Asn Ala Lys
Lys Thr Gly Ser Ile 530 535 540 Ser Phe Asp Phe Arg Thr Thr Glu Pro
Asn Gly Leu Ile Leu Phe Ser 545 550 555 560 His Gly Lys Pro Arg His
Gln Lys Asp Ala Lys His Pro Gln Met Ile 565 570 575 Lys Val Asp Phe
Phe Ala Ile Glu Met Leu Asp Gly His Leu Tyr Leu 580 585 590 Leu Leu
Asp Met Gly Ser Gly Thr Ile Lys Ile Lys Ala Leu Leu Lys 595 600 605
Lys Val Asn Asp Gly Glu Trp Tyr His Val Asp Phe Gln Arg Asp Gly 610
615 620 Arg Ser Gly Thr Ile Ser Val Asn Thr Leu Arg Thr Pro Tyr Thr
Ala 625 630 635 640 Pro Gly Glu Ser Glu Ile Leu Asp Leu Asp Asp Glu
Leu Tyr Leu Gly 645 650 655 Gly Leu Pro Glu Asn Lys Ala Gly Leu Val
Phe Pro Thr Glu Val Trp 660 665 670 Thr Ala Leu Leu Asn Tyr Gly Tyr
Val Gly Cys Ile Arg Asp Leu Phe 675 680 685 Ile Asp Gly Gln Ser Lys
Asp Ile Arg Gln Met Ala Glu Val Gln Ser 690 695 700 Thr Ala Gly Val
Lys Pro Ser Cys Ser Lys Glu Thr Ala Lys Pro Cys 705 710 715 720 Leu
Ser Asn Pro Cys Lys Asn Asn Gly Met Cys Arg Asp Gly Trp Asn 725 730
735 Arg Tyr Val Cys Asp Cys Ser Gly Thr Gly Tyr Leu Gly Arg Ser Cys
740 745 750 Glu Arg Glu Ala Thr Val Leu Ser Tyr Asp Gly Ser Met Phe
Met Lys 755 760 765 Ile Gln Leu Pro Val Val Met His Thr Glu Ala Glu
Asp Val Ser Leu 770 775 780 Arg Phe Arg Ser Gln Arg Ala Tyr Gly Ile
Leu Met Ala Thr Thr Ser 785 790 795 800 Arg Asp Ser Ala Asp Thr Leu
Arg Leu Glu Leu Asp Ala Gly Arg Val 805 810 815 Lys Leu Thr Val Asn
Leu Asp Cys Ile Arg Ile Asn Cys Asn Ser Ser 820 825 830 Lys Gly Pro
Glu Thr Leu Phe Ala Gly Tyr Asn Leu Asn Asp Asn Glu 835 840 845 Trp
His Thr Val Arg Val Val Arg Arg Gly Lys Ser Leu Lys Leu Thr 850 855
860 Val Asp Asp Gln Gln Ala Met Thr Gly Gln Met Ala Gly Asp His Thr
865 870 875 880 Arg Leu Glu Phe His Asn Ile Glu Thr Gly Ile Ile Thr
Glu Arg Arg 885 890 895 Tyr Leu Ser Ser Val Pro Ser Asn Phe Ile Gly
His Leu Gln Ser Leu 900 905 910 Thr Phe Asn Gly Met Ala Tyr Ile Asp
Leu Cys Lys Asn Gly Asp Ile 915 920 925 Asp Tyr Cys Glu Leu Asn Ala
Arg Phe Gly Phe Arg Asn Ile Ile Ala 930 935 940 Asp Pro Val Thr Phe
Lys Thr Lys Ser Ser Tyr Val Ala Leu Ala Thr 945 950 955 960 Leu Gln
Ala Tyr Thr Ser Met His Leu Phe Phe Gln Phe Lys Thr Thr 965 970 975
Ser Leu Asp Gly Leu Ile Leu Tyr Asn Ser Gly Asp Gly Asn Asp Phe 980
985 990 Ile Val Val Glu Leu Val Lys Gly Tyr Leu His Tyr Val Phe Asp
Leu 995 1000 1005 Gly Asn Gly Ala Asn Leu Ile Lys Gly Ser Ser Asn
Lys Pro Leu 1010 1015 1020 Asn Asp Asn Gln Trp His Asn Val Met Ile
Ser Arg Asp Thr Ser 1025 1030 1035 Asn Leu His Thr Val Lys Ile Asp
Thr Lys Ile Thr Thr Gln Ile 1040 1045 1050 Thr Ala Gly Ala Arg Asn
Leu Asp Leu Lys Ser Asp Leu Tyr Ile 1055 1060 1065 Gly Gly Val Ala
Lys Glu Thr Tyr Lys Ser Leu Pro Lys Leu Val 1070 1075 1080 His Ala
Lys Glu Gly Phe Gln Gly Cys Leu Ala Ser Val Asp Leu 1085 1090 1095
Asn Gly Arg Leu Pro Asp Leu Ile Ser Asp Ala Leu Phe Cys Asn 1100
1105 1110 Gly Gln Ile Glu Arg Gly Cys Glu Gly Pro Ser Thr Thr Cys
Gln 1115 1120 1125 Glu Asp Ser Cys Ser Asn Gln Gly Val Cys Leu Gln
Gln Trp Asp 1130 1135 1140 Gly Phe Ser Cys Asp Cys Ser Met Thr Ser
Phe Ser Gly Pro Leu 1145 1150 1155 Cys Asn Asp Pro Gly Thr Thr Tyr
Ile Phe Ser Lys Gly Gly Gly 1160 1165 1170 Gln Ile Thr Tyr Lys Trp
Pro Pro Asn Asp Arg Pro Ser Thr Arg 1175 1180 1185 Ala Asp Arg Leu
Ala Ile Gly Phe Ser Thr Val Gln Lys Glu Ala 1190 1195 1200 Val Leu
Val Arg Val Asp Ser Ser Ser Gly Leu Gly Asp Tyr Leu 1205 1210 1215
Glu Leu His Ile His Gln Gly Lys Ile Gly Val Lys Phe Asn Val 1220
1225 1230 Gly Thr Asp Asp Ile Ala Ile Glu Glu Ser Asn Ala Ile Ile
Asn 1235 1240 1245 Asp Gly Lys Tyr His Val Val Arg Phe Thr Arg Ser
Gly Gly Asn 1250 1255 1260 Ala Thr Leu Gln Val Asp Ser Trp Pro Val
Ile Glu Arg Tyr Pro 1265 1270 1275 Ala Gly Asn Asn Asp Asn Glu Arg
Leu Ala Ile Ala Arg Gln Arg 1280 1285 1290 Ile Pro Tyr Arg Leu Gly
Arg Val Val Asp Glu Trp Leu Leu Asp 1295 1300 1305 Lys Gly Arg Gln
Leu Thr Ile Phe Asn Ser Gln Ala Thr Ile Ile 1310 1315 1320 Ile Gly
Gly Lys Glu Gln Gly Gln Pro Phe Gln Gly Gln Leu Ser 1325 1330 1335
Gly Leu Tyr Tyr Asn Gly Leu Lys Val Leu Asn Met Ala Ala Glu 1340
1345 1350 Asn Asp Ala Asn Ile Ala Ile Val Gly Asn Val Arg Leu Val
Gly 1355 1360 1365 Glu Val Pro Ser Ser Met Thr Thr Glu Ser Thr Ala
Thr Ala Met 1370 1375 1380 Gln Ser Glu Met Ser Thr Ser Ile Met Glu
Thr Thr Thr Thr Leu 1385 1390 1395 Ala Thr Ser Thr Ala Arg Arg Gly
Lys Pro Pro Thr Lys Glu Pro 1400 1405
1410 Ile Ser Gln Thr Thr Asp Asp Ile Leu Val Ala Ser Ala Glu Cys
1415 1420 1425 Pro Ser Asp Asp Glu Asp Ile Asp Pro Cys Glu Pro Ser
Ser Gly 1430 1435 1440 Gly Leu Ala Asn Pro Thr Arg Ala Gly Gly Arg
Glu Pro Tyr Pro 1445 1450 1455 Gly Ser Ala Glu Val Ile Arg Glu Ser
Ser Ser Thr Thr Gly Met 1460 1465 1470 Val Val Gly Ile Val Ala Ala
Ala Ala Leu Cys Ile Leu Ile Leu 1475 1480 1485 Leu Tyr Ala Met Tyr
Lys Tyr Arg Asn Arg Asp Glu Gly Ser Tyr 1490 1495 1500 His Val Asp
Glu Ser Arg Asn Tyr Ile Ser Asn Ser Ala Gln Ser 1505 1510 1515 Asn
Gly Ala Val Val Lys Glu Lys Gln Pro Ser Ser Ala Lys Ser 1520 1525
1530 Ser Asn Lys Asn Lys Lys Asn Lys Asp Lys Glu Tyr Tyr Val 1535
1540 1545 7635PRTArtificial SequenceNLGn4 extracellular domain 7Gln
Ala Gln Tyr Pro Val Val Asn Thr Asn Tyr Gly Lys Ile Arg Gly 1 5 10
15 Leu Arg Thr Pro Leu Pro Asn Glu Ile Leu Gly Pro Val Glu Gln Tyr
20 25 30 Leu Gly Val Pro Tyr Ala Ser Pro Pro Thr Gly Glu Arg Arg
Phe Gln 35 40 45 Pro Pro Glu Pro Pro Ser Ser Trp Thr Gly Ile Arg
Asn Thr Thr Gln 50 55 60 Phe Ala Ala Val Cys Pro Gln His Leu Asp
Glu Arg Ser Leu Leu His 65 70 75 80 Asp Met Leu Pro Ile Trp Phe Thr
Ala Asn Leu Asp Thr Leu Met Thr 85 90 95 Tyr Val Gln Asp Gln Asn
Glu Asp Cys Leu Tyr Leu Asn Ile Tyr Val 100 105 110 Pro Thr Glu Asp
Asp Ile His Asp Gln Asn Ser Lys Lys Pro Val Met 115 120 125 Val Tyr
Ile His Gly Gly Ser Tyr Met Glu Gly Thr Gly Asn Met Ile 130 135 140
Asp Gly Ser Ile Leu Ala Ser Tyr Gly Asn Val Ile Val Ile Thr Ile 145
150 155 160 Asn Tyr Arg Leu Gly Ile Leu Gly Phe Leu Ser Thr Gly Asp
Gln Ala 165 170 175 Ala Lys Gly Asn Tyr Gly Leu Leu Asp Gln Ile Gln
Ala Leu Arg Trp 180 185 190 Ile Glu Glu Asn Val Gly Ala Phe Gly Gly
Asp Pro Lys Arg Val Thr 195 200 205 Ile Phe Gly Ser Gly Ala Gly Ala
Ser Cys Val Ser Leu Leu Thr Leu 210 215 220 Ser His Tyr Ser Glu Gly
Leu Phe Gln Lys Ala Ile Ile Gln Ser Gly 225 230 235 240 Thr Ala Leu
Ser Ser Trp Ala Val Asn Tyr Gln Pro Ala Lys Tyr Thr 245 250 255 Arg
Ile Leu Ala Asp Lys Val Gly Cys Asn Met Leu Asp Thr Thr Asp 260 265
270 Met Val Glu Cys Leu Arg Asn Lys Asn Tyr Lys Glu Leu Ile Gln Gln
275 280 285 Thr Ile Thr Pro Ala Thr Tyr His Ile Ala Phe Gly Pro Val
Ile Asp 290 295 300 Gly Asp Val Ile Pro Asp Asp Pro Gln Ile Leu Met
Glu Gln Gly Glu 305 310 315 320 Phe Leu Asn Tyr Asp Ile Met Leu Gly
Val Asn Gln Gly Glu Gly Leu 325 330 335 Lys Phe Val Asp Gly Ile Val
Asp Asn Glu Asp Gly Val Thr Pro Asn 340 345 350 Asp Phe Asp Phe Ser
Val Ser Asn Phe Val Asp Asn Leu Tyr Gly Tyr 355 360 365 Pro Glu Gly
Lys Asp Thr Leu Arg Glu Thr Ile Lys Phe Met Tyr Thr 370 375 380 Asp
Trp Ala Asp Lys Glu Asn Pro Glu Thr Arg Arg Lys Thr Leu Val 385 390
395 400 Ala Leu Phe Thr Asp His Gln Trp Val Ala Pro Ala Val Ala Thr
Ala 405 410 415 Asp Leu His Ala Gln Tyr Gly Ser Pro Thr Tyr Phe Tyr
Ala Phe Tyr 420 425 430 His His Cys Gln Ser Glu Met Lys Pro Ser Trp
Ala Asp Ser Ala His 435 440 445 Gly Asp Glu Val Pro Tyr Val Phe Gly
Ile Pro Met Ile Gly Pro Thr 450 455 460 Glu Leu Phe Ser Cys Asn Phe
Ser Lys Asn Asp Val Met Leu Ser Ala 465 470 475 480 Val Val Met Thr
Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn 485 490 495 Gln Pro
Val Pro Gln Asp Thr Lys Phe Ile His Thr Lys Pro Asn Arg 500 505 510
Phe Glu Glu Val Ala Trp Ser Lys Tyr Asn Pro Lys Asp Gln Leu Tyr 515
520 525 Leu His Ile Gly Leu Lys Pro Arg Val Arg Asp His Tyr Arg Ala
Thr 530 535 540 Lys Val Ala Phe Trp Leu Glu Leu Val Pro His Leu His
Asn Leu Asn 545 550 555 560 Glu Ile Phe Gln Tyr Val Ser Thr Thr Thr
Lys Val Pro Pro Pro Asp 565 570 575 Met Thr Ser Phe Pro Tyr Gly Thr
Arg Arg Ser Pro Ala Lys Ile Trp 580 585 590 Pro Thr Thr Lys Arg Pro
Ala Ile Thr Pro Ala Asn Asn Pro Lys His 595 600 605 Ser Lys Asp Pro
His Lys Thr Gly Pro Glu Asp Thr Thr Val Leu Ile 610 615 620 Glu Thr
Lys Arg Asp Tyr Ser Thr Glu Leu Ser 625 630 635 8119PRTArtificial
SequenceNLGn4 intracellular domain 8Tyr Lys Lys Asp Lys Arg Arg His
Glu Thr His Arg Arg Pro Ser Pro 1 5 10 15 Gln Arg Asn Thr Thr Asn
Asp Ile Ala His Ile Gln Asn Glu Glu Ile 20 25 30 Met Ser Leu Gln
Met Lys Gln Leu Glu His Asp His Glu Cys Glu Ser 35 40 45 Leu Gln
Ala His Asp Thr Leu Arg Leu Thr Cys Pro Pro Asp Tyr Thr 50 55 60
Leu Thr Leu Arg Arg Ser Pro Asp Asp Ile Pro Leu Met Thr Pro Asn 65
70 75 80 Thr Ile Thr Met Ile Pro Asn Thr Leu Thr Gly Met Gln Pro
Leu His 85 90 95 Thr Phe Asn Thr Phe Ser Gly Gly Gln Asn Ser Thr
Asn Leu Pro His 100 105 110 Gly His Ser Thr Thr Arg Val 115
921PRTArtificial SequenceNLGn4 transmembrane domain 9Val Thr Ile
Ala Val Gly Ala Ser Leu Leu Phe Leu Asn Ile Leu Ala 1 5 10 15 Phe
Ala Ala Leu Tyr 20 10313PRTArtificial SequenceNrx1b extracellular
domain 10Ala Ser Ser Leu Gly Ala His His Ile His His Phe His Gly
Ser Ser 1 5 10 15 Lys His His Ser Val Pro Ile Ala Ile Tyr Arg Ser
Pro Ala Ser Leu 20 25 30 Arg Gly Gly His Ala Gly Thr Thr Tyr Ile
Phe Ser Lys Gly Gly Gly 35 40 45 Gln Ile Thr Tyr Lys Trp Pro Pro
Asn Asp Arg Pro Ser Thr Arg Ala 50 55 60 Asp Arg Leu Ala Ile Gly
Phe Ser Thr Val Gln Lys Glu Ala Val Leu 65 70 75 80 Val Arg Val Asp
Ser Ser Ser Gly Leu Gly Asp Tyr Leu Glu Leu His 85 90 95 Ile His
Gln Gly Lys Ile Gly Val Lys Phe Asn Val Gly Thr Asp Asp 100 105 110
Ile Ala Ile Glu Glu Ser Asn Ala Ile Ile Asn Asp Gly Lys Tyr His 115
120 125 Val Val Arg Phe Thr Arg Ser Gly Gly Asn Ala Thr Leu Gln Val
Asp 130 135 140 Ser Trp Pro Val Ile Glu Arg Tyr Pro Ala Gly Arg Gln
Leu Thr Ile 145 150 155 160 Phe Asn Ser Gln Ala Thr Ile Ile Ile Gly
Gly Lys Glu Gln Gly Gln 165 170 175 Pro Phe Gln Gly Gln Leu Ser Gly
Leu Tyr Tyr Asn Gly Leu Lys Val 180 185 190 Leu Asn Met Ala Ala Glu
Asn Asp Ala Asn Ile Ala Ile Val Gly Asn 195 200 205 Val Arg Leu Val
Gly Glu Val Pro Ser Ser Met Thr Thr Glu Ser Thr 210 215 220 Ala Thr
Ala Met Gln Ser Glu Met Ser Thr Ser Ile Met Glu Thr Thr 225 230 235
240 Thr Thr Leu Ala Thr Ser Thr Ala Arg Arg Gly Lys Pro Pro Thr Lys
245 250 255 Glu Pro Ile Ser Gln Thr Thr Asp Asp Ile Leu Val Ala Ser
Ala Glu 260 265 270 Cys Pro Ser Asp Asp Glu Asp Ile Asp Pro Cys Glu
Pro Ser Ser Gly 275 280 285 Gly Leu Ala Asn Pro Thr Arg Ala Gly Gly
Arg Glu Pro Tyr Pro Gly 290 295 300 Ser Ala Glu Val Ile Arg Glu Ser
Ser 305 310 1156PRTArtificial SequenceNrx1b intracellular domain
11Met Tyr Lys Tyr Arg Asn Arg Asp Glu Gly Ser Tyr His Val Asp Glu 1
5 10 15 Ser Arg Asn Tyr Ile Ser Asn Ser Ala Gln Ser Asn Gly Ala Val
Val 20 25 30 Lys Glu Lys Gln Pro Ser Ser Ala Lys Ser Ser Asn Lys
Asn Lys Lys 35 40 45 Asn Lys Asp Lys Glu Tyr Tyr Val 50 55
1223PRTArtificial SequenceNrx1b transmembrane domain 12Ser Thr Thr
Gly Met Val Val Gly Ile Val Ala Ala Ala Ala Leu Cys 1 5 10 15 Ile
Leu Ile Leu Leu Tyr Ala 20 136PRTArtificial Sequencelinker peptide
13Ile Glu Gly Arg Met Asp 1 5
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