U.S. patent application number 15/881339 was filed with the patent office on 2019-02-07 for pharmaceutical compositions and methods useful for modulating angiogenesis, inhibiting metastasis and tumor fibrosis, and assessing the malignancy of colon cancer tumors.
The applicant listed for this patent is TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD.. Invention is credited to Gal AKIRI, Stela GENGRINOVITCH, Gera NEUFELD, Zahava VADASZ.
Application Number | 20190040470 15/881339 |
Document ID | / |
Family ID | 32392444 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190040470 |
Kind Code |
A1 |
NEUFELD; Gera ; et
al. |
February 7, 2019 |
PHARMACEUTICAL COMPOSITIONS AND METHODS USEFUL FOR MODULATING
ANGIOGENESIS, INHIBITING METASTASIS AND TUMOR FIBROSIS, AND
ASSESSING THE MALIGNANCY OF COLON CANCER TUMORS
Abstract
Methods and compositions suitable for modulating angiogenesis in
a mammalian tissue are provided. Further provided are methods
suitable for inhibiting metastasis and fibrosis in a mammalian
tissue and for assessing the malignancy of colon cancer tumors.
Inventors: |
NEUFELD; Gera; (Haifa,
IL) ; AKIRI; Gal; (Haifa, IL) ; VADASZ;
Zahava; (Haifa, IL) ; GENGRINOVITCH; Stela;
(Merom Galil, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD. |
Technion City |
|
IL |
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Family ID: |
32392444 |
Appl. No.: |
15/881339 |
Filed: |
January 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15647017 |
Jul 11, 2017 |
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15881339 |
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14311110 |
Jun 20, 2014 |
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15647017 |
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13412544 |
Mar 5, 2012 |
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14311110 |
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10536440 |
Nov 14, 2005 |
8163494 |
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PCT/IL03/01008 |
Nov 27, 2003 |
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13412544 |
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10305348 |
Nov 27, 2002 |
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10536440 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/04 20180101;
C12N 9/0022 20130101; A61P 9/00 20180101; G01N 33/57419 20130101;
G01N 2500/00 20130101; A61K 38/00 20130101; C12Y 104/03013
20130101; A61P 35/00 20180101; C07K 16/40 20130101; C07K 16/30
20130101; C12Q 1/6886 20130101; A61K 2039/505 20130101; G01N 33/574
20130101; C12Q 1/26 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; G01N 33/574 20060101 G01N033/574; C07K 16/30 20060101
C07K016/30; C12Q 1/26 20060101 C12Q001/26; C12N 9/06 20060101
C12N009/06; C07K 16/40 20060101 C07K016/40 |
Claims
1. A method of determining that a colon tumor is metastatic,
comprising: (a) obtaining a first sample of colon tumor tissue; (b)
obtaining a second sample of colon tumor tissue, which is
non-metastatic; and (c) determining that a tissue level and/or an
activity level of a polypeptide having an amino acid sequence set
forth in SEQ ID NO: 2 is higher in the first colon tumor tissue
sample than in the second colon tumor tissue sample; thereby
determining that the tumor from which the first sample was derived
is metastatic.
2. The method of claim 1, wherein said determining in step (c) is
effected by performing a method selected from the group consisting
of an immunological detection method, an RNA detection method, and
an enzymatic activity detection method.
3. The method of claim 2, wherein said determining in step (c) is
effected by performing the immunological detection method, which is
selected from the group consisting of a radio-immunoassay (RIA), an
enzyme linked immunosorbent assay (ELISA), a Western blot analysis,
and an immunohistochemical analysis.
4. The method of claim 2, wherein said determining in step (c) is
effected by performing the RNA detection method, which is selected
from the group consisting of a Northern blot analysis, an RNA in
situ hybridization stain, an RT-PCR analysis, and an in situ RT-PCR
stain.
5. The method of claim 2, wherein said determining in step (c) is
effected by performing the enzymatic activity detection method,
which is selected from the group consisting of a cytochemical
stain, an in vitro activity assay, and an activity gel.
6. The method of claim 1, wherein said determining in step (c)
comprises contacting the samples with an antibody that specifically
binds the polypeptide, whereby a polypeptide in one or both of the
samples forms a complex with the antibody, and detecting the
antibody-polypeptide complex or complexes so formed.
7. The method of claim 1, wherein said determining in step (c)
comprises contacting the samples with a nucleic acid probe
homologous to a human LOR-1 gene, whereby an LOR-1 mRNA in one or
both of the samples forms a hybrid with the probe, and detecting
the hybrid or hybrids so formed.
8. A method of determining whether a colon tumor is metastatic,
comprising: determining a tissue level and/or an activity level of
a polypeptide having an amino acid sequence set forth in SEQ ID NO:
2 in a test colon tumor tissue sample; wherein a higher tissue
level and/or activity level of said polypeptide in the test colon
tumor tissue sample, compared with that of a non-metastatic colon
tumor tissue sample, indicates that the tumor from which the test
colon tumor tissue sample was derived is metastatic.
9. The method of claim 8, wherein said determining is effected by
performing a method selected from the group consisting of an
immunological detection method, an RNA detection method, and an
enzymatic activity detection method.
10. The method of claim 9, wherein: said immunological detection
method is selected from the group consisting of a radio-immunoassay
(RIA), an enzyme linked immunosorbent assay (ELISA), a Western blot
analysis, and an immunohistochemical analysis; said RNA detection
method is selected from the group consisting of a Northern blot
analysis, an RNA in situ hybridization stain, an RT-PCR analysis,
and an in situ RT-PCR stain; or said enzymatic activity detection
method is selected from the group consisting of a cytochemical
stain, an in vitro activity assay, and an activity gel.
11. The method of claim 8, wherein said determining comprises
contacting the test colon tumor sample with an antibody that
specifically binds the polypeptide, whereby a polypeptide in the
sample forms a complex with the antibody, and detecting the
antibody-polypeptide complex so formed.
12. The method of claim 8, wherein said determining comprises
contacting the test colon tumor sample with a nucleic acid probe
homologous to a human LOR-1 gene, whereby an LOR-1 mRNA in the
sample forms a hybrid with the probe, and detecting the hybrid so
formed.
13. The method of claim 8, further comprising first obtaining the
test colon tumor sample from a subject.
14. A method of determining that a colon tumor is metastatic,
comprising: determining that a tissue level and/or activity level
of a polypeptide having an amino acid sequence set forth in SEQ ID
NO: 2 in a sample from the colon tumor is higher than a control
tissue and/or activity level of the polypeptide, thereby
determining that the colon tumor is metastatic.
15. The method of claim 14, wherein the control tissue and/or
activity level is a tissue and/or activity level of the polypeptide
in a non-metastatic colon tumor sample.
16. The method of claim 14, wherein said determining is effected by
assaying the sample from the colon tumor.
17. The method of claim 16, wherein said assaying is effected by a
method selected from the group consisting of an immunological
detection method, an RNA detection method, and an enzymatic
activity detection method.
18. The method of claim 17, wherein: said immunological detection
method is selected from the group consisting of a radio-immunoassay
(RIA), an enzyme linked immunosorbent assay (ELISA), a Western blot
analysis, and an immunohistochemical analysis; said RNA detection
method is selected from the group consisting of a Northern blot
analysis, an RNA in situ hybridization stain, an RT-PCR analysis,
and an in situ RT-PCR stain; or said enzymatic activity detection
method is selected from the group consisting of a cytochemical
stain, an in vitro activity assay, and an activity gel.
19. The method of claim 16, wherein said assaying comprises
contacting the sample from the colon tumor with an antibody that
specifically binds the polypeptide, whereby a polypeptide in the
sample forms a complex with the antibody, and detecting the
antibody-polypeptide complex so formed.
20. The method of claim 16, wherein said assaying comprises
contacting the sample from the colon tumor with a nucleic acid
probe homologous to a human LOR-1 gene, whereby an LOR-1 mRNA in
the sample forms a hybrid with the probe, and detecting the hybrid
so formed.
Description
RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/647,017, filed 11 Jul. 2017, pending, which
application is a continuation of U.S. patent application Ser. No.
14/311,110, filed 20 Jun. 2014, now abandoned, which application is
a continuation of U.S. patent application Ser. No. 13/412,544,
filed 5 Mar. 2012, now abandoned, which application is a
continuation of U.S. patent application Ser. No. 10/536,440, filed
14 Nov. 2005, now issued as U.S. Pat. No. 8,163,494, which is a
national phase application of PCT/IL03/01008 having an
international filing date of 27 Nov. 2003, which claims benefit of
U.S. patent application Ser. No. 10/305,348, filed 27 Nov. 2002,
now abandoned. The entire contents of these applications are
incorporated herein by this reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
GILE-051_09US_ST25.txt. The text file is 45 KB, was created on Jan.
26, 2018, and is being submitted electronically via EFS-Web.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention relates to pharmaceutical compositions
and methods useful for modulating angiogenesis and for inhibiting
metastasis and fibrosis in a mammalian tissue. The present
invention further relates to a method of assessing the malignancy
of colon tumors and predicting the prognosis of colon cancer.
Angiogenesis
[0004] In an adult, formation of new blood vessels in normal or
diseased tissues is regulated by two processes, recapitulated
vasculogenesis (the transformation of pre-existing arterioles into
small muscular arteries) and angiogenesis, the sprouting of
existing blood vessels (which occurs both in the embryo and in the
adult).
[0005] The process of angiogenesis is regulated by biomechanical
and biochemical stimuli. Angiogenic factors such as vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF) are released by vascular cells, macrophages, and cells
surrounding blood vessels. These angiogenic factors activate
specific proteases that are involved in degradation of the basement
membrane. As a result of this degradation, vascular cells migrate
and proliferate thus leading to new blood vessel formation.
Peri-endothelial cells, such as pericytes in the capillaries,
smooth muscle cells in larger vessels and cardiac myocytes in the
heart are recruited to provide maintenance and modulatory functions
to the forming vessel.
[0006] The establishment and remodeling of blood vessels is
controlled by paracrine signals, many of which are mediated by
protein ligands which modulate the activity of transmembrane
tyrosine kinase receptors. Among these molecules are vascular
endothelial growth factor (VEGF) and its receptor families
(VEGFR-1, VEGFR-2, neuropilin-1 and neuropilin-2), Angiopoietins
1-4 (Ang-1, Ang-2 etc.) and their respective receptors (Tie-1 and
Tie-2), basic fibroblast growth factor (bFGF), platelet derived
growth factor (PDGF), and transforming growth factor .beta.
(TGF-.beta.).
[0007] The growth of solid tumors is limited by the availability of
nutrients and oxygen. When cells within solid tumors start to
produce angiogenic factors or when the levels of angiogenesis
inhibitors decline, the balance between anti-angiogenic and
angiogenic influences is perturbed, initiating the growth of new
blood vessels from the existing vascular bed into the tumor. This
event in tumor progression is known as the angiogenic switch
(Folkman, 1990; Hanahan and Folkman 1996). It had been demonstrated
that inhibitors of tumor angiogenesis are able to completely
inhibit tumor growth in mice (Boehm et al., 1997; Bergers et al.,
1999) and also inhibit tumor metastasis, a process that relies upon
close contact between the vasculature and tumor cells (Zetter,
1998). It has also been demonstrated that angiogenesis plays an
important role in the progression of breast cancer (Weidner, N.
1998; Degani et al., 1997; Guidi et al., 1997; Balsari et al.,
1999).
[0008] Such findings have prompted the use of known anti-angiogenic
factors in breast cancer therapy (Klauber et al., 1997; Harris et
al., 1996; Weinstatsaslow et al., 1994) and a search for novel
angiogenesis inhibitors.
[0009] During the past decade several novel inhibitors of
angiogenesis have been isolated including inhibitors of VEGF
signaling (Neufeld et al., 1999) and inhibitors of processes which
lead to the maturation and stabilization of new blood vessels.
Anti-integrin antibodies have been used as inhibitors of blood
vessel maturation (Brooks et al., 1994; Brooks et al., 1998).
[0010] Although several anti-angiogenic drugs are now available
commercially, the anti-angiogenic mechanisms of most of these drugs
(e.g., angiostatin and endostatin) remain unclear (O'Reilly et al.,
1997; Oreilly et al., 1996).
[0011] Since angiogenesis can be initiated by many (possibly
compensatory) angiogenic factors it stands to reason that
anti-angiogenic factors which target later processes in the
angiogenic response such as vessel maturation or a combination of
anti-angiogenic factors would be most effective in arresting vessel
formation.
[0012] Platelet factor-4 (PF4) is an anti-angiogenic protein
normally sequestered in platelets (Tanaka et al., 1997; Maione et
al., 1990; Neufeld et al., 2000). PF4 inhibits angiogenesis using
poorly defined mechanisms (Gengrinovitch et al., 1995; Brown, and
Parish, 1994; Gupta, and Singh, 1994; Watson et al., 1994). It was
previously speculated that PF4 binds to cell surface
heparan-sulfate proteoglycans and in this manner inhibits the
activity of angiogenic growth factors such as basic fibroblast
growth factor (Watson et al., 1994).
Tumor Metastasis and Staging
[0013] The transition from a localized tumor to an invasive and
metastatic tumor represents a landmark in the development of
malignant disease, since it is usually associated with a markedly
worse prognosis. The understanding of the processes that govern
this transition is therefore of prime importance.
Breast Cancer
[0014] In breast cancer, the transition from a localized to an
invasive/metastatic tumor is associated in many cases with the
formation of fibrotic foci and desmoplasia, which is the presence
of unusually dense collagenous stroma, within the primary tumor
(Colpaert et al., 2001; Hasebe et al., 2000). A similar correlation
may exist in other types of cancers such as colon and pancreatic
cancers (Nishimura et al., 1998; Ellenrieder et al., 2000). These
observations represent apparent paradoxes at first glance, since
invasiveness has long been associated with the destruction of
extracellular matrix by extracellular matrix degrading enzymes like
metalo-proteases (Stamenkovic, 2000; Duffy et al., 2000) and
heparanase (Vlodaysky and Friedmann, 2001). However, it is possible
that deposition of excess extracellular matrix may stimulate in
turn expression of matrix degrading enzymes that will contribute
under certain circumstances to tumor invasion. In fact, there is
some evidence that an increase in extracellular matrix deposition
can indeed influence the production of extracellular matrix
degrading enzymes (Schuppan et al., 2001; Swada et al., 2001).
[0015] Several prior art studies have attempted to develop agents
to treat breast cancer metastases (Sauer et al., 2002) including a
study by Kim et al., (2000) that described apicidin [cyclo
(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodec-
anoyl)], a fungal metabolite that was identified as an
antiprotozoal agent known to inhibit parasite histone deacetylase
(HDAC), that can inhibit the H-ras-induced invasive phenotype of
MCF10A human breast epithelial cells. Another agent is the
polymeric form of fibronectin that was shown to reduce tumor growth
and to posses antimetastatic activity when administered
systemically to tumor-bearing mice (Yi and Ruoslahti, 2001).
Colon Cancer
[0016] Cancer of the gastrointestinal (GI) tract, especially colon
cancer, is a highly treatable and often a curable disease when
localized to the bowel. Surgery is the primary treatment and
results in cure in approximately 50% of patients. Recurrence
following surgery is a major problem and often is the ultimate
cause of death. Nearly all cases of colorectal cancer arise from
adenomatous polyps, some of which mature into large polyps, undergo
abnormal growth and development, and ultimately progress into
cancer. This progression would appear to take at least 10 years in
most patients, rendering it a readily treatable form of cancer if
diagnosed early, when the cancer is localized.
[0017] The standard procedures currently used for establishing a
definitive diagnosis for a GI tract cancer include barium studies,
endoscopy, biopsy and computed tomography [M. F. Brennan, et al.
In: Cancer: Principles and Practice of Oncology, Fourth Edition,
pp. 849-882, Philadelphia, Pa.: J. B. Lippincott Co. (1993)].
[0018] The prognosis of colon cancer is clearly related to the
degree of penetration of the tumor through the bowel wall and the
presence or absence of nodal involvement. These two characteristics
form the basis for all staging systems developed for this disease.
Staging is usually performed by a pathologist on tissue sections
obtained via biopsy and/or surgery and it aims to determine the
anatomic extent of the disease. Accurate staging is critical for
predicting patient outcome and providing criteria for designing
optimal therapy. Inaccurate staging can result in poor therapeutic
decisions and is a major clinical problem in colon cancer.
[0019] Thus, to increase the accuracy of therapy and the survival
rate of colon cancer patients there is a need to develop sensitive
and accurate methods of staging of colon cancer.
SUMMARY OF THE INVENTION
[0020] While reducing the present invention to practice, the
present inventors have uncovered a novel PF4 binding protein, a
lysyl oxidase protein, LOR-1, which participates in modulating
angiogenesis and breast cancer metastases and as such can be used
as a target for inhibiting metastasis and reducing tumor
invasiveness. Moreover, the present inventors have uncovered that
the expression of LOR-1 is correlated with colon cancer progression
and as such can be used for accurate staging of colon cancer.
[0021] According to one aspect of the present invention there is
provided a method of modulating angiogenesis in a mammalian tissue,
the method comprising administering into the mammalian tissue a
molecule capable of modifying a tissue level and/or activity of at
least one type of lysyl oxidase to thereby modulate angiogenesis in
the mammalian tissue.
[0022] According to another aspect of the present invention there
is provided a method of modulating angiogenesis in a mammalian
tissue, the method comprising administering into the mammalian
tissue a nucleic acid construct being capable of expressing a
polypeptide having lysyl oxidase activity to thereby modulate
angiogenesis within the mammalian tissue.
[0023] According to yet another aspect of the present invention
there is provided a pharmaceutical composition useful for
modulating angiogenesis in mammalian tissue comprising, as an
active ingredient, a molecule capable of modifying a level and/or
activity of at least one type of lysyl oxidase of the mammalian
tissue and a pharmaceutically effective carrier.
[0024] According to another aspect of the present invention there
is provided a method of modulating angiogenesis in a mammalian
tissue, the method comprising administering into the mammalian
tissue a molecule capable of modifying a tissue level and/or
activity of a polypeptide at least 75% homologous to the
polypeptide set forth in SEQ ID:2 or 9 to thereby modulate
angiogenesis within the mammalian tissue. According to further
features in preferred embodiments of the invention described below,
the molecule is an antibody or an antibody fragment capable of
binding with, and at least partially inhibiting the activity of,
the at least one polypeptide.
[0025] According to still further features in the described
preferred embodiments the antibody or the antibody fragment is
directed against at least a portion of the polypeptide set forth in
SEQ ID NO:2, 3, 6, 8 or 9.
[0026] According to still further features in the described
preferred embodiments the molecule is a polynucleotide capable of
down regulating expression of the at least one type of lysyl
oxidase.
[0027] According to still further features in the described
preferred embodiments the polynucleotide is at least partially
complementary with the polynucleotide set forth in SEQ ID NO:1, 4,
5 or 7.
[0028] According to still further features in the described
preferred embodiments the molecule is a polypeptide having lysyl
oxidase activity.
[0029] According to still further features in the described
preferred embodiments the polypeptide is as set forth in SEQ ID
NO:2, 3, 6, 8 or 9.
[0030] According to another aspect of the present invention there
is provided a method of modulating angiogenesis in a mammalian
tissue, the method comprising administering into the mammalian
tissue a nucleic acid construct being capable of expressing a
polypeptide having lysyl oxidase activity to thereby modulate
angiogenesis within the mammalian tissue.
[0031] According to still further features in the described
preferred embodiments the polypeptide is at least 75% homologous to
the polypeptide set forth in SEQ ID NO:2, 3, 6, 8 or 9.
[0032] According to still another aspect of the present invention
there is provided method of identifying molecules capable of
modulating angiogenesis, the method comprising: (a) isolating
molecules which exhibit specific reactivity with at least one type
of lysyl oxidase; and (b) testing the molecules within mammalian
tissue so as to determine the angiogenesis modulation activity
thereof.
[0033] According to still further features in the described
preferred embodiments step (a) is effected by binding assays and/or
lysyl oxidase activity assays.
[0034] According to an additional aspect of the present invention
there is provided method of determining the malignancy of cancerous
tissue, the method comprising (a) determining a lysyl oxidase
expression level and/or activity of the cancerous tissue; and (b)
comparing the lysyl oxidase expression level and/or activity with
that determined for control tissue to thereby determine the
malignancy of the cancerous tissue.
[0035] According to another aspect of the present invention there
is provided method of inhibiting metastasis and fibrosis in a
mammalian tissue, the method comprising administering into the
mammalian tissue a molecule capable of downregulating a tissue
level and/or activity of at least one type of lysyl oxidase to
thereby inhibit metastasis in the mammalian tissue
[0036] According to still another aspect of the present invention
there is provided a pharmaceutical composition useful for
inhibiting metastasis and fibrosis in mammalian tissue comprising,
as an active ingredient, a molecule capable of downregulating a
level and/or activity of at least one type of lysyl oxidase of the
mammalian tissue and a pharmaceutically effective carrier.
[0037] According to further features in preferred embodiments of
the invention described below, the molecule is an antibody or an
antibody fragment capable of binding with, and at least partially
inhibiting the activity of, the at least one polypeptide.
[0038] According to still further features in the described
preferred embodiments the antibody or the antibody fragment is
directed against at least a portion of the polypeptide set forth in
SEQ ID NO:2, 3, 6, 8 or 9.
[0039] According to still further features in the described
preferred embodiments the molecule is a polynucleotide capable of
downregulating expression of the at least one type of lysyl
oxidase.
[0040] According to still further features in the described
preferred embodiments the polynucleotide is at least partially
complementary with the polynucleotide set forth in SEQ ID NO:1, 4,
5 or 7.
[0041] According to still another aspect of the present invention
there is provided method of identifying molecules capable of
inhibiting metastasis and fibrosis, the method comprising: (a)
screening and identifying molecules which exhibit specific
reactivity with at least one type of lysyl oxidase; and (b) testing
the metastasis and fibrosis inhibitory potential of the said
molecules.
[0042] According to still further features in the described
preferred embodiments step (a) is effected by binding assays and/or
lysyl oxidase activity assays.
[0043] According to yet another aspect of the present invention
there is provided a method for inhibiting metastasis and fibrosis
in a mammalian tissue, the method comprising administering to the
mammalian tissue a molecule capable of downregulating a tissue
level and/or activity of a polypeptide at least 75% homologous to
the polypeptide set forth in ID NO: 2 or 9, to thereby inhibit
metastasis and fibrosis in a mammalian tissue.
[0044] According to yet another aspect of the present invention
there is provided a method of assessing a malignancy of a colon
tumor comprising determining a tissue level and/or an activity
level of a polypeptide at least 75% homologous to the polypeptide
set forth in SEQ ID NO:2 or 9 in a tissue of the colon tumor,
thereby assessing the malignancy of the colon tumor.
[0045] According to yet another aspect of the present invention
there is provided a method of predicting a prognosis of an
individual diagnosed with colon cancer comprising: (a) obtaining a
colon tumor tissue from the individual, and; (b) determining a
tissue level and/or an activity level of a polypeptide at least 75%
homologous to the polypeptide set forth in SEQ ID NO:2 or 9 in the
colon tumor tissue to thereby assess the malignancy of the colon
tumor tissue and predict the prognosis of the individual diagnosed
with colon cancer.
[0046] According to still further features in the described
preferred embodiments the tissue of the colon tumor is obtained
using a colon biopsy and/or a colon surgery.
[0047] According to still further features in the described
preferred embodiments determining the tissue level of the
polypeptide is effected by an immunological detection method and/or
an RNA detection method.
[0048] According to still further features in the described
preferred embodiments determining the activity level of the
polypeptide is effected by an enzymatic activity detection
method.
[0049] According to still further features in the described
preferred embodiments the immunological detection method is
selected from the group consisting of a radio-immunoassay (RIA), an
enzyme linked immunosorbent assay (ELISA), a Western blot analysis,
and an immunohistochemical analysis.
[0050] According to still further features in the described
preferred embodiments the RNA detection method is selected from the
group consisting of a Northern blot analysis, an RNA in situ
hybridization stain, an RT-PCR analysis, and an in situ RT-PCR
stain.
[0051] According to still further features in the described
preferred embodiments the enzymatic activity detection method is
selected from the group consisting of a cytochemical stain, an in
vitro activity assay, and an activity gel.
[0052] According to still further features in the described
preferred embodiments the malignancy of the colon tumor is assessed
by comparing the tissue level and/or the activity level of the
polypeptide in the tissue of the colon tumor with a tissue level
and/or activity level of the polypeptide in a normal colon
tissue.
[0053] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
pharmaceutical compositions and methods that can be used to treat
metastatic cancer, formation of tumor fibrosis and other disorders
characterized by excessive or insufficient blood vessel formation
as well as by providing a method of assessing the malignancy of
colon cancer.
[0054] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0056] In the drawings:
[0057] FIG. 1 illustrates SDS-PAGE analysis of extracts of Porcine
aortic endothelial cells (PAE Cells) which were transfected with
vector along (lane 1) or with vector containing the LOR-1 cDNA
(lane 3) and metabolically labeled with .sup.355-methionine.
Extracts from the vector transfected cells (lane 2), from vector
containing LOR-1 cDNA transfected cells (lane 4) or from
.sup.35S-methionine labeled human umbilical vein endothelial cells
(HUVEC) (lane 5) were purified on a PF4 affinity column. A Band
corresponding in size to the original band observed in the HUVEC is
evident (compare lanes 4 and 5); this band is absent in extracts of
vector transfected cells.
[0058] FIG. 2 illustrates differential expression of LOR-1 in
breast cancer derived cells of different metastatic potential. The
metastatic potential of the cells increases from left to right, and
is correlated to increased LOR-1 mRNA expression. The results
correspond to northern blot analysis of LOR-1 mRNA expression. Data
regarding the relative metastatic potential of the cell lines was
derived from the literature.
[0059] FIG. 3 illustrates expression of recombinant LOR-1 in MCF-7
breast cancer cells (lane 1). Vector transfected MCF-7 cells (lane
2) and two clones of MCF-7 expressing recombinant LOR-1 (lane 3,
clone 12, lane 4, clone 22) were grown for two days in serum free
medium. The medium from an equal number of cells was collected,
concentrated 30 fold using Centricon.TM., and 10 .mu.l aliquots
were electrophoresed using an SDS/PAGE gel. Proteins were blotted
onto nitrocellulose, and LOR-1 protein was identified using an
antibody directed against the C-terminal of LOR-1. A secondary
alkaline-phosphatase coupled antibody and NBT-BICP staining were
used to detect the primary bound antibody.
[0060] FIG. 4 illustrates tumor size as correlated to LOR-1
expression. Parental MCF-7 cells (par), MCF-7 cells transfected
with pCDNA3 vector alone (vec) and two recombinant LOR-1 expressing
MCF-7 cells (clones 12 and 24) were deposited under the skin of
immune deficient mice (10.sup.7/injection site) along with an
estrogen slow release pellet. Six animals were used for each cell
type implanted. The area of the tumors was measured every few days.
Bars represent standard deviation from the mean.
[0061] FIGS. 5a-b illustrate anti-factor-8 immunostaining of tumors
generated by MCF-7 cells transfected with the expression vector
alone (FIG. 5a) or with an expression vector containing the LOR-1
cDNA (FIG. 5b). Counter staining was performed with
Hematoxylin-eosin (blue). Invasion of blood vessels into the tumor
mass is more abundant in LOR-1 expressing tumors (FIG. 5b) as
compared to tumors generated by control cells which do not express
LOR-1 (FIG. 5a).
[0062] FIGS. 6a-d illustrate liver sections of Wilson's disease
patients (FIGS. 6c-d) and normal patients (FIGS. 6a-b) probed with
a LOR-1 sense probe (FIGS. 6a, 6c) and antisense probe (FIGS. 6b,
6d).
[0063] FIG. 7 illustrates results of a whole mount in-situ
hybridization using a LOR-1 cDNA probe and a 4 day old chick
embryo. Strong expression of LOR-1 mRNA is observed in amniotic
blood vessels (arrow).
[0064] FIG. 8 illustrates sequence alignment of several lysyl
oxidases including LOR-1.
[0065] FIGS. 9a-b. illustrate the expression of LOR-1 and LOR-2 in
human breast cancer derived cells: Total RNA was prepared from
confluent MCF-7 cells (MCF-7), MDA-MB-231 cells (MDA-231) and
MDA-MB-435 cells (MDA-435) and was subjected to Northern blot
analysis using a LOR-1 (FIG. 9a) and LOR-2 (FIG. 9b) specific cDNA
probes. LOR-1 specific hybridization signal is seen in tumors
derived from both MDA-231 and MDA-435 cells but not in tumors
derived from MCF-7 cells. LOR-2 specific hybridization signal is
seen only in tumors derived from MDA-435 cells. LOR-1 and LOR-2
non-specific hybridization signals are seen in all tumors' RNA in a
band that corresponds to the 28S rRNA.
[0066] FIGS. 9c-f illustrate the expression pattern of LOR-1 in
normal human breast (FIG. 9c), in in-situ non-invasive breast
carcinoma (FIG. 9d), in grade-1 invasive ductal carcinoma (FIG. 9e)
and in grade-3 invasive ductal carcinoma (FIG. 9f). A polyclonal
affinity purified rabbit antibody directed against the C-terminal
of LOR-1 was used to detect the expression of LOR-1. High level
expression of LOR-1 protein is seen in the epithelium of the normal
duct (FIG. 9c, open arrow); magnification .times.100. In in-situ
non-invasive breast carcinoma (FIG. 9d) the cancer cells have
filled the duct but are still confined to it. Many of the cells
located at periphery of the tumor have lost their ability to
express LOR-1, while at the center, the cells still express high
levels of LOR-1 (FIG. 9d, empty arrow); magnification .times.200.
In grade-1 invasive breast carcinoma the tumorigenic cells have
formed pseudo-ducts (FIG. 9e, black arrows) but they do not express
LOR-1 anymore. However, cells found in a nearby carcinoma in-situ
express LOR-1 (FIG. 9e, empty arrow); magnification .times.200. In
grade-3 invasive breast carcinoma the tumorigenic cells express
large amounts of LOR-1 and the morphology is completely disorderly
(FIG. 9f); magnification .times.200.
[0067] FIG. 10a illustrates the expression of recombinant LOR-1 in
MCF-7 cells transfected with the expression vector alone (vec) or
with an expression vector containing the LOR-1 cDNA clone 12 or 24
(#12 or #24, respectively). LOR-1 proteins were detected by Western
blot with an antibody directed against the C-terminal of human
LOR-1.
[0068] FIG. 10b illustrates post-translational processes of LOR-1.
MCF-7/Tet-LOR1 cells grown in the presence of tetracycline were
trypsinized and seeded into a 24-well dish (5.times.10.sup.4
cells/well) in a serum free medium in the absence of tetracycline.
Cell aliquots collected at the noted times following tetracycline
removal and were analyzed for the presence of LOR-1 by Western blot
as described in FIG. 10a.
[0069] FIG. 10c illustrates the growth rate of tumors derived from
control or LOR-1 expressing MCF-7 cells. MCF-7 cells transfected
with the expression vector alone (vec) or with an expression vector
containing the LOR-1 cDNA clone 12 or 24 were injected into the
mammary fat pads of female athymic nude mice. Each cell type was
implanted in 8 animals. Tumor area was measured at the indicated
times. Error bars represent the standard error of the mean. The
experiment was terminated and the mice sacrificed when the tumor
reached a diameter of about 1 cm.
[0070] FIG. 10d illustrates the relative size of tumor in mice 25
days following injection of MCF-7 cells. Shown are mice harboring
tumors that developed from cells transfected with the expression
vector alone (left) or with the expression vector containing LOR-1
cDNA clone 24 (center) or 12 (right).
[0071] FIGS. 11a-h illustrate fibrotic foci and collagen deposits
in tumors derived from MCF-7 cells expressing recombinant LOR-1.
Hematoxylin-eosin staining demonstrate a few necrotic foci in a
tumor derived from MCF-7 cells transfected with expression vector
alone (FIG. 11a, arrow, magnification .times.20) and numerous
necrotic foci in a tumor derived from MCF-7 cells transfected with
expression vector containing LOR-1 cDNA clone 12 (FIG. 11b, arrows,
magnification .times.20). FIG. 11c illustrates human keratin-7
immunostaining of tumors generated by MCF-7 cells transfected with
expression vector containing clone 12. Counter staining was
performed with Hematoxylin-eosin (blue). Arrow points to nuclei of
host cells concentrated in the fibrotic foci. No necrosis can be
seen; magnification .times.40. FIGS. 11d-f illustrate collagen
deposits in tumor cells as viewed by Masson's Trichrome stain. A
few collagen deposits are seen in tumors generated from MCF-7 cells
transfected with the expression vector alone (FIG. 11d, arrows,
magnification .times.200). Thick collagen bundles are seen between
tumor cells generated from MCF-7 cells transfected with expression
vector containing clone 12 (FIG. 11e, magnification .times.200).
The fibrotic area is full with collagen fibers and interspaced with
host derived cells in tumors generated from MCF-7 cells transfected
with expression vector containing clone 24 (FIG. 11f, magnification
.times.200). FIGS. 11g-h illustrate blood vessels stained with
Masson's Trichrome in tumors generated from MCF-7 cells transfected
with the expression vector alone (FIG. 11g) or expression vector
containing clone 12 (FIG. 11h); magnification .times.400.
[0072] FIGS. 12a-d illustrate the deposition of collagen type-3 by
reticulum stain in tumors generated from MCF-7 and C6 glioma cells
transfected with expression vector alone (FIGS. 12a, c) or vector
expressing LOR-1 cDNA clone 12 (FIGS. 12b, d); magnification
.times.200.
[0073] FIGS. 13a-h illustrate the invasiveness of tumors derived
from MCF-7 cells expressing recombinant LOR-1. Shown are
histological sections of tumors generated from MCF-7 cells
transfected with an expression vector alone (FIGS. 13a, b) or a
vector expressing LOR-1 (FIGS. 13c-h). Sections were labeled with
either a monoclonal antibody specific to human keratin-7 (FIGS.
13a-d and f-h, blue purple stain) or with an antibody directed
against LOR-1 (FIG. 13e, red stain). Counterstain was with
Hematoxylin (light blue) in all sections. Black arrows designate
cytokeratin-7 positive tumor cells invading the tumor
pseudo-capsule (FIG. 13c), infiltrating between adjacent muscle
bundles (FIG. 13d), or invading the vasculature (FIGS. 13f, g) and
peri-neural space of nerves (FIG. 13h). White arrows designate
LOR-1 positive tumor cells infiltrating muscles located next to the
tumor (FIG. 13g). Blood vessels v; nerves n; muscle fibers m;
magnification .times.100 for FIGS. 13a-c, f, h and .times.200 for
FIGS. 13d, e, g.
[0074] FIGS. 14a-d illustrate the expression pattern of LOR-1 in
normal human colon (FIG. 14a), in a colon tumor including
hyperplasia (FIG. 14b, diamond arrows) and adenoma (FIG. 14b,
circled arrows), in a low-grade colon adenocarcinoma (FIG. 14c,
arrows, brown stain) and in a high-grade colon adenocarcinoma (FIG.
14d, brown stain). A polyclonal affinity purified rabbit antibody
directed against the C-terminal of LOR-1 was used to detect the
expression of LOR-1 by immunohistochemistry in tissue sections
obtained from various colon cancer tumors. Note the faint LOR-1
staining in a few cells of the normal colon (FIG. 14a, arrows,
magnification .times.10), the moderate LOR-1 staining in
hyperplasia tumor (FIG. 14b, diamond circles) and the high LOR-1
staining in cells of the colon adenoma (FIG. 14b, circle arrows,
magnification .times.4). Also note the significant LOR-1 staining
in low-grade adenocarcinoma (FIG. 14c, arrows, magnification
.times.10) and in high-grade carcinoma (FIG. 14d, magnification
.times.20).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0075] The present invention is of pharmaceutical compositions and
methods that can be used to modulate angiogenesis and to inhibit
tumor invasiveness and tumor fibrosis. Specifically, the present
invention can be used to suppress tumor growth and metastasis as
well as to treat disorders such as, for example, arthritis,
diabetic retinopathy, psoriasis and vasculitis. Moreover, the
present invention is of a method of determining the malignancy of
colon cancer tumors which can be used for colon cancer staging.
Specifically, the present invention can be used to predict the
prognosis of colon cancer patients based on the tissue level and/or
activity level of LOR-1 in the colon tumors.
[0076] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0077] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings described in the
Examples section. The invention is capable of other embodiments or
of being practiced or carried out in various ways. Also, it is to
be understood that the phraseology and terminology employed herein
is for the purpose of description and should not be regarded as
limiting.
[0078] As described in the Examples section which follows, the
present inventors have uncovered a novel protein constituent of the
angiogenic process.
[0079] This protein, which is termed LOR-1 herein (SEQ ID NO:2)
belongs to the lysyl oxidase family of enzymes which catalyze the
formation of covalent crosslinks between lysine residues on
adjacent collagen or elastin fibrils. The lysyl oxidase family
includes four genes (Saito et al., 1997; Kim et al., 1995; Reiser
et al., 1992; Jang et al., 1999), the protein sequences of which
are presented in SEQ ID NOs:3, 6, 8 and 9. A homology comparison
between several lysyl oxidase family members is presented in FIG. 8
which is further described in the Examples section which
follows.
[0080] Each member of the lysyl oxidase family of enzymes includes
a highly conserved lysyl oxidase domain, the activity of which is
highly dependent on the presence of copper.
[0081] It should be noted that prior art studies have shown that
removal of copper from tumor tissues leads to inhibition of
angiogenesis (Rabinovitz, 1999; Yoshida et al., 1995). This further
substantiates the role of the lysyl oxidase family of enzymes in
angiogenesis since presumably, removal of copper leads to
inhibition of lysyl oxidases.
[0082] Further support to the angiogenic activity of lysyl oxidases
is provided by the PF4-LOR-1 binding assays presented herein. As
mentioned hereinabove, PF4 is an inhibitor of angiogenesis. As
such, the anti-angiogenic activity exhibited by PF4 may be effected
through LOR-1 inhibition, which, as demonstrated in the Examples
section which follows, is highly expressed in the endothelial cells
lining blood vessels.
[0083] Thus according to one aspect of the present invention there
is provided a method of modulating angiogenesis
[0084] The method is effected by administering into the mammalian
tissue a molecule capable of modifying a tissue level and/or
activity of at least one type of lysyl oxidase to thereby modulate
angiogenesis in the mammalian tissue.
[0085] As used herein, the phrase "tissue level" refers to the
level of lysyl oxidase protein present in active form in the tissue
at a given time point. Protein levels are determined by factors
such as, transcription and/or translation rates, RNA or protein
turnover and/or protein localization within the cell. As such any
molecule which effects any of these factors can modify the tissue
level of the lysyl oxidase.
[0086] As used herein the term "activity" refers to an enzymatic
activity of the lysyl oxidase. A molecule which can modify the
enzymatic activity may directly or indirectly alter substrate
specificity of the enzyme or activity of the catalytic site
thereof.
[0087] There are numerous examples of molecules which can
specifically modify the tissue level and/or activity of a lysyl
oxidase. Such molecules can be categorized into lysyl oxidase
"downregulators" or "upregulators".
Downregulators
[0088] One example of an agent capable of downregulating a lysyl
oxidase protein is an antibody or antibody fragment capable of
specifically binding lysyl oxidase or at least part of the lysyl
oxidase protein (e.g., region spanning the catalytic site) and
inhibiting its activity when introduced into the mammalian tissue.
As such, an antibody or an antibody fragment directed at a lysyl
oxidase can be used to suppress or arrest the formation of blood
vessels, and to inhibit tumor fibrosis and metastasis.
[0089] Numerous examples of antibody inhibitors are known in the
art, including inhibitors of angiogenesis which target angiogenic
factors (Brooks et al., 1994; Brooks et al., 1998).
[0090] Preferably, the antibody specifically binds to at least one
epitope of a lysyl oxidase. As used herein, the term "epitope"
refers to any antigenic determinant on an antigen to which the
paratope of an antibody binds.
[0091] Epitopic determinants usually consist of chemically active
surface groupings of molecules such as amino acids or carbohydrate
side chains and usually have specific three-dimensional structural
characteristics, as well as specific charge characteristics.
[0092] The term "antibody" as used in this invention includes
intact molecules as well as functional fragments thereof, such as
Fab, F(ab')2, and Fv that are capable of binding to macrophages.
These functional antibody fragments are defined as follows: (1)
Fab, the fragment which contains a monovalent antigen-binding
fragment of an antibody molecule, can be produced by digestion of
whole antibody with the enzyme papain to yield an intact light
chain and a portion of one heavy chain; (2) Fab', the fragment of
an antibody molecule that can be obtained by treating whole
antibody with pepsin, followed by reduction, to yield an intact
light chain and a portion of the heavy chain; two Fab' fragments
are obtained per antibody molecule; (3) (Fab')2, the fragment of
the antibody that can be obtained by treating whole antibody with
the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer
of two Fab' fragments held together by two disulfide bonds; (4) Fv,
defined as a genetically engineered fragment containing the
variable region of the light chain and the variable region of the
heavy chain expressed as two chains; and (5) Single chain antibody
("SCA"), a genetically engineered molecule containing the variable
region of the light chain and the variable region of the heavy
chain, linked by a suitable polypeptide linker as a genetically
fused single chain molecule.
[0093] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0094] Antibody fragments according to the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli or mammalian cells (e.g. Chinese hamster ovary cell
culture or other protein expression systems) of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
This fragment can be further cleaved using a thiol reducing agent,
and optionally a blocking group for the sulfhydryl groups resulting
from cleavage of disulfide linkages, to produce 3.5S Fab'
monovalent fragments. Alternatively, an enzymatic cleavage using
pepsin produces two monovalent Fab' fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained
therein, which patents are hereby incorporated by reference in
their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126
(1959)]. Other methods of cleaving antibodies, such as separation
of heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0095] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al.
[Proc. Nat. Acad. Sci. USA 69: 2659-62 (1972)]. Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as gluteraldehyde. Preferably,
the Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow and Filpula, Methods 2:
97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et
al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778,
which is hereby incorporated by reference in its entirety.
[0096] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry [Methods, 2: 106-10
(1991)].
[0097] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0098] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues 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)1, by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable 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.
[0099] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et
al., J. Mol. Biol., 222: 581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J.
Immunol., 147(1): 86-95 (1991)]. Similarly, human antibodies can be
made by introduction of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368:
812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg
and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0100] As is described below, various approaches can be used to
reduce or abolish transcription or translation of a lysyl oxidase.
These include antisense oligonucleotides, Ribozyme, DNAzyme, siRNA
and triple helix forming oligonucleotide approaches.
Antisense Polynucleotide
[0101] Downregulation of a lysyl oxidase can be effected using an
antisense polynucleotide capable of specifically hybridizing with
an mRNA transcript encoding the lysyl oxidase protein.
[0102] Design of antisense molecules which can be used to
efficiently downregulate lysyl oxidase must be effected while
considering two aspects important to the antisense approach. The
first aspect is delivery of the oligonucleotide into the cytoplasm
of the appropriate cells, while the second aspect is design of an
oligonucleotide which specifically binds the designated mRNA within
cells in a way which inhibits translation thereof
[0103] Several considerations must be taken into account when
designing antisense oligonucleotides. For efficient in vivo
inhibition of gene expression using antisense oligonucleotides or
analogs, the oligonucleotides or analogs must fulfill the following
requirements (i) sufficient specificity in binding to the target
sequence; (ii) solubility in water; (iii) stability against intra-
and extracellular nucleases; (iv) capability of penetration through
the cell membrane; and (v) when used to treat an organism, low
toxicity. Algorithms for identifying those sequences with the
highest predicted binding affinity for their target mRNA based on a
thermodynamic cycle that accounts for the energy of structural
alterations in both the target mRNA and the oligonucleotide are
also available [see, for example, Walton et al. Biotechnol Bioeng
65: 1-9 (1999)].
[0104] Such algorithms have been successfully used to implement an
antisense approach in cells. For example, the algorithm developed
by Walton et al. enabled scientists to successfully design
antisense oligonucleotides for rabbit beta-globin (RBG) and mouse
tumor necrosis factor-alpha (TNF alpha) transcripts. The same
research group has more recently reported that the antisense
activity of rationally selected oligonucleotides against three
model target mRNAs (human lactate dehydrogenase A and B and rat
gp130) in cell culture as evaluated by a kinetic PCR technique
proved effective in almost all cases, including tests against three
different targets in two cell types with phosphodiester and
phosphorothioate oligonucleotide chemistries.
[0105] In addition, several approaches for designing and predicting
efficiency of specific oligonucleotides using an in vitro system
were also published (Matveeva et al., Nature Biotechnology 16:
1374-1375 (1998)].
[0106] An antisense molecule which can be used with the present
invention includes a polynucleotide or a polynucleotide analog of
at least 10 bases, preferably between 10 and 15, more preferably
between 15 and 20 bases, most preferably at least 17, at least 18,
at least 19, at least 20, at least 22, at least 25, at least 30 or
at least 40 bases which is hybridizable in vivo, under
physiological conditions, with a portion of a polynucleotide strand
encoding a polypeptide at least 50% homologous to SEQ ID NO:1, 4, 5
or 7 or at least 75% homologous to an N-terminal portion thereof as
determined using the BestFit software of the Wisconsin sequence
analysis package, utilizing the Smith and Waterman algorithm, where
gap creation penalty equals 8 and gap extension penalty equals
2.
[0107] The antisense oligonucleotides used by the present invention
can be expressed from a nucleic acid construct administered into
the tissue, in which case inducible promoters are preferably used
such that antisense expression can be switched on and off, or
alternatively such oligonucleotides can be chemically synthesized
and administered directly into the tissue, as part of, for example,
a pharmaceutical composition.
[0108] The ability of chemically synthesizing oligonucleotides and
analogs thereof having a selected predetermined sequence offers
means for downmodulating gene expression. Four types of gene
expression modulation strategies may be considered.
[0109] At the transcription level, antisense or sense
oligonucleotides or analogs that bind to the genomic DNA by strand
displacement or the formation of a triple helix, may prevent
transcription. At the transcript level, antisense oligonucleotides
or analogs that bind target mRNA molecules lead to the enzymatic
cleavage of the hybrid by intracellular RNase H. In this case, by
hybridizing to the targeted mRNA, the oligonucleotides or
oligonucleotide analogs provide a duplex hybrid recognized and
destroyed by the RNase H enzyme. Alternatively, such hybrid
formation may lead to interference with correct splicing. As a
result, in both cases, the number of the target mRNA intact
transcripts ready for translation is reduced or eliminated.
[0110] At the translation level, antisense oligonucleotides or
analogs that bind target mRNA molecules prevent, by steric
hindrance, binding of essential translation factors (ribosomes), to
the target mRNA, a phenomenon known in the art as hybridization
arrest, disabling the translation of such mRNAs.
[0111] Unmodified oligonucleotides are typically impractical for
use as antisense sequences since they have short in vivo
half-lives, during which they are degraded rapidly by nucleases.
Furthermore, they are difficult to prepare in more than milligram
quantities. In addition, such oligonucleotides are poor cell
membrane penetrants.
[0112] Thus it is apparent that in order to meet all the above
listed requirements, oligonucleotide analogs need to be devised in
a suitable manner.
[0113] For example, problems arising in connection with
double-stranded DNA (dsDNA) recognition through triple helix
formation have been diminished by a clever "switch back" chemical
linking, whereby a sequence of polypurine on one strand is
recognized, and by "switching back", a homopurine sequence on the
other strand can be recognized. Also, good helix formation has been
obtained by using artificial bases, thereby improving binding
conditions with regard to ionic strength and pH.
[0114] In addition, in order to improve half-life as well as
membrane penetration, a large number of variations in
polynucleotide backbones have been done, nevertheless with little
success.
[0115] Oligonucleotides can be modified either in the base, the
sugar or the phosphate moiety. These modifications include, for
example, the use of methylphosphonates, monothiophosphates,
dithiophosphates, phosphoramidates, phosphate esters, bridged
phosphorothioates, bridged phosphoramidates, bridged
methylenephosphonates, dephospho intemucleotide analogs with
siloxane bridges, carbonate bridges, carboxymethyl ester bridges,
carbonate bridges, carboxymethyl ester bridges, acetamide bridges,
carbamate bridges, thioether bridges, sulfoxy bridges, sulfono
bridges, various "plastic" DNAs, anomeric bridges and borane
derivatives [Cook (1991) Medicinal chemistry of antisense
oligonucleotides-future opportunities. Anti-Cancer Drug Design 6:
585].
[0116] International patent application WO 89/12060 discloses
various building blocks for synthesizing oligonucleotide analogs,
as well as oligonucleotide analogs formed by joining such building
blocks in a defined sequence. The building blocks may be either
"rigid" (i.e., containing a ring structure) or "flexible" (i.e.,
lacking a ring structure). In both cases, the building blocks
contain a hydroxy group and a mercapto group, through which the
building blocks are said to join to form oligonucleotide analogs.
The linking moiety in the oligonucleotide analogs is selected from
the group consisting of sulfide (--S--), sulfoxide (--SO--), and
sulfone (--SO2-).
[0117] International patent application WO 92/20702 describe an
acyclic oligonucleotide which includes a peptide backbone on which
any selected chemical nucleobases or analogs are stringed and serve
as coding characters as they do in natural DNA or RNA. These new
compounds, known as peptide nucleic acids (PNAs), are not only more
stable in cells than their natural counterparts, but also bind
natural DNA and RNA 50 to 100 times more tightly than the natural
nucleic acids cling to each other. PNA oligomers can be synthesized
from the four protected monomers containing thymine, cytosine,
adenine and guanine by Merrifield solid-phase peptide synthesis. In
order to increase solubility in water and to prevent aggregation, a
lysine amide group is placed at the C-terminal region.
[0118] RNA oligonucleotides may also be used for antisense
inhibition as they form a stable RNA-RNA duplex with the target,
suggesting efficient inhibition. However, due to their low
stability RNA oligonucleotides are typically expressed inside the
cells using vectors designed for this purpose. This approach is
favored when attempting to target an mRNA that encodes an abundant
and long-lived protein.
[0119] The prior art teaches of a number of delivery strategies
which can be used to efficiently deliver oligonucleotides into a
wide variety of cell types [see, for example, Luft J Mol Med 76:
75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et
al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys
Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem
Biophys Res Commun 231: 540-5 (1997)].
[0120] Antisense therapeutics has the potential to treat many
life-threatening diseases with a number of advantages over
traditional drugs. Traditional drugs intervene after a
disease-causing protein is formed. Antisense therapeutics, however,
block mRNA transcription/translation and intervene before a protein
is formed, and since antisense therapeutics target only one
specific mRNA, they should be more effective with fewer side
effects than current protein-inhibiting therapy.
[0121] Several clinical trials have demonstrated safety,
feasibility and activity of antisense oligonucleotides. For
example, antisense oligonucleotides suitable for the treatment of
cancer have been successfully used [Holmund et al., Curr Opin Mol
Ther 1: 372-85 (1999)], while treatment of hematological
malignancies via antisense oligonucleotides targeting c-myb gene,
p53 and Bcl-2 had entered clinical trials and had been shown to be
tolerated by patients [Gerwitz Curr Opin Mol Ther 1: 297-306
(1999)].
[0122] More recently, antisense-mediated suppression of human
heparanase gene expression has been reported to inhibit pleural
dissemination of human cancer cells in a mouse model [Uno et al.,
Cancer Res 61: 7855-60 (2001)].
[0123] The first antisense drug was recently approved by the FDA.
The drug, Fomivirsen, was developed by Isis, and is indicated for
local treatment of cytomegalovirus in patients with AIDS who are
intolerant of or have a contraindication to other treatments for
CMV retinitis or who were insufficiently responsive to previous
treatments for CMV retinitis (Pharmacotherapy News Network).
[0124] Thus, the current consensus is that recent developments in
the field of antisense technology which, as described above, have
led to the generation of highly accurate antisense design
algorithms and a wide variety of oligonucleotide delivery systems,
enable an ordinarily skilled artisan to design and implement
antisense approaches suitable for downregulating expression of
known sequences without having to resort to undue trial and error
experimentation.
Ribozyme
[0125] Another agent capable of downregulating a lysyl oxidase is a
ribozyme molecule capable of specifically cleaving an mRNA
transcript encoding a lysyl oxidase. Ribozymes are being
increasingly used for the sequence-specific inhibition of gene
expression by the cleavage of mRNAs encoding proteins of interest
[Welch et al., Curr Opin Biotechnol. 9: 486-96 (1998)]. The
possibility of designing ribozymes to cleave any specific target
RNA has rendered them valuable tools in both basic research and
therapeutic applications. In the therapeutics area, ribozymes have
been exploited to target viral RNAs in infectious diseases,
dominant oncogenes in cancers and specific somatic mutations in
genetic disorders [Welch et al., Clin Diagn Virol. 10: 163-71
(1998)]. Most notably, several ribozyme gene therapy protocols for
HIV patients are already in Phase 1 trials. More recently,
ribozymes have been used for transgenic animal research, gene
target validation and pathway elucidation. Several ribozymes are in
various stages of clinical trials. ANGIOZYME was the first
chemically synthesized ribozyme to be studied in human clinical
trials. ANGIOZYME specifically inhibits formation of the VEGF-r
(Vascular Endothelial Growth Factor receptor), a key component in
the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well
as other firms have demonstrated the importance of
anti-angiogenesis therapeutics in animal models. HEPTAZYME, a
ribozyme designed to selectively destroy Hepatitis C Virus (HCV)
RNA, was found effective in decreasing Hepatitis C viral RNA in
cell culture assays (Ribozyme Pharmaceuticals, Incorporated--WEB
home page).
DNAzyme
[0126] Another agent capable of downregulating a lysyl oxidase is a
DNAzyme molecule capable of specifically cleaving an mRNA
transcript or DNA sequence of the lysyl oxidase. DNAzymes are
single-stranded polynucleotides which are capable of cleaving both
single and double stranded target sequences (Breaker, R. R. and
Joyce, G. Chemistry and Biology 1995; 2: 655; Santoro, S. W. &
Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943: 4262). A general
model (the "10-23" model) for the DNAzyme has been proposed.
"10-23" DNAzymes have a catalytic domain of 15
deoxyribonucleotides, flanked by two substrate-recognition domains
of seven to nine deoxyribonucleotides each. This type of DNAzyme
can effectively cleave its substrate RNA at purine:pyrimidine
junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci.
USA 1997; 943: 4262; for rev of DNAzymes see Khachigian, L M [Curr
Opin Mol Ther 4: 119-21 (2002)].
[0127] Examples of construction and amplification of synthetic,
engineered DNAzymes recognizing single and double-stranded target
cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to
Joyce et al. DNAzymes of similar design directed against the human
Urokinase receptor were recently observed to inhibit Urokinase
receptor expression, and successfully inhibit colon cancer cell
metastasis in vivo (Itoh et al., 2002, Abstract 409, Ann Meeting
Am. Soc. Gen. Ther., located on the World Wide Web at asgt.org). In
another application, DNAzymes complementary to bcr-abl oncogenes
were successful in inhibiting the oncogenes expression in leukemia
cells, and lessening relapse rates in autologous bone marrow
transplant in cases of CML and ALL.
siRNA
[0128] Another mechanism of down regulating a lysyl oxidase at the
transcript level is RNA interference (RNAi), an approach which
utilizes small interfering dsRNA (siRNA) molecules that are
homologous to the target mRNA and lead to its degradation [Carthew
R W. Gene silencing by double-stranded RNA. Curr Opin Cell Biol
2001 April; 13(2):244-8].
[0129] RNA interference is a two-step process. In the first step,
which is termed as the initiation step, input dsRNA is digested
into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably
by the action of Dicer, a member of the RNase III family of
dsRNA-specific ribonucleases, which processes (cleaves) dsRNA
(introduced directly or via a transgene or a virus) in an
ATP-dependent manner. Successive cleavage events degrade the RNA to
19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs
[Hutvagner and Zamore Curr. Opin. Genetics and Development 12:
225-232 (2002); and Bernstein Nature 409: 363-366 (2001)].
[0130] In the effector step, the siRNA duplexes bind to a nuclease
complex to form the RNA-induced silencing complex (RISC). An
ATP-dependent unwinding of the siRNA duplex is required for
activation of the RISC. The active RISC then targets the homologous
transcript by base pairing interactions and cleaves the mRNA into
12 nucleotide fragments from the 3' terminus of the siRNA
[Hutvagner and Zamore Curr. Opin. Genetics and Development 12:
225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2: 110-119
(2001); and Sharp Genes. Dev. 15: 485-90 (2001)]. Although the
mechanism of cleavage is still to be elucidated, research indicates
that each RISC contains a single siRNA and an RNase [Hutvagner and
Zamore Curr. Opin. Genetics and Development 12: 225-232
(2002)].
[0131] Because of the remarkable potency of RNAi, an amplification
step within the RNAi pathway has been suggested. Amplification
could occur by copying of the input dsRNAs which would generate
more siRNAs, or by replication of the siRNAs formed. Alternatively
or additionally, amplification could be effected by multiple
turnover events of the RISC [Hammond et al. Nat. Rev. Gen. 2:
110-119 (2001), Sharp Genes. Dev. 15: 485-90 (2001); Hutvagner and
Zamore Curr. Opin. Genetics and Development 12: 225-232 (2002)].
For more information on RNAi see the following reviews Tuschl Chem
Biochem. 2: 239-245 (2001); Cullen Nat. Immunol. 3: 597-599 (2002);
and Brantl Biochem. Biophys. Act. 1575: 15-25 (2002).
[0132] Synthesis of RNAi molecules suitable for use with the
present invention can be effected as follows. First, the lysyl
oxidase mRNA sequence is scanned downstream of the AUG start codon
for AA dinucleotide sequences. Occurrence of each AA and the 3'
adjacent 19 nucleotides is recorded as potential siRNA target
sites. Preferably, siRNA target sites are selected from the open
reading frame, as untranslated regions (UTRs) are richer in
regulatory protein binding sites. UTR-binding proteins and/or
translation initiation complexes may interfere with binding of the
siRNA endonuclease complex [Tuschl Chem Biochem. 2: 239-245, 2001].
It will be appreciated though, that siRNAs directed at untranslated
regions may also be effective, as demonstrated for GAPDH wherein
siRNA directed at the 5' UTR mediated about 90% decrease in
cellular GAPDH mRNA and completely abolished protein level (located
on the World Wide Web at ambion.com/techlib/tn/91/912.html).
[0133] Second, potential target sites are compared to an
appropriate genomic database (e.g., human, mouse, rat etc.) using
any sequence alignment software, such as the BLAST software
available from the NCBI server (located on the World Wide Web at
ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit
significant homology to other coding sequences are filtered
out.
[0134] Qualifying target sequences are selected as template for
siRNA synthesis. Preferred sequences are those including low G/C
content as these have proven to be more effective in mediating gene
silencing as compared to those with G/C content higher than 55%.
Several target sites are preferably selected along the length of
the target gene for evaluation. For better evaluation of the
selected siRNAs, a negative control is preferably used in
conjunction. Negative control siRNA preferably include the same
nucleotide composition as the siRNAs but lack significant homology
to the genome. Thus, a scrambled nucleotide sequence of the siRNA
is preferably used, provided it does not display any significant
homology to any other gene.
[0135] The siRNA molecules of the present invention are preferably
transcribed from expression vectors which can facilitate stable
expression of the siRNA transcripts once introduced into a host
cell. These vectors are engineered to express small hairpin RNAs
(shRNAs), which are processed in vivo into siRNA molecules capable
of carrying out gene-specific silencing [Brummelkamp, T. R., et
al., (2002) A system for stable expression of short interfering
RNAs in mammalian cells. Science 296: 550-53; Paddison, P. J., et
al. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific
silencing in mammalian cells. Genes Dev. 16:948-58; Paul et al.
(2002) Nature Biotech. 20: 505-08; Yu, J. Y., et al. (2002) RNA
interference by expression of short-interfering RNAs and hairpin
RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99:
6047-52]
[0136] An example of a suitable expression vector is the
pSUPER.TM., which includes the polymerase-III H1-RNA gene promoter
with a well defined start of transcription and a termination signal
consisting of five thymidines in a row (T5) [Brummelkamp, T. R. et
al. (2002), Science 296: 550-53]. Most importantly, the cleavage of
the transcript at the termination site is at a site following the
second uridine, thus yielding a transcript which resembles the ends
of synthetic siRNAs, which also contain nucleotide overhangs. siRNA
is cloned such that it includes the sequence of interest, i.e.,
lysyl oxidase separated by a short spacer from the reverse
complement of the same sequence. The resulting transcript folds
back on itself to form a stem-loop structure, which mediates lysyl
oxidase RNAi.
[0137] Another suitable siRNA expression vector encodes the sense
and antisense siRNA under the regulation of separate polIII
promoters [Miyagishi and Taira (2002) Nature Biotech. 20: 497-500].
The siRNA, generated by this vector also includes a five thymidine
(T5) termination signal.
[0138] Since approaches for introducing synthetic siRNA into cells
by lipofection can result in low transfection efficiencies in some
cell types and/or short-term persistence of silencing effects,
vector mediated methods have been developed.
[0139] Thus, siRNA molecules utilized by the present invention are
preferably delivered into cell using retroviruses. Delivery of
siRNA using retroviruses provides several advantages over methods,
such as lipofection, since retroviral delivery is more efficient,
uniform and immediately selects for stable "knock-down" cells
[Devroe, E. and Silver, P. A. (2002). Retrovirus-delivered siRNA.
BMC Biotechnol. 2: 15]
[0140] Recent scientific publications have validated the efficacy
of such short double stranded RNA molecules in inhibiting target
mRNA expression and thus have clearly demonstrated the therapeutic
potential of such molecules. For example, RNAi has been utilized to
inhibit expression of hepatitis C (McCaffrey, A. P., et al., 2002,
Gene expression: RNA interference in adult mice. Nature 418,
38-39), HIV-1 (Jacque, J-M., et al. 2002, Modulation of HIV-1
replication by RNA interference. Nature 418, 435-438), cervical
cancer cells (Jiang, M., and Milner, J. 2002, Selective silencing
of viral gene expression in HPV-positive human cervical carcinoma
cells treated with siRNA, a primer of RNA interference. Oncogene
21, 6041-8) and leukemic cells [Wilda, M., et al., 2002, Killing of
leukemic cells with a BCR/ABL fusion gene by RNA interference
(RNAi). Oncogene 21, 5716-24].
Triple Helix Forming Oligonucleotide (TFO)
[0141] An additional method of regulating the expression of a lysyl
oxidase in cells is via triplex forming oligonucleotides (TFOs).
Recent studies have shown that TFOs can be designed which can
recognize and bind to polypurine/polypyrimidine regions in
double-stranded helical DNA in a sequence-specific manner. These
recognition rules are outlined by Maher III, L. J., et al., Science
(1989), 245: 725-730; Moser, H. E., et al., Science (1987), 238:
645-630; Beal, P. A., et al, Science (1992), 251: 1360-1363;
Cooney, M., et al., Science (1988), 241: 456-459; and Hogan, M. E.,
et al., EP Publication 375408. Modification of the
oligonucleotides, such as the introduction of intercalators and
backbone substitutions, and optimization of binding conditions (pH
and cation concentration) have aided in overcoming inherent
obstacles to TFO activity such as charge repulsion and instability,
and it was recently shown that synthetic oligonucleotides can be
targeted to specific sequences [for a recent review see Seidman and
Glazer, J. Clin. Invest. (2003), 112: 487-94].
[0142] In general, the triplex-forming oligonucleotide has the
sequence correspondence:
TABLE-US-00001 oligo 3'-AGGT duplex 5'-AGCT duplex 3'-TCGA
[0143] However, it has been shown that the A-AT and G-GC triplets
have the greatest triple helical stability (Reither and Jeltsch,
BMC Biochem, 2002, Sep. 12, Epub). The same authors have
demonstrated that TFOs designed according to the A-AT and G-GC rule
do not form non-specific triplexes, indicating that the triplex
formation is indeed sequence specific.
[0144] Thus for any given sequence in the lysyl oxidase regulatory
region a triplex forming sequence may be devised. Triplex-forming
oligonucleotides preferably are at least 15, more preferably 25,
still more preferably 30 or more nucleotides in length, up to 50 or
100 bp.
[0145] Transfection of cells (for example, via cationic liposomes)
with TFOs, and formation of the triple helical structure with the
target DNA induces steric and functional changes, blocking
transcription initiation and elongation, allowing the introduction
of desired sequence changes in the endogenous DNA and resulting in
the specific downregulation of gene expression. Examples of such
suppression of gene expression in cells treated with TFOs include
knockout of episomal supFG1 and endogenous HPRT genes in mammalian
cells (Vasquez et al., Nucl Acids Res. 1999; 27: 1176-81, and Puri,
et al, J Biol Chem, 2001; 276: 28991-98), and the sequence- and
target specific downregulation of expression of the Ets2
transcription factor, important in prostate cancer etiology
(Carbone, et al, Nucl Acid Res. 2003; 31: 833-43), and the
pro-inflammatory ICAM-1 gene (Besch et al, J Biol Chem, 2002; 277:
32473-79). In addition, Vuyisich and Beal have recently shown that
sequence specific TFOs can bind to dsRNA, inhibiting activity of
dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and
Beal, Nuc. Acids Res 2000; 28: 2369-74).
[0146] Additionally, TFOs designed according to the abovementioned
principles can induce directed mutagenesis capable of effecting DNA
repair, thus providing both downregulation and upregulation of
expression of endogenous genes (Seidman and Glazer, J Clin Invest
2003; 112: 487-94). Detailed description of the design, synthesis
and administration of effective TFOs can be found in U.S. Pat.
Appl. Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002
0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No.
5,721,138 to Lawn.
[0147] The downregulators described hereinabove would be
particularly useful for inhibiting angiogenesis in tumor tissue. It
has been shown that PF4, a lysyl oxidase binding protein which
inhibits angiogenesis in tumor tissue specifically accumulates in
newly formed blood vessels of tumors (angiogenic vessels) but not
in established blood vessels (Hansell, P., et al., 1995, Selective
binding of platelet factor 4 to regions of active angiogenesis in
vivo. Amer. J. Physiol-Heart. Circ. Phy. 38, H829-H836; Reiser, K.,
et al., 1992, Enzymatic and nonenzymatic cross-linking of collagen
and elastin. FASEB J. 6, 2439-2449).
[0148] Newly formed angiogenic blood vessels are more permeable to
proteins than established blood vessels because the major inducer
of angiogenesis in many angiogenic diseases is VEGF, a growth
factor which also functions as a potent blood vessel permeabilizing
factor (VPF) [Neufeld et al., 1999, Vascular endothelial growth
factor (VEGF) and its receptors. FASEB J. 13(1): 9-22]. Tumor
associated blood vessels are therefore in a permanent state of
hyperpermeability due to deregulated over-expression of VEGF
(Shweiki et al., 1995; Rak et al., 1995) and as such, a
downregulator molecule used by the method of the present invention
would be able to extravasate efficiently from tumor blood vessels
but much less efficiently from normal stabilized blood vessels.
Upregulators
[0149] Several approaches can be utilized to increase the levels of
lysyl oxidase and as such to enhance the formation of blood
vessels.
[0150] For example, a nucleic acid construct including a
constitutive, inducible or tissue specific promoter positioned
upstream of a polynucleotide encoding a polypeptide having lysyl
oxidase activity, such as the polypeptide set forth in SEQ ID NO:2,
3, 6, 8 or 9 can be administered into a mammalian tissue. The lysyl
oxidase expressed from this construct would substantially increase
the levels of lysyl oxidase within the cells of the tissue and as
such enhance angiogenesis.
[0151] The polynucleotide segments encoding the lysyl oxidase can
be ligated into a commercially available expression vector. Such an
expression vector includes a promoter sequence for directing
transcription of the polynucleotide sequence in the cell in a
constitutive or inducible manner. A suitable promoter can be, for
example, a Tie-2 promoter which is capable of directing lysyl
oxidase specific gene expression in endothelial cells (see
Schlaeger, T. M., Bartunkova, S., Lawitts, J. A., Teichmann, G.,
Risau, W., Deutsch, U., and Sato, T. N. (1997). Uniform
vascular-endothelial-cell-specific gene expression in both
embryonic and adult transgenic mice. Proc. Natl. Acad. Sci. U.S.A
94, 3058-3063). The expression vector of the present invention can
further include additional polynucleotide sequences such as for
example, sequences encoding selection markers or reporter
polypeptides, sequences encoding origin of replication in bacteria,
sequences that allow for translation of several proteins from a
single mRNA such as an internal ribosome entry site (IRES),
sequences for genomic integration of the promoter-chimeric
polypeptide encoding region and/or sequences generally included in
mammalian expression vector such as pcDNA3, pcDNA3.1(+/-),
pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto,
pCR3.1, which are available from Invitrogen, pCI which is available
from Promega, pBK-RSV and pBK-CMV which are available from
Strategene, pTRES which is available from Clontech, and their
derivatives.
[0152] It will be appreciated that such commercially available
vector systems can easily be modified via commonly used recombinant
techniques in order to replace, duplicate or mutate existing
promoter or enhancer sequences and/or introduce any additional
polynucleotide sequences.
[0153] An agent capable of upregulating a lysyl oxidase may also be
any compound which is capable of increasing the transcription
and/or translation of an endogenous DNA or mRNA encoding a lysyl
oxidase using for example gene "knock in" techniques.
[0154] Enhancer elements can be "knocked-in" adjacent to endogenous
lysyl oxidase coding sequences to thereby increase transcription
therefrom.
[0155] Further details relating to the construction and use of
knock-out and knock-in constructs is provided elsewhere [Fukushige
S. and Ikeda, J. E. Trapping of mammalian promoters by Cre-lox
site-specific recombination. DNA Res 3 (1996) 73-80; Bedell, M. A.,
et al. Mouse models of human disease. Part I: Techniques and
resources for genetic analysis in mice. Genes and Development 11
(1997) 1-11; Bermingham, J. J., et al. Tst-1/Oct-6/SCIP regulates a
unique step in peripheral myelination and is required for normal
respiration. Genes Dev 10 (1996) 1751-62].
[0156] It will be appreciated that direct administration of a
polypeptide exhibiting a lysyl oxidase activity can also be
utilized for enhancing angiogenesis.
[0157] Thus, affinity binding assays and/or activity assays, the
principles of which are well known in the art, can be used to
screen for novel compounds (e.g., substrate analogs) which can
specifically regulate the activity of a lysyl oxidase and as such
can be used with the present invention.
[0158] An assay suitable for use with this aspect of the present
invention has been previously described in a study conducted by
Bedell-Hogan et al., 1993.
[0159] As is clearly illustrated in the Examples section which
follows, the present study also correlated expression levels of
LOR-1 to the metastatic properties of breast cancer derived cell
lines, indicating that LOR-1 may play additional roles in tumor
invasiveness in addition to its role in angiogenesis.
[0160] Thus, the present invention also provides a method of
inhibiting metastasis and/or fibrosis in a mammalian tissue. The
method is effected by administering to the mammalian tissue a
molecule capable of downregulating a tissue level and/or an
activity of at least one type of a lysyl oxidase.
[0161] The method of the present invention can be used to treat
human patients that have been diagnosed with cancerous tumors, by
administering any of the downregulating molecules described herein
above, in order to reduce the tissue level and/or activity of at
least one type of a lysyl oxidase.
[0162] As used herein, the phrase "cancerous tumor" refers to any
malignant tumor within a human body including, but not limiting to,
tumors with metastases. In addition, and without being bound to any
particular type of cancerous tumor, the present invention is useful
to treat breast cancer tumors, with or without metastases.
[0163] As used herein, the phrase "administering" refers to all
modes of administration described hereinbelow with respect to the
pharmaceutical compositions of the present invention.
[0164] These include, but not limit to, local administration at the
tumor tissue, an organ where the cancerous tumor was diagnosed
and/or related tissues that typically form metastases [Hortobagyi,
2002, Semin Oncol 29 (3 Suppl 11): 134-44; Morrow and Gradishar,
2002, 324: 410-4]. Examples of related tissue include lymph nodes
adjacent to, for example, breast tissue and bones.
[0165] Administration can also be effected in a systemic manner in
order to treat the affected tissue, i.e., the tissue where the
cancerous tumor was formed and where metastases are present or
likely to be formed with tumor progression.
[0166] Since any molecule capable of downregulating a lysyl oxidase
activity can be utilized by the methods described hereinabove, the
present invention also provides a method of identifying molecules
capable of inhibiting metastasis and/or fibrosis.
[0167] This method is effected by screening and identifying
molecules which exhibit specific reactivity with at least one type
of lysyl oxidase and testing a metastasis and/or fibrosis
inhibitory potential of these molecules.
[0168] Numerous types of molecules can be screened for reactivity
with at least one type of lysyl oxidase, examples include, but are
not limited to, molecules such as antisense oligonucleotides,
siRNA, DNAzymes, ribozymes and triple helix forming
oligonucleotides (TFOs) that interact with a polynucleotide
expressing a lysyl oxidase activity or molecules such as antibodies
that interact with polypeptides having a lysyl oxidase activity. In
addition, short peptides and other small molecules can also be
screened by this method of the present invention.
[0169] Screening for cross reactivity can be effected by lysyl
oxidase enzymatic activity assays, by binding assays and the like.
Examples of suitable assays are provided in Rodriguez et al., 2002,
Arterioscler Thromb Vasc Biol 22: 1409-14; Wilson and Nock, 2002,
Curr Opin Chem Biol 6: 81-5; Uetz, 2002, Curr Opin Chem Biol 6:
57-62; Stoll et al., 2002, Front Biosci 2002 7: c13-32).
[0170] Testing a metastatic phenotype of transformed tumor cells
can be performed in vitro since nearly all steps of the metastatic
process, including attachment, matrix degradation and migration,
can be modeled experimentally in vitro by measuring invasion of a
reconstituted basement membrane (RBM). Metastatic invasiveness of
tumor cell can be modeled by migration of tumor cells into
reconstituted basement membrane (RBM) in the presence and absence
of a chemoattractant, such as fibroblast conditioned medium (FCM).
The assay determines cells that have attached to the RBM, degraded
the RBM enzymatically and, finally, cells that have penetrated the
FCM side of the membrane.
[0171] Since in vitro metastasis events correspond to steps
observed in the metastatic spread of tumor cells through the
basement membrane in vivo, in vitro invasiveness of cells can be
assayed by the methods described in Albini et al., 1987 Cancer
Research 47: 3239-3245, which is incorporated herein by reference
in its entirety. Invasiveness assays and other methods for
assessing metastatic affects, are described in Leyton et al., 1994
Cancer Research 54: 3696-3699, which is incorporated by reference
herein in its entirety. Reconstituted basement membrane
preparations for use in accordance with the hereinabove described
assays are readily available from numerous commercial suppliers.
One suitable example membrane in this regard is "MATRIGEL"
available from Collaborative Biomedical Products of Bedford,
Mass.
[0172] In vitro evaluation of tumor cell metastatic phenotype can
also be effected by determining level and pattern of expression of
one or more metastasis associated markers such protease markers,
which are considered to be an integral part of tumor metastasis
(see U.S. Pat. No. 6,303,318). One example is the arachidonic acid,
the release of which in cells can serve to indicate metastatic
potential of a tumor (U.S. Pat. No. 6,316,416). In this regard,
determining phospholipase A-2 (PLA2) activity, and the activity or
abundance of factors that affect the activity of PLA2, such as
uteroglobin protein (U.S. Pat. No. 6,316,416) can serve as an
indication of metastatic potential.
[0173] Determining pattern and level of expression of
metastasis-associated markers can be effected by one of several
methods known in the art.
[0174] The presence or level of proteins indicative of metastatic
potential of tumors can be determined in cells by conventional
methods well known to those of skill in the art. For instance, the
techniques for making and using antibody and other immunological
reagents and for detecting particular proteins in samples using
such reagents are described in current protocols in immunology,
Coligan et al., Eds., John Wiley & Sons, New York (1995), which
is incorporated by reference herein in parts pertinent to making
and using reagents useful for determining specific proteins in
samples. As another example, immunohistochemical methods for
determining proteins in cells in tissues are described in Volume 2,
Chapter 14 of current protocols in molecular biology, Ausubel et
al., Eds., John Wiley & Sons, Inc. (1994), which is
incorporated by reference herein in part pertinent to carrying out
such determinations. Finally, Linnoila et al., A.J.C.P. 97(2):
235-243 (1992) and Peri et al., J. Clin. Invest. 92: 2099-2109
(1992), incorporated herein as referred to above, describe
techniques that may need, in part, in this aspect of the present
invention.
[0175] Metastatic potential can also be determined in vivo at the
mRNA level. The presence and/or level of mRNA transcripts can be
determined by a variety of methods known to those of skill in the
art. A given mRNA may be detected in cells by hybridization to a
specific probe. Such probes may be cloned DNAs or fragments
thereof, RNA, typically made by in vitro transcription, or
oligonucleotide probes, usually generated by solid phase synthesis.
Methods for generating and using probes suitable for specific
hybridization are well known and used in the art.
[0176] A variety of controls may be usefully employed to improve
accuracy in mRNA detection assays. For instance, samples may be
hybridized to an irrelevant probe and treated with RNAse A prior to
hybridization, to assess false hybridization.
[0177] In order to modulate angiogenesis or inhibit metastasis or
tumor fibrosis, the molecules used by the present invention can be
administered to the individual per se, or in a pharmaceutical
composition where it is mixed with suitable carriers or
excipients.
[0178] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration/targeting of a compound
to a mammal.
[0179] As used herein the term "active ingredients", refers to the
preparation accountable for the biological effect, i.e. the
upregulator/downregulator molecules used by the present invention
to modulate angiogenesis and the downregulators molecules used by
the present invention to inhibit metastasis and tumor fibrosis.
[0180] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" are
interchangeably used to refer to a carrier, such as, for example, a
liposome, a virus, a micelle, or a protein, or a diluent which do
not cause significant irritation to the mammal and do not abrogate
the biological activity and properties of the active ingredient. An
adjuvant is included under these phrases.
[0181] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients, include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0182] Techniques for formulation and administration of
compositions may be found in "Remington's Pharmaceutical Sciences"
Mack Publishing Co., Easton, Pa., latest edition, which is
incorporated herein by reference.
[0183] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, transnasal, intestinal or parenteral
delivery, including intramuscular, subcutaneous and intramedullary
injections as well as intrathecal, direct intraventricular,
intravenous, inrtaperitoneal, intranasal, or intraocular
injections.
[0184] For injection, the active ingredients of the invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0185] For oral administration, the compounds can be formulated
readily by combining the active ingredient with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
active ingredient of the invention to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0186] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0187] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0188] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0189] The preparations described herein may be formulated for
parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0190] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0191] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0192] The preparation of the present invention may also be
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0193] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose.
[0194] The pharmaceutical composition may form a part of an article
of manufacturing which also includes a packaging material for
containing the pharmaceutical composition and a leaflet which
provides indications of use for the pharmaceutical composition.
[0195] Thus, the present invention provides a method and
pharmaceutical compositions useful modulating angiogenesis.
[0196] Such modulation activity can be used to treat arthritis
(Koch, 1998; Paleolog and Fava, 1998), diabetic retinopathy (Miller
et al., 1997), psoriasis (Detmar et al., 1994; Creamer et al.,
1997) and vasculitis (Lie, 1992; Klipple and Riordan, 1989).
[0197] In addition, the present invention can also be used to treat
disease characterized by fragile blood vessels, including Marfans
syndrome, Kawasaki, Ehlers-Danlos, cutis-laxa, and takysu (Lie,
1992; Klipple and Riordan, 1989; Brahn et al., 1999; Cid et al.,
1993; Hoffman et al., 1991).
[0198] It is possible that some of these diseases result from
reduced or abolished lysyl oxidase activity which leads to the
synthesis of a fragile extracellular matrix, and consequently,
fragile blood vessels.
[0199] As such, administration of lysyl oxidase encoding sequences
or polypeptides can be used to correct some of the manifestations
of these diseases.
[0200] The present invention can also be used to treat diseases
which are characterized by changes in the wall of blood vessels.
For example, restenosis which is a common complication following
balloon therapy, Fibromuscular dysplasia (Begelman and Olin, 2000)
and aortic stenosis (Palta et al., 2000) are all potentially
treatable by the method of the present invention.
[0201] In addition, as is illustrated in the Examples section which
follows, LOR-1 is more highly expressed in metastatic tumors and
cell lines than in non-metastatic tumors and cell lines (FIGS. 3,
9, and 13). This suggests that levels of LOR-1 expression can be
used as a diagnostic tool to determine the malignancy of cancer
cells, as well as, to determine and implement suitable treatment
regimens.
[0202] Colon cancer is a highly treatable and often a curable
disease when localized to the bowel. However, in many cases, due to
mis-diagnosis, a pre-malignant colon hyperplasia progress into
colon adenoma which further develop into more malignant forms of
low-grade and high-grade colon adenocarcinoma. Once an individual
is diagnosed with colon cancer the malignancy of the tumor needs to
be assessed in order to select for suitable treatment regimens. The
current practice for assessing the malignancy of a colon tumor is
based on the tumor-node-metastases (TNM) staging system developed
by the American Joint Committee on Cancer (AJCC). According to this
method staging is based on scoring for the presence or absence of
cancerous cells in the tumor itself, in the submucosa of the bowel
wall, in the muscular layer of the bowel wall (muscularis propria),
and/or in the subserosa, pericolic or perirectal tissues, as well
as in regional lymph nodes and distance metastases. Thus, staging
of colon tumors involves multiple tissue biopsies and complex
pathological evaluations which are time consuming and can result in
misdiagnosis.
[0203] While reducing the present invention to practice, the
present inventors have also uncovered that LOR-1 expression in
epithelial and/or connective tissue cells in a colon tissue is
indicative of a malignant colon cancer thus providing a new method
of assessing a malignancy of colon cancer tumors devoid of the
above limitations.
[0204] As described in Example 3 of the Examples section which
follows, the expression of LOR-1 is correlated with the formation
of benign colon tumors (FIG. 14b) and is increased in more
malignant forms of colon cancer tumors (FIGS. 14c and 14d) thus
suggesting the use of LOR-1 in determining the stage of colon
cancer tumors.
[0205] Thus according to another aspect of the present invention
there is provided a method of assessing a malignancy of a colon
tumor. The method is effected by determining a tissue level and/or
an activity level of a polypeptide at least 75% homologous to the
polypeptide set forth in SEQ ID NO:2 or 9 in the colon tumor
tissue, thereby assessing the malignancy of the colon tumor.
[0206] As is used herein, the phrase "assessing a malignancy of a
colon tumor" refers to determining the stage of the colon tumor,
i.e., the progress of the colon tumor from a benign colon tumor to
a highly malignant colon cancer which invades the surrounding
tissue.
[0207] The polypeptide detected by the present invention is at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%
homologous to SEQ ID NO:2 or 9, as determined using the BestFit
software of the Wisconsin sequence analysis package, utilizing the
Smith and Waterman algorithm, where gap creation penalty equals 8
and gap extension penalty equals 2.
[0208] Preferably, the polypeptide of the present invention is
LOR-1 (SEQ ID NO:2), a member of the lysyl oxidase family which are
fully described herein above.
[0209] According to the method of the present invention, a colon
tumor tissue is obtained using a colon biopsy and/or a colon
surgery using methods know in the art. Once obtained, the tissue
level and/or activity level of the polypeptide of the present
invention is determined in the colon tumor tissue.
[0210] Determination of the tissue level of the polypeptide of the
present invention may be accomplished directly using immunological
methods.
[0211] The immunological detection methods used in context of the
present invention are fully explained in, for example, "Using
Antibodies: A Laboratory Manual" (Ed Harlow, David Lane eds., Cold
Spring Harbor Laboratory Press (1999)) and those familiar with the
art will be capable of implementing the various techniques
summarized hereinbelow as part of the present invention. All of the
immunological techniques require antibodies specific to at least
one epitope of the polypeptide of the present invention.
Immunological detection methods suited for use as part of the
present invention include, but are not limited to,
radio-immunoassay (RIA), enzyme linked immunosorbent assay (ELISA),
western blot, immunohistochemical analysis.
[0212] Radio-immunoassay (RIA): In one version, this method
involves precipitation of the desired substrate, e.g., LOR-1, with
a specific antibody and radiolabelled antibody binding protein
(e.g., protein A labeled with I.sup.125) immobilized on a
precipitable carrier such as agarose beads. The number of counts in
the precipitated pellet is proportional to the amount of
substrate.
[0213] In an alternate version of the RIA, a labeled substrate and
an unlabelled antibody binding protein are employed. A sample
containing an unknown amount of substrate is added in varying
amounts. The decrease in precipitated counts from the labeled
substrate is proportional to the amount of substrate in the added
sample.
[0214] Enzyme linked immunosorbent assay (ELISA): This method
involves fixation of a sample (e.g., fixed cells or a proteinaceous
solution) containing a protein substrate (e.g., LOR-1) to a surface
such as a well of a microtiter plate. A substrate specific antibody
coupled to an enzyme is applied and allowed to bind to the
substrate. Presence of the antibody is then detected and
quantitated by a colorimetric reaction employing the enzyme coupled
to the antibody. Enzymes commonly employed in this method include
horseradish peroxidase and alkaline phosphatase. If well calibrated
and within the linear range of response, the amount of substrate
present in the sample is proportional to the amount of color
produced. A substrate standard is generally employed to improve
quantitative accuracy.
[0215] Western blot analysis: This method involves separation of a
substrate (e.g., LOR-1 protein) from other proteins by means of an
acrylamide gel followed by transfer of the substrate to a membrane
(e.g., nylon or PVDF). Presence of the substrate is then detected
by antibodies specific to the substrate, which are in turn detected
by antibody binding reagents. Antibody binding reagents may be, for
example, protein A, or other antibodies. Antibody binding reagents
may be radiolabelled or enzyme linked as described hereinabove.
Detection may be by autoradiography, colorimetric reaction or
chemiluminescence. This method allows both quantitation of an
amount of substrate and determination of its identity by a relative
position on the membrane which is indicative of a migration
distance in the acrylamide gel during electrophoresis.
[0216] Immunohistochemical analysis: This method involves detection
of a substrate in situ in fixed tissue by substrate specific
antibodies. The substrate specific antibodies may be enzyme linked
or linked to fluorophores. Detection is by microscopy and
subjective evaluation. If enzyme linked antibodies are employed, a
colorimetric reaction may be required.
[0217] Since tissue levels of a polypeptide can be inferred from
the levels of mRNA encoding such a polypeptide, the method
according to this aspect of the present invention can also employ
various polynucleotide detection approaches for determining the
tissue level of the polypeptide of the present invention.
[0218] RNA molecules can be detected using methods known in the art
including for example, Northern blot analysis, RT-PCR analyses, RNA
in situ hybridization stain and in situ RT-PCR stain.
[0219] Northern Blot analysis: This method involves the detection
of a particular RNA (e.g., the RNA molecule encoding LOR-1) in a
mixture of RNAs. An RNA sample is denatured by treatment with an
agent (e.g., formaldehyde) that prevents hydrogen bonding between
base pairs, ensuring that all the RNA molecules have an unfolded,
linear conformation. The individual RNA molecules are then
separated according to size by gel electrophoresis and transferred
to a nitrocellulose or a nylon-based membrane to which the
denatured RNAs adhere. The membrane is then exposed to labeled DNA
probes. Probes may be labeled using radio-isotopes or enzyme linked
nucleotides. Detection may be using autoradiography, colorimetric
reaction or chemiluminescence as described hereinabove. This method
allows both quantitation of an amount of particular RNA molecules
and determination of its identity by a relative position on the
membrane which is indicative of a migration distance in the gel
during electrophoresis.
[0220] RT-PCR analysis: This method uses PCR amplification of
relatively rare RNAs molecules. First, RNA molecules from a
particular tissue (e.g., a colon tumor tissue) are purified and
converted into complementary DNA (cDNA) using a reverse
transcriptase enzyme (such as an MMLV-RT) and primers such as,
oligo dT, random hexamers or gene specific primers, all of which
are available from Invitrogen Life Technologies, Frederick, Md.,
USA. Then by applying gene specific primers and Taq DNA polymerase,
a PCR amplification reaction is carried out in a PCR machine. Those
of skills in the art are capable of selecting the length and
sequence of the gene specific primers and the PCR conditions (i.e.,
annealing temperatures, number of cycles and the like) which are
suitable for detecting specific RNA molecules.
[0221] RNA in situ hybridization stain: In this method DNA or RNA
probes are attached to the RNA molecules present in the tissue.
Generally, a tissue sample (e.g., a colon tissue) is fixed to
preserve its structure and to prevent the RNA from being degraded
and then sectioned for microscopy and placed on a slide.
Alternatively, frozen tissue samples can be first sectioned and put
on a slide and then subject to fixation prior to hybridization.
Hybridization conditions include reagents such as formamide and
salts (e.g., sodium chloride and sodium citrate) which enable
specific hybridization of the DNA or RNA probes with their target
mRNA molecules in situ while avoiding non-specific binding of
probe. Those of skills in the art are capable of adjusting the
hybridization conditions (i.e., temperature, concentration of salts
and formamide and the like) to specific probes and types of cells.
Following hybridization, any unbound probe is washed off and the
slide is subjected to either a photographic emulsion which reveals
signals generated using radio-labeled probes or to a colorimetric
reaction which reveals signals generated using enzyme-linked
labeled probes as described hereinabove.
[0222] In situ RT-PCR stain: This method is described in Nuovo G J,
et al. (Intracellular localization of polymerase chain reaction
(PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17:
683-90) and Komminoth P, et al. [Evaluation of methods for
hepatitis C virus detection in archival liver biopsies. Comparison
of histology, immunohistochemistry, in situ hybridization, reverse
transcriptase polymerase chain reaction (RT-PCR) and in situ
RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR
reaction is performed on fixed tissue sections by incorporating
labeled nucleotides to the PCR reaction. The reaction is carried on
using a specific in situ RT-PCR apparatus such as the laser-capture
microdissection PixCell I LCM system available from Arcturus
Engineering (Mountainview, Calif.).
[0223] Determination of an activity level of the polypeptide of the
present invention (e.g., LOR-1) in a colon tumor tissue may be
effected using suitable substrates in a cytochemical stain and/or
in vitro activity assays.
[0224] Cytochemical stain: According to this method, a chromogenic
substrate is applied on the colon tumor tissue containing an active
enzyme (e.g., LOR-1). The enzyme catalyzes a reaction in which the
substrate is decomposed to produce a chromogenic product visible by
a light or a fluorescent microscope.
[0225] In vitro activity assays: In these methods the activity of a
particular enzyme is measured in a protein mixture extracted from
the tissue of interest (e.g., a colon tumor tissue). The activity
can be measured in a spectrophotometer well using colorimetric
methods (see for example, Wande Li, et al. Localization and
activity of lysyl oxidase within nuclei of fibrogenic cells. Proc.
Natl. Acad. Sci. USA. 1997, 94: 12817-12822) or can be measured in
a non-denaturing acrylamide gel (i.e., activity gel). Following
electrophoresis the gel is soaked in a solution containing a
substrate and colorimetric reagents. The resulting stained band
corresponds to the enzymatic activity of the polypeptide of
interest (e.g., LOR-1). If well calibrated and within the linear
range of response, the amount of enzyme present in the sample is
proportional to the amount of color produced. An enzyme standard is
generally employed to improve quantitative accuracy.
[0226] Once the tissue level and/or the activity level of the
polypeptide (or mRNA) of the present invention (e.g., LOR-1) is
determined in the colon tumor tissue the malignancy of the tumor is
assessed by comparing the expression level and/or activity in the
colon tumor tissue to that of a normal colon tissue.
[0227] It will be appreciated that the normal colon tissue may be
obtained from a biopsy and/or a surgery of a colon tissue obtained
form a healthy individual. Alternatively, the normal colon tissue
can be obtained from an unaffected segment of the colon of the same
individual. Methods of determining the status of a normal colon
tissue are known to skilled in the art and include for example, a
morphological evaluation of tissue sections.
[0228] Once malignancy of colon cancer is determined as described
above, tissue level and/or activity level of the polypeptide (or
mRNA thereof) of the present invention can also be utilized to
stage the colon tumor and to thereby predict the prognosis of an
individual diagnosed with colon cancer.
[0229] Such staging can be effected by assessing the tissue level
and/or activity level of the polypeptide and correlating it to
results obtained from colon cancer tissue at various stages
(obtainable through pathological evaluation of colon tumors). It
will be appreciated that such accurate and rapid staging will
enable accurate and rapid prognosis of an individual afflicted with
colon cancer and timely administration of suitable treatment
regimen.
[0230] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0231] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0232] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (Eds.) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (Eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (Eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
The Role of LOR1 in Angiogenesis
[0233] A study was conducted in efforts to further substantiate and
characterize the role of LOR-1 in angiogenesis.
Materials and Methods
[0234] Human recombinant platelet factor-4 (PF4, GenBank Accession
number M20901) which was produced in bacteria and subsequently
refolded was supplied by Dr. Maione of Repligen Corp. (Boston,
USA). Estrogen slow release pellets were obtained from Innovative
Research of America, Sarasota, Fla., USA.
[0235] Construction of LOR-1 expression vector, transfection into
MCF-7 cells, and expression: The LOR-1 cDNA (SEQ ID NO:1) was
cloned into a pCDNA3.1-hygro expression vector (Invitrogen Inc.,
USA) under the control of a CMV promoter. Clones of cells
expressing LOR-1 were selected using hygromycine and assayed for
LOR-1 expression using the polyclonal antisera described below.
[0236] Construction of platelet factor-4 affinity columns and
purification of LOR-1 on such columns: PF4 was coupled to sepharose
using a modification of the method of Miron and Wilchek as
previously described for vascular endothelial growth factor. Serum
free conditioned medium was collected from .sup.35S-methionine
labeled MCF-7 cells which over-expressed LOR-1. The conditioned
medium was passed through the column twice. The column was washed
with phosphate buffered saline (300 mM NaCl, pH-7.2) and eluted
with PBS (containing 2M NaCl).
[0237] Experiments with nude mice: Modified or parental MCF-7 cells
(10.sup.7 cells per animal) were implanted under the skin of nude
mice. A pellet of slow release estrogen was implanted 1 cm away as
previously described. Tumors were measured periodically, following
which, tumors at least 1 cm in size were removed and
immuno-histologically analyzed using a commercial antibody directed
against a factor 8 like antigen which served as a specific marker
for endothelial cells.
[0238] In-situ hybridization: Fragments encompassing nucleotides
922-1564 of LOR-1, nucleotides 976-1391 of LOL, nucleotides 400-950
of LO and nucleotides 1061-1590 of LOR-2 (as numbered from the ATG
codon of these sequences) where each independently subcloned into
the Bluescript SK and KS (Strategene) vectors. A DIG cRNA labeling
kit of Boehringer-Mannheim was used to transcribe sense (s) and
antisense (as) digoxigenin-labeled cRNA probes from the T7 promoter
of the Bluescript constructs. Hybridization and subsequent
detection of hybridized probes was carried out essentially as
previously described (Cohen et al., 2001).
[0239] Anti-LOR-1 polyclonal antisera: Antisera was generated by
injecting a recombinant peptide containing the C-terminal 200
amino-acids of LOR-1 (amino acids 540-744 of SEQ ID NO:2) into
female rabbits. Serum was collected 10 days following each
injection and an immunoglobulin fraction was purified using a
protein A Sepharose affinity column (Pharmacia).
Results
[0240] LOR-1 purification: A PF4 affinity column was used to detect
endothelial cell proteins which specifically interact with PF4.
[0241] Two PF4 binding proteins were detected in conditioned medium
of human umbilical vein endothelial cells (HUVEC), whereas PF4
binding proteins were not detected in detergent extracts of
endothelial cells.
[0242] Two liters of conditioned medium enabled a partial
purification of one such binding protein which eluted from the
column at a relatively high salt concentrations (0.4-0.5 M
NaCl).
[0243] Further purification of this protein was performed using
reverse phase high pressure liquid chromatography and SDS/PAGE
chromatography. The PF4 binding protein did not bind to heparin nor
was it a heparan-sulfate proteoglycan since heparinase digestion
failed to change its mobility in SDS/PAGE experiments.
[0244] Partial sequencing and database comparison revealed that the
PF4 binding protein of the present invention (LOR-1) belongs to a
family of proteins containing a lysyl oxidase like domain (Kim et
al., 1995; Kim et al., 1999). Lysyl oxidases are copper dependent
enzymes that participate in the synthesis of the extracellular
matrix by catalyzing the formation of covalent bonds between
lysines of adjacent collagen or elastin fibers.
[0245] The full length amino acid sequence of LOR-1 (as deduced
from the isolated cDNA sequence) displayed a high degree of
identity to WS9-14, a protein over expressed in senescent
fibroblasts, in several types of adherent cells (but not in
non-adherent cells) and in fibroblasts, in which it was correlated
to pro-collagen I-al expression levels, as well as being induced by
TGF-.beta. and inhibited by phorbol esters and retinoic acid (Saito
et al., 1997).
[0246] The role of LOR-1 in tumor development: Recombinant LOR-1
expressed in PAE cells specifically bound with the PF4 affinity
column (FIG. 1). Since LOR-1 is a member of the LO family it was
hypothesized that it participates in ECM formation during
angiogenesis. Furthermore it was also hypothesized that PF4
suppresses or inhibits the pro-angiogenic activity of LOR-1 thus
inhibiting the later stages of blood vessel formation and as a
result limiting tumor growth.
[0247] LOR-1 expression: In-situ hybridization demonstrated that
LOR-1 is expressed in a wide variety of tissues and cell types
including fibroblasts, adipocytes, nerve cells, endothelial cells
and a variety of epithelial cells. Several cell types, such as
liver hepatocytes, did not express LOR-1; of the 4 LO family
members examined (all except for LoxC) LOR-1 was the only one
expressed in endothelial cells of blood vessels.
[0248] LOR-1 and cancer: as shown in FIG. 2, a direct correlation
between the expression levels of LOR-1 and the metastatic
properties of breast cancer derived cell lines was demonstrated
herein.
[0249] Since the epithelial cells which line the milk ducts of
normal breast tissue (from which most breast tumors arise) express
large amounts of LOR-1 it is possible that the less metastatic
lines lost LOR-1 expression rather than gained it.
[0250] To substantiate its role in metastasis, LOR-1 cDNA was
expressed in non-metastatic breast cancer derived MCF-7 cell lines
which do not normally express LOR-1. The expression of LOR-1 was
examined using rabbit polyclonal antibodies generated as described
above (FIG. 3).
[0251] A control cell line which was transfected with an empty
expression vector, and an MCF-7 cell line expressing LOR-1 were
implanted under the skin of immune deficient mice along with an
estrogen slow release pellet as described above. Estrogen was added
since the development of tumors from this non-metastatic cell line
is estrogen dependent.
[0252] The rate of tumor development in the mice was continuously
monitored (FIG. 4); tumors 1 cm in size were excised and subjected
to histological analysis as described above. Interestingly, the
rate of tumor development varied between the two cell lines, with
some tumors exhibiting slower growth in the LOR-1 expressing MCF-7
and yet other exhibiting slower growth in the control cells.
[0253] In order to overcome expression level problems, LOR-1 cDNA
was placed under the control of a tetracycline induced promoter
(The TET-off system). Such a construct will enable to determine
conclusively whether the reduced rate of tumor growth observed in
LOR-1 expressing cells is indeed caused by LOR-1.
[0254] Tumors expressing large amounts of LOR-1 were sectioned and
stained with an antibody directed against factor 8 like antigen, a
specific marker of endothelial cells. Control tumor tissue
predominantly stained at the capsule around the tumor while in the
LOR-1 expressing tumors pronounced staining was observed in inner
regions of the tumor tissue (FIG. 5a and FIG. 5b respectively).
[0255] The role of LOR-1 in Wilson's disease and in other chronic
liver diseases: Normal and diseased liver tissue were probed with
LOR-1 sense (FIGS. 6a and 6c) and antisense probes (FIGS. 6b and
6d). Normal liver tissues expresses very low levels of LOR-1 (FIG.
6b). However, fibrotic liver tissues such as those observed in
Wilson's disease, exhibit a strong increase in hepatocyte
expression of LOR-1 (FIG. 6d).
[0256] Expression of LOR-1 in chick embryos: FIG. 7 illustrates
LOR-1 expression of LOR-1 mRNA in blood vessels of a developing
chick embryo. Whole-mount in-situ hybridization of 4 day old chick
embryos revealed LOR-1 mRNA expression in blood vessels located in
the amnion (arrow).
[0257] The Lysyl oxidase family: A homology comparison between five
members of the lysyl oxidase family which includes the LO and LOL
subfamily and the LOR-1 and LOR-2 subfamily revealed a strong
homology at the C-terminal portion which includes the conserved
lysyl oxidase motif. LOR-1 and LOR-2 are characterized by long
N-terminal stretches which are not found in LO and LOL.
Example 2
MCF-7 Breast Cancer Cells Expressing Recombinant Lysyl Oxidase
Related Protein-1 (LOR-1) Form Invasive Tumors Characterized by
Extensive Fibrosis
[0258] A study was conducted in efforts to further substantiate and
characterize the role of LOR-1 in inhibiting metastasis and tumor
fibrosis.
Materials and Methods
[0259] Estrogen pellets (17.beta.-estradiol, 0.72 mg/pellet,
60-days release) were from Innovative Research of America, Fla.,
USA. Masson Trichrome stain kit was purchased from Bio-Optica
(Milano, Italy), Reverse Transcriptase, G418 were from GIBCO BRL
(U.K.), Hygromycin B, Tetracycline hydrochloride, Reticulum stain
kit fast green and Sirius red (direct red 80) were from Sigma
(USA), Restriction enzymes, T4 ligase were from New England Biolabs
(USA). The bacterial expression vector pQE-30 and the nickel
affinity column were obtained from Qiagen (Germany), .sup.32p-dATP
was purchased from NEN (USA). Monoclonal anti cytokeratin-7 (CAM
5.2) coupled to FITC was acquired from Becton Dickinson (USA), and
monoclonal mouse anti vimentin (clone V9) was purchased from DAKO
Denmark). Anti FITC alkaline phosphates-conjugated was purchased
from ROCHE (USA), CAS block, citrate and EDTA antigen retrieval
buffers were from Zymed (USA).
[0260] Cell culture: MCF-7 breast cancer cells were kindly provided
by Dr. Hadasa Degani (Weizmann Institute, Israel). The MDA-MB-435
breast cancer cell line was kindly provided by Dr. Israel Vlodaysky
(Technion, Israel). The MDA-MB-231 cells were kindly provided by
Dr. Michael Klagsbrun (Harvard University, USA). These cell lines
were routinely cultured in Dulbecco's modified eagle medium
supplemented with gentamicin, amphotericin, glutamine and 10% Fetal
Calf Serum (FCS). Human umbilical vein derived endothelial cells
were isolated and cultured as described. Tissue culture media,
sera, and cell culture supplements were from Beth-Haemek Biological
Industries, Israel, or from Gibco-BRL. MCF-7 TetOff cells
(Clontech, USA) containing the tetracycline trans-activator (tTA)
were grown in DMEM medium containing 10% Tet system approved fetal
calf serum (Clontech), in the presence of 100 .mu.g/ml G418, 150
.mu.g/ml Hygromycin B and 1 .mu.g/ml Tetracyclin.
[0261] Cloning of the LOR1 and LOR2 cDNA: Total RNA (4 .mu.g) from
HUVEC cells (for LOR1) or melanoma cells (for LOR2) was reversed
transcribed using MMLV reverse transcriptase (GIBCO BRL) as
described. The LOR-1 and LOR-2 cDNAs were amplified using the
Expand Long High Fidelity PCR system (ROCHE) and the following
pairs of amplification primers: For LOR-1 SEQ ID NOs:10 and 11, and
for LOR-2 SEQ ID NOs:12 and 13. The 2.3 kb cDNA of LOR-1 (SEQ ID
NO:1) and the 2.26KB of LOR-2 (SEQ ID NO:4) were subcloned into the
pGEM-T Easy vector (Promega) by T-A cloning.
[0262] Generation of polyclonal antibodies against human LOR1: A
cDNA fragment containing nucleotides 1641-2253 of LOR1 (SEQ ID
NO:14) was amplified using the Expand High Fidelity PCR kit and a
pair of amplification primers (SEQ ID NOs:15 and 16). The PCR
product was subcloned into the pGEM-T Easy vector (Promega) by T-A
cloning.
[0263] A 613 bp LOR1 cDNA fragment (SEQ ID NO:14) was digested with
the Sph-I and Hind III restriction enzymes and ligated into the
bacterial expression vector pQE-30, which added an in-frame
sequence encoding a six histidine (6.times. His) tag to the 5' end
of the insert. The resulting plasmid was used to produce a
recombinant, 6.times. His tagged 23 kDa peptide (SEQ ID NO:17). The
peptide was purified from bacterial cell extracts using nickel
affinity chromatography, and further purified using SDS-PAGE. The
gel was electroblotted onto nitrocellulose and the band containing
the peptide was cut out from the blot, solubilized in DMSO, and
used to immunize rabbits. Antiserum was affinity purified on
protein-A sepharose followed by affinity purification on a column
to which the recombinant peptide was coupled using a previously
described method (Wilchek and Miron, 1982). The antibody was eluted
from the column using 0.1 M glycine at pH 3.
[0264] Transfections: To constitutively express LOR1, the full
length LOR1 cDNA (SEQ ID NO:1), was digested out of the pGEM-T easy
vector (Promega) with Hind III and XbaI (which were incorporated
into the primers used for the cloning of the LOR1 cDNA) and ligated
into the mammalian expression vector pcDNA3.1 Hygro (Invitrogen,
USA) to generate the expression vector pcDNA-LOR1. Empty pCDNA3.1
Hygro plasmid or pcDNA-LOR1 plasmid (10-20 .mu.g) were stably
transfected into MCF-7 cells using electroporation with a BioRad
gene pulser (960 .mu.F, 0.28 V). Stable transfectants were selected
using 300 .mu.g/ml hygromycin B. Clones expressing recombinant LOR1
were obtained in two consecutive stable transfections and screened
for LOR1 expression using our anti-LOR-1 polyclonal antibodies.
Conditioned medium was collected after 48 hours from transfected
cells and LOR-1 expression was monitored using western blot
analysis (FIG. 10A).
[0265] To inducively express LOR1, full length LOR1 cDNA (SEQ ID
NO:1) was cloned into the pTET-Splice vector (Clontech), which
enables an inducible expression under the control of tetracycline
(Tet off system). The pTET-Splice plasmid DNA was digested with
Hind III and SpeI and ligated to the 2.3 kb hLOR1 cDNA fragment
which was rescued out of the pCDNA-LOR1 plasmid using Hind III and
XbaI. The resultant plasmid, which was designated as pTET-LOR1, was
co-transfected into MCF-7 TetOff cells along with pTK-Hygro at a
ratio of 20:1 respectively. LOR-1 expressing cells were selected in
medium containing 100 .mu.g/ml G418, 150 .mu.g/ml hygromycin B and
1 .mu.g/ml tetracycline. Stable transfectants were screened for
inducible expression of LOR1 using western blot analysis 48 hours
following removal of the tetracycline from the growth media. The
clone having the highest induction levels in the absence of
tetracycline and lowest basal expression levels in the presence of
tetracycline was selected and designated MCF-7/Tet-LOR1.
[0266] C6 glioma cells were transfected and screened for LOR1
expression as described above.
[0267] Northern blot Analysis: Total RNA was extracted from
cultured cells using Tri-Reagent (MRC, Cincinnati) according to the
manufacture's instructions. Total RNA (15 .mu.g) was loaded on a
1.2% agarose gel and Northern blot analysis was carried out as
previously described. LOR-1 and LOR-2 .sup.32P labeled cDNA
fragments; nucleotides 1-660 of SEQ ID NO:18 and 1061-1590 of SEQ
ID NO:19 (respectively) were used as probes.
[0268] Protein blot analysis: Serum free conditioned media (40
.mu.l) was separated on a 8% SDS-PAGE gel and the proteins were
electroblotted onto a nitrocellulose filter using semi-dry
electroblotting. The filter was blocked for 1 hour at room
temperature with TBST buffer containing 10 mM Tris-HCl (pH-7.0),
0.15 M NaCl, and 0.3% Tween-20 supplemented with 10% low fat milk.
The filter was incubated over night at 4.degree. C. with affinity
purified rabbit anti-LOR1 polyclonal antibody in TBST (1:2500). The
blot was subsequently washed 3 times in TBST and incubated with
goat anti-rabbit IgG peroxidase-conjugated secondary antibodies for
1 hour at room temperature. Bound antibody was visualized using the
ECL detection system (Biological Industries, Israel).
[0269] Nude Mice Experiments: Slow release pellets containing
17.beta.-estradiol (0.72 mg/pellet, 60 day release, Innovative
Research) were pre-implanted subcutaneously in 6-8 weeks old female
athymic nude mice (CD1). MCF-7 cells (10.sup.7 cells/mouse) were
injected into the mammary fat pads. Tumor size was measured with a
caliper once or twice a week, and tumor volume was determined using
the following formula: volume=width.sup.2.times.length.times.0.52.
Mice were sacrificed 4 weeks following injection of the MCF-7
cells. In other experiments, the tumors were excised when reaching
a diameter of 0.8 cm. The primary tumor, the liver and the lungs
were removed, weighted, fixed in 10% buffered formalin and embedded
in paraffin.
[0270] The development of tumors from C6 glioma cells expressing
recombinant LOR-1 was also studied. Cells (2.times.10.sup.5
cells/animal) transfected with a control expression vector or with
an expression vector containing LOR1 cDNA were injected
subcutaneously in the hind limb. Mice were sacrificed 3 weeks after
the injection of the cells. The primary tumors were removed, fixed
in 10% buffered formalin, and embedded in paraffin for
analysis.
[0271] Histology and Immunohistochemistry: Formalin-fixed,
paraffin-embedded tissues were cut into serial sections of 5.mu.m
each and used for immunohistochemistry. Sections were
deparaffinized by heating to 60.degree. C. for 1 hour, washed twice
with xylen for 5 min. and rehydrated by consecutive washes in 100%,
95%, and 70% ethanol followed by a wash in water. Endogenous
peroxidase activity was inhibited by a 15 minute incubation with 3%
hydrogen peroxide in methanol, followed by washes with water and
PBS. The sections were then antigen retrieved by heating twice for
10 minutes in a microwave oven to 90.degree. C. in citrate buffer
at pH-6.2 (for cytokeratin and vimentin antibodies) or in 1mM EDTA
buffer (for LOR1 antibody); blocking was performed using CAS block
(Zymed). Following blocking, the sections were incubated for 1.5 hr
at room temperature with the following antibodies, all diluted with
in antibody diluent reagent solution (Zymed): affinity purified
anti-LOR1 antibody (1:30-1:50), monoclonal anti human cytokeratin-7
antibodies-FITC conjugated (1:50), or with monoclonal antibodies
directed against vimentin (1:50). The sections were then washed 3
times with TBST, and secondary detection was applied using anti
FITC alkaline phosphates-conjugate (Roche) at a 1:200 dilution (for
anti cytokeratin) or DAKO Envision detection system (anti rabbit or
anti mouse--HRP). Sections were developed in
3-amino-9-ethylcarbazole (AEC, DAKO) solution, counterstained by
Hematoxylin, and photographed under a microscope. In control
experiments the primary antibodies were omitted. Masson Trichrome
and reticulum stains were according to manufacturer's protocol. For
Sirius red stain sections were incubated for 5 min. with 0.03%
(w/v) fast green solution, rinsed twice with 1% acetic acid,
incubated for 15 min. in 0.1% Sirius red solution, rinsed as above,
dehydrated and examined under polarizing light microscope.
Experimental Results
[0272] LOR-1 is expressed in highly metastatic breast cancer
derived cell types but not in non-metastatic MCF-7 cells:
Desmoplasia and formation of fibrotic foci in breast cancer tumors
is associated with the transition from a localized, relatively
benign tumor to an invasive/metastatic tumor. Lysyl oxidases
contribute to the deposition of collagen by covalently
cross-linking collagen monomers. To find out whether expression of
lysyl oxidases is associated with the invasive/metastatic phenotype
several human breast cancer derived cell types have been screened
for the expression of lysyl oxidases. Northern blot analysis
revealed that the LOR-1 gene is expressed in the highly malignant,
hormone independent MDA-MB-231 and MDA-MB-435 cells but not in
hormone dependent non-metastatic MCF-7 cells (FIG. 9a). LOR-2 on
the other hand was expressed only in MDA-MB-435 cells but not in
the MDA-MB-231 or in MCF-7 cells (FIG. 9b).
[0273] Highly malignant grade 1 breast carcinoma cells do not
express LOR-1 while highly malignant cells of grade 3 carcinomas
express LOR-1: LOR-1 expression was confined to the milk ducts in
both normal breast (FIG. 9c) and in in-situ ductal carcinoma (FIG.
9d). However, in grade 1 well-differentiated ductal breast
carcinoma, wherein the tumor cells migrate out of the ducts to form
pseudo ducts, the cells do not express LOR-1 (FIG. 9e, black
arrows). On the other hand, the cells of grade 3 ductal breast
carcinoma tumors, a highly malignant tumor characterized by
desmoplasia, express high levels of LOR-1 (FIG. 9f).
[0274] Tumors generated in mice from LOR-1 MCF-7 transfected cells
have a slower growth rate: In order to determine whether LOR-1
expression contributes to the progression of breast tumors and to
the invasive/metastatic phenotype, non-invasive MCF-7 cells have
been transfected with an expression plasmid containing LOR-1 cDNA
(SEQ ID NO:1). Several transfectant clones have been isolated and
selected for their LOR-1 expression. The conditioned medium of two
LOR-1 expressing clones (clone 12 and 24) and of a clone
transfected with an expression vector alone (vec) was assayed with
LOR-1 antibodies directed against the C-terminal portion of LOR-1
(SEQ ID NO:17). Western blot analysis revealed higher levels of
LOR-1 expression in clone 12 cells as compared with clone 24 cells,
and no expression in cells transfected with the expression vector
alone (FIG. 10a).
[0275] In addition, both LOR-1 expressing cells displayed extra
protein bands of about 70 kDa (FIGS. 10a, b) suggesting that LOR-1
may undergo proteolytic processing like other members of the lysyl
oxidase family. To verify that the low molecular weight forms are
produced as a result of post-translational processing, LOR-1 cDNA
has been expressed in MCF-7 cells under the control of a
Tetracycline inducible promoter. It can be seen that once the
tetracycline inhibition is removed, the cells start to produce full
length LOR-1 which is then converted into a shorter, 70 kDa
C-terminal containing form (FIG. 10b). It is not yet clear whether
all of these forms are enzymatically active.
[0276] To substantiate its role in tumor growth and metastasis
LOR-1 producing cells and cells transfected with expression vector
alone were pre-implanted subcutaneously in nude mice (described
above). Interestingly, the growth rate of tumors containing LOR-1
expressing cells was retarded as compared with that of tumors
developed from parental cells or cells transfected with the
expression vector alone (FIGS. 10c, d).
[0277] To determine if the decreased tumor growth rate was a result
of slower proliferation, the proliferation rate of empty vector
transfected MCF-7 with that of clone 12 and clone 24 cells were
also compared; significant differences in their rates of
proliferation was not detected.
[0278] Tumors that develop from LOR-1 producing MCF-7 cells contain
many necrotic and fibrotic foci rich in collagen deposits:
Hematoxylin-eosin staining of tumor sections revealed major
necrosis in tumors generated from LOR-1 expressing MCF-7 cells
(FIG. 11b) and only a few necrotic areas in tumors generated from
parental or MCF-7 cells transfected with expression vector alone
(FIG. 11a).
[0279] LOR-1 expressing tumors also contained extensive fibrotic
areas mainly composed of host derived cells such as mouse
fibroblasts rather than MCF-7 cells. These cells are easily
distinguishable from the host cells since they do not react with an
antibody against human keratin 7 (FIG. 11c).
[0280] Since LOR-1 was shown to oxidize lysine residues on
collagen-I it was anticipated that it may induce collagen
cross-linking and deposition. However, this activity is probably
depended on the existence of collagen since LOR-1 expressing MCF-7
cells grown in culture do not produce more collagen than parental
MCF-7 cells. To further understand the involvement of LOR-1 in
tumor progression tumors derived from LOR-1 expressing MCF-7 cells
have been stained with Mason's Trichrome, a reagent that reacts
primarily with collagen-I and produces an azure color (Pinder et
al., 1994). (1) Tumors that developed from parental or MCF-7 cells
transfected by an expression vector alone contained limited amounts
of collagen that was scattered between the tumor cells (FIG. 11d,
arrows). In-contrast, tumors that developed from clone 12 or clone
24 LOR-1 expressing MCF-7 cells contained denser deposits of
collagen between the tumor cells (FIG. 11e). Furthermore, the
fibrotic and necrotic areas in these tumors appeared choke full
with collagen fibers (FIG. 11f). In addition, while the blood
vessels within tumors that developed from vector-transfected MCF-7
cells remained almost unstained (FIG. 11g, arrows), the blood
vessels in tumors derived from LOR-1 expressing MCF-7 cells were
sheathed by a thick layer of collagen (FIG. 11h, arrows).
[0281] These experiments indicate that LOR-1 affects both
collagen-I production and deposition in MCF-7 derived breast cancer
tumors. To substantiate the involvement of LOR-1 in collagen
deposition C6 glioma cells were also subcutaneously injected into
mice. While tumors generated from C6-glioma cells did not contain
large amounts of collagen, tumors generated from LOR-1 expressing
C6-glioma cells were rich with collagen. These results indicate a
more general effect for LOR-1 on collagen-I deposits in tumors. In
addition, Reticulum staining of collagen-III revealed that tumors
generated from LOR-1 expressing MCF-7 or C6-glioma cells contained
thicker and higher concentrations of collagen-III fibers (FIG. 12b,
12d) as compared with tumors generated from cells transfected with
the expression vector alone (FIGS. 12a, c).
[0282] Expression of LOR-1 in MCF-7 cells transforms the cells into
invasive cells in-vivo: It was reported that the appearance of
fibrotic foci in breast cancer tumors correlates with their degree
of invasiveness. To substantiate the involvement of LOR-1 in tumor
invasiveness human keratin-7 staining was employed. Tumors
generated from MCF-7 cells transfected by the expression vector
alone were surrounded by thick capsules with sharp borders. No
staining of human keratin-7 was observed within the capsule (FIG.
13a) or in between blood vessels, nerves and muscles located
adjacent to the capsules (FIG. 13b, arrows). In contrast, in tumors
generated from LOR-1 expressing MCF-7 cells, human keratin-7
positive cells were observed within the capsule (FIG. 13c).
Furthermore, in many areas the tumor cells migrated on-mass through
the capsule and invaded muscles (FIG. 13d), nerves (FIG. 13e) and
blood vessels (FIG. 13f). The invading cells were identified as the
transfected LOR-1 expressing MCF-7 cells using an anti-LOR-1
antibody (FIG. 13g). These observations provide a strong evidence
that the production of LOR-1 by breast cancer tumor cells
contribute to the transition from localized non-invasive tumors to
invasive tumors. Furthermore, aggregates of tumor cells were also
detected inside lymph vessels adjacent to the tumors indicating
that the LOR-1 expressing MCF-7 cells are metastatic (Luna,
1968).
Example 3
LOR-1 Expression is Correlated with the Malignancy of Colon
Tumors
[0283] To study the correlation between LOR-1 expression and the
malignancy of colon tumors various colon tumor sections were
subjected to immunohistochemistry staining using an antibody
directed against LOR-1.
Experimental Results
[0284] LOR-1 is moderately expressed in benign colon tumors: Normal
colon tissues and benign colon tumors including hyperplasia and
adenoma tissues were subjected to immunohistochemistry using an
antibody directed against the C-terminal of human LOR-1. As is
shown in FIGS. 14a-b, while low level of LOR-1 expression was seen
in a few cells of the normal colon tissue (FIG. 14a), a moderate
level of expression was detected in the hyperplasia tissue and a
substantial level of expression was detected in cells comprising
the adenoma tissue of the benign colon tumors (FIG. 14b). Thus,
these results demonstrate that the expression of LOR-1 correlates
with the formation of benign colon tumors.
[0285] Highly malignant colon tumors express high levels of LOR-1:
To further substantiate the correlation between LOR-1 and the
progression of colon tumors, low- and high-grade colon
adenocarcinoma tissues were subjected to LOR-1
immunohistochemistry. As is shown in FIGS. 14c-d, a high level of
LOR-1 expression was detected in both low-grade and high-grade
adenocarcinoma tissues. However, while in low-grade adenocarcinoma
the expression of LOR-1 was mainly confined to the carcinoma
structures, (FIG. 14c, arrows), in the more malignant, high-grade
colon adenocarcinoma high levels of LOR-1 expression was detected
throughout the disorganized tumor tissue. These results demonstrate
that a high level of LOR-1 expression is correlated with more
malignant colon tumors.
[0286] Altogether these results demonstrate that LOR-1 expression
is correlated with the progression of colon cancer and suggest the
use of LOR-1 in determining the staging of colon cancer tumors.
[0287] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0288] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents, patent applications and sequences identified
by their accession numbers mentioned in this specification are
herein incorporated in their entirety by reference into the
specification, to the same extent as if each individual
publication, patent, patent application or sequence identified by
their accession number was specifically and individually indicated
to be incorporated herein by reference. In addition, citation or
identification of any reference in this application shall not be
construed as an admission that such reference is available as prior
art to the present invention.
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Sequence CWU 1
1
1912325DNAHomo sapiens 1atggagaggc ctctgtgctc ccacctctgc agctgcctgg
ctatgctggc cctcctgtcc 60cccctgagcc tggcacagta tgacagctgg ccccattacc
ccgagtactt ccagcaaccg 120gctcctgagt atcaccagcc ccaggccccc
gccaacgtgg ccaagattca gctgcgcctg 180gctgggcaga agaggaagca
cagcgagggc cgggtggagg tgtactatga tggccagtgg 240ggcaccgtgt
gcgatgacga cttctccatc cacgctgccc acgtcgtctg ccgggagctg
300ggctatgtgg aggccaagtc ctggactgcc agctcctcct acggcaaggg
agaagggccc 360atctggttag acaatctcca ctgtactggc aacgaggcga
cccttgcagc atgcacctcc 420aatggctggg gcgtcactga ctgcaagcac
acggaggatg tcggtgtggt gtgcagcgac 480aaaaggattc ctgggttcaa
atttgacaat tcgttgatca accagataga gaacctgaat 540atccaggtgg
aggacattcg gattcgagcc atcctctcaa cctaccgcaa gcgcacccca
600gtgatggagg gctacgtgga ggtgaaggag ggcaagacct ggaagcagat
ctgtgacaag 660cactggacgg ccaagaattc ccgcgtggtc tgcggcatgt
ttggcttccc tggggagagg 720acatacaata ccaaagtgta caaaatgttt
gcctcacgga ggaagcagcg ctactggcca 780ttctccatgg actgcaccgg
cacagaggcc cacatctcca gctgcaagct gggcccccag 840gtgtcactgg
accccatgaa gaatgtcacc tgcgagaatg ggctaccggc cgtggtgggt
900tgtgtgcctg ggcaggtctt cagccctgac ggaccctcaa gattccggaa
agcgtacaag 960ccagagcaac ccctggtgcg actgagaggc ggtgcctaca
tcggggaggg ccgcgtggag 1020gtgctcaaaa atggagaatg ggggaccgtc
tgcgacgaca agtgggacct ggtgtcggcc 1080agtgtggtct gcagagagct
gggctttggg agtgccaaag aggcagtcac tggctcccga 1140ctggggcaag
ggatcggacc catccacctc aacgagatcc agtgcacagg caatgagaag
1200tccattatag actgcaagtt caatgccgag tctcagggct gcaaccacga
ggaggatgct 1260ggtgtgagat gcaacacccc tgccatgggc ttgcagaaga
agctgcgcct gaacggcggc 1320cgcaatccct acgagggccg agtggaggtg
ctggtggaga gaaacgggtc ccttgtgtgg 1380gggatggtgt gtggccaaaa
ctggggcatc gtggaggcca tggtggtctg ccgccagctg 1440ggcctgggat
tcgccagcaa cgccttccag gagacctggt attggcacgg agatgtcaac
1500agcaacaaag tggtcatgag tggagtgaag tgctcgggaa cggagctgtc
cctggcgcac 1560tgccgccacg acggggagga cgtggcctgc ccccagggcg
gagtgcagta cggggccgga 1620gttgcctgct cagaaaccgc ccctgacctg
gtcctcaatg cggagatggt gcagcagacc 1680acctacctgg aggaccggcc
catgttcatg ctgcagtgtg ccatggagga gaactgcctc 1740tcggcctcag
ccgcgcagac cgaccccacc acgggctacc gccggctcct gcgcttctcc
1800tcccagatcc acaacaatgg ccagtccgac ttccggccca agaacggccg
ccacgcgtgg 1860atctggcacg actgtcacag gcactaccac agcatggagg
tgttcaccca ctatgacctg 1920ctgaacctca atggcaccaa ggtggcagag
ggccacaagg ccagcttctg cttggaggac 1980acagaatgtg aaggagacat
ccagaagaat tacgagtgtg ccaacttcgg cgatcagggc 2040atcaccatgg
gctgctggga catgtaccgc catgacatcg actgccagtg ggttgacatc
2100actgacgtgc cccctggaga ctacctgttc caggttgtta ttaaccccaa
cttcgaggtt 2160gcagaatccg attactccaa caacatcatg aaatgcagga
gccgctatga cggccaccgc 2220atctggatgt acaactgcca cataggtggt
tccttcagcg aagagacgga aaaaaagttt 2280gagcacttca gcgggctctt
aaacaaccag ctgtccccgc agtaa 23252774PRTHomo sapiens 2Met Glu Arg
Pro Leu Cys Ser His Leu Cys Ser Cys Leu Ala Met Leu 1 5 10 15 Ala
Leu Leu Ser Pro Leu Ser Leu Ala Gln Tyr Asp Ser Trp Pro His 20 25
30 Tyr Pro Glu Tyr Phe Gln Gln Pro Ala Pro Glu Tyr His Gln Pro Gln
35 40 45 Ala Pro Ala Asn Val Ala Lys Ile Gln Leu Arg Leu Ala Gly
Gln Lys 50 55 60 Arg Lys His Ser Glu Gly Arg Val Glu Val Tyr Tyr
Asp Gly Gln Trp 65 70 75 80 Gly Thr Val Cys Asp Asp Asp Phe Ser Ile
His Ala Ala His Val Val 85 90 95 Cys Arg Glu Leu Gly Tyr Val Glu
Ala Lys Ser Trp Thr Ala Ser Ser 100 105 110 Ser Tyr Gly Lys Gly Glu
Gly Pro Ile Trp Leu Asp Asn Leu His Cys 115 120 125 Thr Gly Asn Glu
Ala Thr Leu Ala Ala Cys Thr Ser Asn Gly Trp Gly 130 135 140 Val Thr
Asp Cys Lys His Thr Glu Asp Val Gly Val Val Cys Ser Asp 145 150 155
160 Lys Arg Ile Pro Gly Phe Lys Phe Asp Asn Ser Leu Ile Asn Gln Ile
165 170 175 Glu Asn Leu Asn Ile Gln Val Glu Asp Ile Arg Ile Arg Ala
Ile Leu 180 185 190 Ser Thr Tyr Arg Lys Arg Thr Pro Val Met Glu Gly
Tyr Val Glu Val 195 200 205 Lys Glu Gly Lys Thr Trp Lys Gln Ile Cys
Asp Lys His Trp Thr Ala 210 215 220 Lys Asn Ser Arg Val Val Cys Gly
Met Phe Gly Phe Pro Gly Glu Arg 225 230 235 240 Thr Tyr Asn Thr Lys
Val Tyr Lys Met Phe Ala Ser Arg Arg Lys Gln 245 250 255 Arg Tyr Trp
Pro Phe Ser Met Asp Cys Thr Gly Thr Glu Ala His Ile 260 265 270 Ser
Ser Cys Lys Leu Gly Pro Gln Val Ser Leu Asp Pro Met Lys Asn 275 280
285 Val Thr Cys Glu Asn Gly Leu Pro Ala Val Val Gly Cys Val Pro Gly
290 295 300 Gln Val Phe Ser Pro Asp Gly Pro Ser Arg Phe Arg Lys Ala
Tyr Lys 305 310 315 320 Pro Glu Gln Pro Leu Val Arg Leu Arg Gly Gly
Ala Tyr Ile Gly Glu 325 330 335 Gly Arg Val Glu Val Leu Lys Asn Gly
Glu Trp Gly Thr Val Cys Asp 340 345 350 Asp Lys Trp Asp Leu Val Ser
Ala Ser Val Val Cys Arg Glu Leu Gly 355 360 365 Phe Gly Ser Ala Lys
Glu Ala Val Thr Gly Ser Arg Leu Gly Gln Gly 370 375 380 Ile Gly Pro
Ile His Leu Asn Glu Ile Gln Cys Thr Gly Asn Glu Lys 385 390 395 400
Ser Ile Ile Asp Cys Lys Phe Asn Ala Glu Ser Gln Gly Cys Asn His 405
410 415 Glu Glu Asp Ala Gly Val Arg Cys Asn Thr Pro Ala Met Gly Leu
Gln 420 425 430 Lys Lys Leu Arg Leu Asn Gly Gly Arg Asn Pro Tyr Glu
Gly Arg Val 435 440 445 Glu Val Leu Val Glu Arg Asn Gly Ser Leu Val
Trp Gly Met Val Cys 450 455 460 Gly Gln Asn Trp Gly Ile Val Glu Ala
Met Val Val Cys Arg Gln Leu 465 470 475 480 Gly Leu Gly Phe Ala Ser
Asn Ala Phe Gln Glu Thr Trp Tyr Trp His 485 490 495 Gly Asp Val Asn
Ser Asn Lys Val Val Met Ser Gly Val Lys Cys Ser 500 505 510 Gly Thr
Glu Leu Ser Leu Ala His Cys Arg His Asp Gly Glu Asp Val 515 520 525
Ala Cys Pro Gln Gly Gly Val Gln Tyr Gly Ala Gly Val Ala Cys Ser 530
535 540 Glu Thr Ala Pro Asp Leu Val Leu Asn Ala Glu Met Val Gln Gln
Thr 545 550 555 560 Thr Tyr Leu Glu Asp Arg Pro Met Phe Met Leu Gln
Cys Ala Met Glu 565 570 575 Glu Asn Cys Leu Ser Ala Ser Ala Ala Gln
Thr Asp Pro Thr Thr Gly 580 585 590 Tyr Arg Arg Leu Leu Arg Phe Ser
Ser Gln Ile His Asn Asn Gly Gln 595 600 605 Ser Asp Phe Arg Pro Lys
Asn Gly Arg His Ala Trp Ile Trp His Asp 610 615 620 Cys His Arg His
Tyr His Ser Met Glu Val Phe Thr His Tyr Asp Leu 625 630 635 640 Leu
Asn Leu Asn Gly Thr Lys Val Ala Glu Gly His Lys Ala Ser Phe 645 650
655 Cys Leu Glu Asp Thr Glu Cys Glu Gly Asp Ile Gln Lys Asn Tyr Glu
660 665 670 Cys Ala Asn Phe Gly Asp Gln Gly Ile Thr Met Gly Cys Trp
Asp Met 675 680 685 Tyr Arg His Asp Ile Asp Cys Gln Trp Val Asp Ile
Thr Asp Val Pro 690 695 700 Pro Gly Asp Tyr Leu Phe Gln Val Val Ile
Asn Pro Asn Phe Glu Val 705 710 715 720 Ala Glu Ser Asp Tyr Ser Asn
Asn Ile Met Lys Cys Arg Ser Arg Tyr 725 730 735 Asp Gly His Arg Ile
Trp Met Tyr Asn Cys His Ile Gly Gly Ser Phe 740 745 750 Ser Glu Glu
Thr Glu Lys Lys Phe Glu His Phe Ser Gly Leu Leu Asn 755 760 765 Asn
Gln Leu Ser Pro Gln 770 3757PRTHomo sapiens 3Met Met Trp Pro Gln
Pro Pro Thr Phe Ser Leu Phe Leu Leu Leu Leu 1 5 10 15 Leu Ser Gln
Ala Pro Ser Ser Arg Pro Gln Ser Ser Gly Thr Lys Lys 20 25 30 Leu
Arg Leu Val Gly Pro Ala Asp Arg Pro Glu Glu Gly Arg Leu Glu 35 40
45 Val Leu His Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asp Phe Ala
50 55 60 Leu Gln Glu Ala Thr Val Ala Cys Arg Gln Leu Gly Phe Glu
Ser Ala 65 70 75 80 Leu Thr Trp Ala His Ser Ala Lys Tyr Gly Gln Gly
Glu Gly Pro Ile 85 90 95 Trp Leu Asp Asn Val Arg Cys Leu Gly Thr
Glu Lys Thr Leu Asp Gln 100 105 110 Cys Gly Ser Asn Gly Trp Gly Ile
Ser Asp Cys Arg His Ser Glu Asp 115 120 125 Val Gly Val Val Cys His
Pro Arg Arg Gln His Gly Tyr His Ser Glu 130 135 140 Lys Val Ser Asn
Ala Leu Gly Pro Gln Gly Arg Arg Leu Glu Glu Val 145 150 155 160 Arg
Leu Lys Pro Ile Leu Ala Ser Ala Lys Arg His Ser Pro Val Thr 165 170
175 Glu Gly Ala Val Glu Val Arg Tyr Asp Gly His Trp Arg Gln Val Cys
180 185 190 Asp Gln Gly Trp Thr Met Asn Asn Ser Arg Val Val Cys Gly
Met Leu 195 200 205 Gly Phe Pro Ser Gln Thr Ser Val Asn Ser His Tyr
Tyr Arg Lys Val 210 215 220 Trp Asn Leu Lys Met Lys Asp Pro Lys Ser
Arg Leu Asn Ser Leu Thr 225 230 235 240 Lys Lys Asn Ser Phe Trp Ile
His Arg Val Asp Cys Phe Gly Thr Glu 245 250 255 Pro His Leu Ala Lys
Cys Gln Val Gln Val Ala Pro Gly Arg Gly Lys 260 265 270 Leu Arg Pro
Ala Cys Pro Gly Gly Met His Ala Val Val Ser Cys Val 275 280 285 Ala
Gly Pro His Phe Arg Arg Gln Lys Pro Lys Pro Thr Arg Lys Glu 290 295
300 Ser His Ala Glu Glu Leu Lys Val Arg Leu Arg Ser Gly Ala Gln Val
305 310 315 320 Gly Glu Gly Arg Val Glu Val Leu Met Asn Arg Gln Trp
Gly Thr Val 325 330 335 Cys Asp His Arg Trp Asn Leu Ile Ser Ala Ser
Val Val Cys Arg Gln 340 345 350 Leu Gly Phe Gly Ser Ala Arg Glu Ala
Leu Phe Gly Ala Gln Leu Gly 355 360 365 Gln Gly Leu Gly Pro Ile His
Leu Ser Glu Val Arg Cys Arg Gly Tyr 370 375 380 Glu Arg Thr Leu Gly
Asp Cys Leu Ala Leu Glu Gly Ser Gln Asn Gly 385 390 395 400 Cys Gln
His Ala Asn Asp Ala Ala Val Arg Cys Asn Ile Pro Asp Met 405 410 415
Gly Phe Gln Asn Lys Val Arg Leu Ala Gly Gly Arg Asn Ser Glu Glu 420
425 430 Gly Val Val Glu Val Gln Val Glu Val Asn Gly Gly Pro Arg Trp
Gly 435 440 445 Thr Val Cys Ser Asp His Trp Gly Leu Thr Glu Ala Met
Val Thr Cys 450 455 460 Arg Gln Leu Gly Leu Gly Phe Ala Asn Phe Ala
Leu Lys Asp Thr Trp 465 470 475 480 Tyr Trp Gln Gly Thr Pro Glu Ala
Lys Glu Val Val Met Ser Gly Val 485 490 495 Arg Cys Ser Gly Thr Glu
Met Ala Leu Gln Gln Cys Gln Arg His Gly 500 505 510 Pro Val His Cys
Ser His Gly Pro Gly Arg Phe Ser Ala Gly Val Ala 515 520 525 Cys Met
Asn Ser Ala Pro Asp Leu Val Met Asn Ala Gln Leu Val Gln 530 535 540
Glu Thr Ala Tyr Leu Glu Asp Arg Pro Leu Ser Met Leu Tyr Cys Ala 545
550 555 560 His Glu Glu Asn Cys Leu Ser Lys Ser Ala Asp His Met Asp
Trp Pro 565 570 575 Tyr Gly Tyr Arg Arg Leu Leu Arg Phe Ser Ser Gln
Ile Tyr Asn Leu 580 585 590 Gly Arg Ala Asp Phe Arg Pro Lys Ala Gly
Arg His Ser Trp Ile Trp 595 600 605 His Gln Cys His Arg His Asn His
Ser Ile Glu Val Phe Thr His Tyr 610 615 620 Asp Leu Leu Thr Leu Asn
Gly Ser Lys Val Ala Glu Gly His Lys Ala 625 630 635 640 Ser Phe Cys
Leu Glu Asp Thr Asn Cys Pro Ser Gly Val Gln Arg Arg 645 650 655 Tyr
Ala Cys Ala Asn Phe Gly Glu Gln Gly Val Ala Val Gly Cys Trp 660 665
670 Asp Thr Tyr Arg His Asp Ile Asp Cys Gln Trp Val Asp Ile Thr Asp
675 680 685 Val Gly Pro Gly Asp Tyr Ile Phe Gln Val Val Val Asn Pro
Thr Asn 690 695 700 Asp Val Ala Glu Ser Asp Phe Ser Asn Asn Met Ile
Arg Cys Arg Cys 705 710 715 720 Lys Tyr Asp Gly Gln Arg Val Trp Leu
His Asn Cys His Thr Gly Asp 725 730 735 Ser Tyr Arg Ala Asn Ala Glu
Leu Ser Leu Glu Gln Glu Gln Arg Leu 740 745 750 Arg Asn Asn Leu Ile
755 42262DNAHomo sapiens 4atgcgacctg tcagtgtctg gcagtggagc
ccctgggggc tgctgctgtg cctgctgtgc 60agttcgtgct tggggtctcc gtccccttcc
acgggccctg agaagaaggc cgggagccag 120gggcttcggt tccggctggc
tggcttcccc aggaagccct acgagggccg cgtggagata 180cagcgagctg
gtgaatgggg caccatctgc gatgatgact tcacgctgca ggctgcccac
240atcctctgcc gggagctggg cttcacagag gccacaggct ggacccacag
tgccaaatat 300ggccctggaa caggccgcat ctggctggac aacttgagct
gcagtgggac cgagcagagt 360gtgactgaat gtgcctcccg gggctggggg
aacagtgact gtacgcacga tgaggatgct 420ggggtcatct gcaaagacca
gcgcctccct ggcttctcgg actccaatgt cattgaggta 480gagcatcacc
tgcaagtgga ggaggtgcga attcgacccg ccgttgggtg gggcagacga
540cccctgcccg tgacggaggg gctggtggaa gtcaggcttc ctgacggctg
gtcgcaagtg 600tgcgacaaag gctggagcgc ccacaacagc cacgtggtct
gcgggatgct gggcttcccc 660agcgaaaaga gggtcaacgc ggccttctac
aggctgctag cccaacggca gcaacactcc 720tttggtctgc atggggtggc
gtgcgtgggc acggaggccc acctctccct ctgttccctg 780gagttctatc
gtgccaatga caccgccagg tgccctgggg ggggccctgc agtggtgagc
840tgtgtgccag gccctgtcta cgcggcatcc agtggccaga agaagcaaca
acagtcgaag 900cctcaggggg aggcccgtgt ccgtctaaag ggcggcgccc
accctggaga gggccgggta 960gaagtcctga aggccagcac atggggcaca
gtctgtgacc gcaagtggga cctgcatgca 1020gccagcgtgg tgtgtcggga
gctgggcttc gggagtgctc gagaagctct gagtggcgct 1080cgcatggggc
agggcatggg tgctatccac ctgagtgaag ttcgctgctc tggacaggag
1140ctctccctct ggaagtgccc ccacaagaac atcacagctg aggattgttc
acatagccag 1200gatgccgggg tccggtgcaa cctaccttac actggggcag
agaccaggat ccgactcagt 1260gggggccgca gccaacatga ggggcgagtc
gaggtgcaaa tagggggacc tgggcccctt 1320cgctggggcc tcatctgtgg
ggatgactgg gggaccctgg aggccatggt ggcctgtagg 1380caactgggtc
tgggctacgc caaccacggc ctgcaggaga cctggtactg ggactctggg
1440aatataacag aggtggtgat gagtggagtg cgctgcacag ggactgagct
gtccctggat 1500cagtgtgccc atcatggcac ccacatcacc tgcaagagga
cagggacccg cttcactgct 1560ggagtcatct gttctgagac tgcatcagat
ctgttgctgc actcagcact ggtgcaggag 1620accgcctaca tcgaagaccg
gcccctgcat atgttgtact gtgctgcgga agagaactgc 1680ctggccagct
cagcccgctc agccaactgg ccctatggtc accggcgtct gctccgattc
1740tcctcccaga tccacaacct gggacgagct gacttcaggc ccaaggctgg
gcgccactcc 1800tgggtgtggc acgagtgcca tgggcattac cacagcatgg
acatcttcac tcactatgat 1860atcctcaccc caaatggcac caaggtggct
gagggccaca aagctagttt ctgtctcgaa 1920gacactgagt gtcaggagga
tgtctccaag cggtatgagt gtgccaactt tggagagcaa 1980ggcatcactg
tgggttgctg ggatctctac cggcatgaca ttgactgtca gtggattgac
2040atcacggatg tgaagccagg aaactacatt ctccaggttg tcatcaaccc
aaactttgaa 2100gtagcagaga gtgactttac caacaatgca atgaaatgta
actgcaaata tgatggacat 2160agaatctggg tgcacaactg ccacattggt
gatgccttca gtgaagaggc caacaggagg 2220tttgaacgct accctggcca
gaccagcaac cagattatct aa 226251725DNAHomo sapiens 5atggctctgg
cccgaggcag ccggcagctg ggggccctgg tgtggggcgc ctgcctgtgc 60gtgctggtgc
acgggcagca ggcgcagccc gggcagggct cggaccccgc ccgctggcgg
120cagctgatcc agtgggagaa caacgggcag gtgtacagct tgctcaactc
gggctcagag 180tacgtgccgg ccggacctca gcgctccgag agtagctccc
gggtgctgct ggccggcgcg 240ccccaggccc agcagcggcg cagccacggg
agcccccggc gtcggcaggc gccgtccctg 300cccctgccgg ggcgcgtggg
ctcggacacc gtgcgcggcc aggcgcggca
cccattcggc 360tttggccagg tgcccgacaa ctggcgcgag gtggccgtcg
gggacagcac gggcatggcc 420ctggcccgca cctccgtctc ccagcaacgg
cacgggggct ccgcctcctc ggtctcggct 480tcggccttcg ccagcaccta
ccgccagcag ccctcctacc cgcagcagtt cccctacccg 540caggcgccct
tcgtcagcca gtacgagaac tacgaccccg cgtcgcggac ctacgaccag
600ggtttcgtgt actaccggcc cgcgggcggc ggcgtgggcg cgggggcggc
ggccgtggcc 660tcggcggggg tcatctaccc ctaccagccc cgggcgcgct
acgaggagta cggcggcggc 720gaagagctgc ccgagtaccc gcctcagggc
ttctacccgg cccccgagag gccctacgtg 780ccgccgccgc cgccgccccc
cgacggcctg gaccgccgct actcgcacag tctgtacagc 840gagggcaccc
ccggcttcga gcaggcctac cctgaccccg gtcccgaggc ggcgcaggcc
900catggcggag acccacgcct gggctggtac ccgccctacg ccaacccgcc
gcccgaggcg 960tacgggccgc cgcgcgcgct ggagccgccc tacctgccgg
tgcgcagctc cgacacgccc 1020ccgccgggtg gggagcggaa cggcgcgcag
cagggccgcc tcagcgtagg cagcgtgtac 1080cggcccaacc agaacggccg
cggtctccct gacttggtcc cagaccccaa ctatgtgcaa 1140gcatccactt
atgtgcagag agcccacctg tactccctgc gctgtgctgc ggaggagaag
1200tgtctggcca gcacagccta tgcccctgag gccaccgact acgatgtgcg
ggtgctactg 1260cgcttccccc agcgcgtgaa gaaccagggc acagcagact
tcctccccaa ccggccacgg 1320cacacctggg agtggcacag ctgccaccag
cattaccaca gcatggacga gttcagccac 1380tacgacctac tggatgcagc
cacaggcaag aaggtggccg agggccacaa ggccagtttc 1440tgcctggagg
acagcacctg tgacttcggc aacctcaagc gctatgcatg cacctctcat
1500acccagggcc tgagcccagg ctgctatgac acctacaatg cggacatcga
ctgccagtgg 1560atcgacataa ccgacgtgca gcctgggaac tacatcctca
aggtgcacgt gaacccaaag 1620tatattgttt tggagtctga cttcaccaac
aacgtggtga gatgcaacat tcactacaca 1680ggtcgctacg tttctgcaac
aaactgcaaa attgtccaat cctga 17256574PRTHomo sapiens 6Met Ala Leu
Ala Arg Gly Ser Arg Gln Leu Gly Ala Leu Val Trp Gly 1 5 10 15 Ala
Cys Leu Cys Val Leu Val His Gly Gln Gln Ala Gln Pro Gly Gln 20 25
30 Gly Ser Asp Pro Ala Arg Trp Arg Gln Leu Ile Gln Trp Glu Asn Asn
35 40 45 Gly Gln Val Tyr Ser Leu Leu Asn Ser Gly Ser Glu Tyr Val
Pro Ala 50 55 60 Gly Pro Gln Arg Ser Glu Ser Ser Ser Arg Val Leu
Leu Ala Gly Ala 65 70 75 80 Pro Gln Ala Gln Gln Arg Arg Ser His Gly
Ser Pro Arg Arg Arg Gln 85 90 95 Ala Pro Ser Leu Pro Leu Pro Gly
Arg Val Gly Ser Asp Thr Val Arg 100 105 110 Gly Gln Ala Arg His Pro
Phe Gly Phe Gly Gln Val Pro Asp Asn Trp 115 120 125 Arg Glu Val Ala
Val Gly Asp Ser Thr Gly Met Ala Leu Ala Arg Thr 130 135 140 Ser Val
Ser Gln Gln Arg His Gly Gly Ser Ala Ser Ser Val Ser Ala 145 150 155
160 Ser Ala Phe Ala Ser Thr Tyr Arg Gln Gln Pro Ser Tyr Pro Gln Gln
165 170 175 Phe Pro Tyr Pro Gln Ala Pro Phe Val Ser Gln Tyr Glu Asn
Tyr Asp 180 185 190 Pro Ala Ser Arg Thr Tyr Asp Gln Gly Phe Val Tyr
Tyr Arg Pro Ala 195 200 205 Gly Gly Gly Val Gly Ala Gly Ala Ala Ala
Val Ala Ser Ala Gly Val 210 215 220 Ile Tyr Pro Tyr Gln Pro Arg Ala
Arg Tyr Glu Glu Tyr Gly Gly Gly 225 230 235 240 Glu Glu Leu Pro Glu
Tyr Pro Pro Gln Gly Phe Tyr Pro Ala Pro Glu 245 250 255 Arg Pro Tyr
Val Pro Pro Pro Pro Pro Pro Pro Asp Gly Leu Asp Arg 260 265 270 Arg
Tyr Ser His Ser Leu Tyr Ser Glu Gly Thr Pro Gly Phe Glu Gln 275 280
285 Ala Tyr Pro Asp Pro Gly Pro Glu Ala Ala Gln Ala His Gly Gly Asp
290 295 300 Pro Arg Leu Gly Trp Tyr Pro Pro Tyr Ala Asn Pro Pro Pro
Glu Ala 305 310 315 320 Tyr Gly Pro Pro Arg Ala Leu Glu Pro Pro Tyr
Leu Pro Val Arg Ser 325 330 335 Ser Asp Thr Pro Pro Pro Gly Gly Glu
Arg Asn Gly Ala Gln Gln Gly 340 345 350 Arg Leu Ser Val Gly Ser Val
Tyr Arg Pro Asn Gln Asn Gly Arg Gly 355 360 365 Leu Pro Asp Leu Val
Pro Asp Pro Asn Tyr Val Gln Ala Ser Thr Tyr 370 375 380 Val Gln Arg
Ala His Leu Tyr Ser Leu Arg Cys Ala Ala Glu Glu Lys 385 390 395 400
Cys Leu Ala Ser Thr Ala Tyr Ala Pro Glu Ala Thr Asp Tyr Asp Val 405
410 415 Arg Val Leu Leu Arg Phe Pro Gln Arg Val Lys Asn Gln Gly Thr
Ala 420 425 430 Asp Phe Leu Pro Asn Arg Pro Arg His Thr Trp Glu Trp
His Ser Cys 435 440 445 His Gln His Tyr His Ser Met Asp Glu Phe Ser
His Tyr Asp Leu Leu 450 455 460 Asp Ala Ala Thr Gly Lys Lys Val Ala
Glu Gly His Lys Ala Ser Phe 465 470 475 480 Cys Leu Glu Asp Ser Thr
Cys Asp Phe Gly Asn Leu Lys Arg Tyr Ala 485 490 495 Cys Thr Ser His
Thr Gln Gly Leu Ser Pro Gly Cys Tyr Asp Thr Tyr 500 505 510 Asn Ala
Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Gln Pro 515 520 525
Gly Asn Tyr Ile Leu Lys Val His Val Asn Pro Lys Tyr Ile Val Leu 530
535 540 Glu Ser Asp Phe Thr Asn Asn Val Val Arg Cys Asn Ile His Tyr
Thr 545 550 555 560 Gly Arg Tyr Val Ser Ala Thr Asn Cys Lys Ile Val
Gln Ser 565 570 71254DNAHomo sapiens 7atgcgcttcg cctggaccgt
gctcctgctc gggcctttgc agctctgcgc gctagtgcac 60tgcgcccctc ccgccgccgg
ccaacagcag cccccgcgcg agccgccggc ggctccgggc 120gcctggcgcc
agcagatcca atgggagaac aacgggcagg tgttcagctt gctgagcctg
180ggctcacagt accagcctca gcgccgccgg gacccgggcg ccgccgtccc
tggtgcagcc 240aacgcctccg cccagcagcc ccgcactccg atcctgctga
tccgcgacaa ccgcaccgcc 300gcggcgcgaa cgcggacggc cggctcatct
ggagtcaccg ctggccgccc caggcccacc 360gcccgtcact ggttccaagc
tggctactcg acatctagag cccgcgaagc tggcgcctcg 420cgcgcggaga
accagacagc gccgggagaa gttcctgcgc tcagtaacct gcggccgccc
480agccgcgtgg acggcatggt gggcgacgac ccttacaacc cctacaagta
ctctgacgac 540aacccttatt acaactacta cgatacttat gaaaggccca
gacctggggg caggtaccgg 600cccggatacg gcactggcta cttccagtac
ggtctcccag acctggtggc cgacccctac 660tacatccagg cgtccacgta
cgtgcagaag atgtccatgt acaacctgag atgcgcggcg 720gaggaaaact
gtctggccag tacagcatac agggcagatg tcagagatta tgatcacagg
780gtgctgctca gatttcccca aagagtgaaa aaccaaggga catcagattt
cttacccagc 840cgaccaagat attcctggga atggcacagt tgtcatcaac
attaccacag tatggatgag 900tttagccact atgacctgct tgatgccaac
acccagagga gagtggctga aggccacaaa 960gcaagtttct gtcttgaaga
cacatcctgt gactatggct accacaggcg atttgcatgt 1020actgcacaca
cacagggatt gagtcctggc tgttatgata cctatggtgc agacatagac
1080tgccagtgga ttgatattac agatgtaaaa cctggaaact atatcctaaa
ggtcagtgta 1140aaccccagct acctggttcc tgaatctgac tataccaaca
atgttgtgcg ctgtgacatt 1200cgctacacag gacatcatgc gtatgcctca
ggctgcacaa tttcaccgta ttag 12548417PRTHomo sapiens 8Met Arg Phe Ala
Trp Thr Val Leu Leu Leu Gly Pro Leu Gln Leu Cys 1 5 10 15 Ala Leu
Val His Cys Ala Pro Pro Ala Ala Gly Gln Gln Gln Pro Pro 20 25 30
Arg Glu Pro Pro Ala Ala Pro Gly Ala Trp Arg Gln Gln Ile Gln Trp 35
40 45 Glu Asn Asn Gly Gln Val Phe Ser Leu Leu Ser Leu Gly Ser Gln
Tyr 50 55 60 Gln Pro Gln Arg Arg Arg Asp Pro Gly Ala Ala Val Pro
Gly Ala Ala 65 70 75 80 Asn Ala Ser Ala Gln Gln Pro Arg Thr Pro Ile
Leu Leu Ile Arg Asp 85 90 95 Asn Arg Thr Ala Ala Ala Arg Thr Arg
Thr Ala Gly Ser Ser Gly Val 100 105 110 Thr Ala Gly Arg Pro Arg Pro
Thr Ala Arg His Trp Phe Gln Ala Gly 115 120 125 Tyr Ser Thr Ser Arg
Ala Arg Glu Ala Gly Ala Ser Arg Ala Glu Asn 130 135 140 Gln Thr Ala
Pro Gly Glu Val Pro Ala Leu Ser Asn Leu Arg Pro Pro 145 150 155 160
Ser Arg Val Asp Gly Met Val Gly Asp Asp Pro Tyr Asn Pro Tyr Lys 165
170 175 Tyr Ser Asp Asp Asn Pro Tyr Tyr Asn Tyr Tyr Asp Thr Tyr Glu
Arg 180 185 190 Pro Arg Pro Gly Gly Arg Tyr Arg Pro Gly Tyr Gly Thr
Gly Tyr Phe 195 200 205 Gln Tyr Gly Leu Pro Asp Leu Val Ala Asp Pro
Tyr Tyr Ile Gln Ala 210 215 220 Ser Thr Tyr Val Gln Lys Met Ser Met
Tyr Asn Leu Arg Cys Ala Ala 225 230 235 240 Glu Glu Asn Cys Leu Ala
Ser Thr Ala Tyr Arg Ala Asp Val Arg Asp 245 250 255 Tyr Asp His Arg
Val Leu Leu Arg Phe Pro Gln Arg Val Lys Asn Gln 260 265 270 Gly Thr
Ser Asp Phe Leu Pro Ser Arg Pro Arg Tyr Ser Trp Glu Trp 275 280 285
His Ser Cys His Gln His Tyr His Ser Met Asp Glu Phe Ser His Tyr 290
295 300 Asp Leu Leu Asp Ala Asn Thr Gln Arg Arg Val Ala Glu Gly His
Lys 305 310 315 320 Ala Ser Phe Cys Leu Glu Asp Thr Ser Cys Asp Tyr
Gly Tyr His Arg 325 330 335 Arg Phe Ala Cys Thr Ala His Thr Gln Gly
Leu Ser Pro Gly Cys Tyr 340 345 350 Asp Thr Tyr Gly Ala Asp Ile Asp
Cys Gln Trp Ile Asp Ile Thr Asp 355 360 365 Val Lys Pro Gly Asn Tyr
Ile Leu Lys Val Ser Val Asn Pro Ser Tyr 370 375 380 Leu Val Pro Glu
Ser Asp Tyr Thr Asn Asn Val Val Arg Cys Asp Ile 385 390 395 400 Arg
Tyr Thr Gly His His Ala Tyr Ala Ser Gly Cys Thr Ile Ser Pro 405 410
415 Tyr 9752PRTHomo sapiens 9Met Arg Pro Val Ser Val Trp Gln Trp
Ser Pro Trp Gly Leu Leu Leu 1 5 10 15 Cys Leu Leu Cys Ser Ser Cys
Leu Gly Ser Pro Ser Pro Ser Thr Gly 20 25 30 Pro Glu Lys Lys Ala
Gly Ser Gln Gly Leu Arg Phe Arg Leu Ala Gly 35 40 45 Phe Pro Arg
Lys Pro Tyr Glu Gly Arg Val Glu Ile Gln Arg Ala Gly 50 55 60 Glu
Trp Gly Thr Ile Cys Asp Asp Asp Phe Thr Leu Gln Ala Ala His 65 70
75 80 Ile Leu Cys Arg Glu Leu Gly Phe Thr Glu Ala Thr Gly Trp Thr
His 85 90 95 Ser Ala Lys Tyr Gly Pro Gly Thr Gly Arg Ile Trp Leu
Asp Asn Leu 100 105 110 Ser Cys Ser Gly Thr Glu Gln Ser Val Thr Glu
Cys Ala Ser Arg Gly 115 120 125 Trp Gly Asn Ser Asp Cys Thr His Asp
Glu Asp Ala Gly Val Ile Cys 130 135 140 Lys Asp Gln Arg Leu Pro Gly
Phe Ser Asp Ser Asn Val Ile Glu Val 145 150 155 160 Glu His His Leu
Gln Val Glu Glu Val Arg Ile Arg Pro Ala Val Gly 165 170 175 Trp Gly
Arg Arg Pro Leu Pro Val Thr Glu Gly Leu Val Glu Val Arg 180 185 190
Leu Pro Asp Gly Trp Ser Gln Val Cys Asp Lys Gly Trp Ser Ala His 195
200 205 Asn Ser His Val Val Cys Gly Met Leu Gly Phe Pro Ser Glu Lys
Arg 210 215 220 Val Asn Ala Ala Phe Tyr Arg Leu Leu Ala Gln Arg Gln
Gln His Ser 225 230 235 240 Phe Gly Leu His Gly Val Ala Cys Val Gly
Thr Glu Ala His Leu Ser 245 250 255 Leu Cys Ser Leu Glu Phe Tyr Arg
Ala Asn Asp Thr Ala Arg Cys Pro 260 265 270 Gly Gly Gly Pro Ala Val
Val Ser Cys Val Pro Gly Pro Val Tyr Ala 275 280 285 Ala Ser Ser Gly
Gln Lys Lys Gln Gln Gln Ser Lys Pro Gln Gly Glu 290 295 300 Ala Arg
Val Arg Leu Lys Gly Gly Ala His Pro Gly Glu Gly Arg Val 305 310 315
320 Glu Val Leu Lys Ala Ser Thr Trp Gly Thr Val Cys Asp Arg Lys Trp
325 330 335 Asp Leu His Ala Ala Ser Val Val Cys Arg Glu Leu Gly Phe
Gly Ser 340 345 350 Ala Arg Glu Ala Leu Ser Gly Ala Arg Met Gly Gln
Gly Met Gly Ala 355 360 365 Ile His Leu Ser Glu Val Arg Cys Ser Gly
Gln Glu Leu Ser Leu Trp 370 375 380 Lys Cys Pro His Lys Asn Ile Thr
Ala Glu Asp Cys Ser His Ser Gln 385 390 395 400 Asp Ala Gly Val Arg
Cys Asn Leu Pro Tyr Thr Gly Ala Glu Thr Arg 405 410 415 Ile Arg Leu
Ser Gly Gly Arg Ser Gln His Glu Gly Arg Val Glu Val 420 425 430 Gln
Ile Gly Gly Pro Gly Pro Leu Arg Trp Gly Leu Ile Cys Gly Asp 435 440
445 Asp Trp Gly Thr Leu Glu Ala Met Val Ala Cys Arg Gln Leu Gly Leu
450 455 460 Gly Tyr Ala Asn His Gly Leu Gln Glu Thr Trp Tyr Trp Asp
Ser Gly 465 470 475 480 Asn Ile Thr Glu Val Val Met Ser Gly Val Arg
Cys Thr Gly Thr Glu 485 490 495 Leu Ser Leu Asp Gln Cys Ala His His
Gly Thr His Ile Thr Cys Lys 500 505 510 Arg Thr Gly Thr Arg Phe Thr
Ala Gly Val Ile Cys Ser Glu Thr Ala 515 520 525 Ser Asp Leu Leu Leu
His Ser Ala Leu Val Gln Glu Thr Ala Tyr Ile 530 535 540 Glu Asp Arg
Pro Leu His Met Leu Tyr Cys Ala Ala Glu Glu Asn Cys 545 550 555 560
Leu Ala Ser Ser Ala Arg Ser Ala Asn Trp Pro Tyr Gly His Arg Arg 565
570 575 Leu Leu Arg Phe Ser Ser Gln Ile His Asn Leu Gly Arg Ala Asp
Phe 580 585 590 Arg Pro Lys Ala Gly Arg His Ser Trp Val Trp His Glu
Cys His Gly 595 600 605 His Tyr His Ser Met Asp Ile Phe Thr His Tyr
Asp Ile Leu Thr Pro 610 615 620 Asn Gly Thr Lys Val Ala Glu Gly His
Lys Ala Ser Phe Cys Leu Glu 625 630 635 640 Asp Thr Glu Cys Gln Glu
Asp Val Ser Lys Arg Tyr Glu Cys Ala Asn 645 650 655 Phe Gly Glu Gln
Gly Ile Thr Val Gly Cys Trp Asp Leu Tyr Arg His 660 665 670 Asp Ile
Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Lys Pro Gly Asn 675 680 685
Tyr Ile Leu Gln Val Val Ile Asn Pro Asn Phe Glu Val Ala Glu Ser 690
695 700 Asp Phe Thr Asn Asn Ala Met Lys Cys Asn Cys Lys Tyr Asp Gly
His 705 710 715 720 Arg Ile Trp Val His Asn Cys His Ile Gly Asp Ala
Phe Ser Glu Glu 725 730 735 Ala Asn Arg Arg Phe Glu Arg Tyr Pro Gly
Gln Thr Ser Asn Gln Ile 740 745 750 1036DNAArtificial
sequenceSynthetically constructed single strand DNA oligonucleotide
10cgcaagcttg gatccgggat ggagaggcct ctgtgc 361137DNAArtificial
sequenceSynthetically constructed single strand DNA oligonucleotide
11cgctctagag gatccttact gcggggacag ctggttg 371221DNAArtificial
sequenceSynthetically constructed single strand DNA oligonucleotide
12gccatgcgac ctgtcagtgt c 211318DNAArtificial sequenceSynthetically
constructed single strand DNA oligonucleotide 13gggcagtggc acttagat
1814613DNAHomo sapiens 14ccctgacctg gtcctcaatg cggagatggt
gcagcagacc acctacctgg aggaccggcc 60catgttcatg ctgcagtgtg ccatggagga
gaactgcctc tcggcctcag ccgcgcagac 120cgaccccacc acgggctacc
gccggctcct gcgcttctcc tcccagatcc acaacaatgg 180ccagtccgac
ttccggccca agaacggccg ccacgcgtgg atctggcacg actgtcacag
240gcactaccac agcatggagg tgttcaccca ctatgacctg ctgaacctca
atggcaccaa 300ggtggcagag ggccacaagg ccagcttctg cttggaggac
acagaatgtg aaggagacat 360ccagaagaat tacgagtgtg
ccaacttcgg cgatcagggc atcaccatgg gctgctggga 420catgtaccgc
catgacatcg actgccagtg ggttgacatc actgacgtgc cccctggaga
480ctacctgttc caggttgtta ttaaccccaa cttcgaggtt gcagaatccg
attactccaa 540caacatcatg aaatgcagga gccgctatga cggccaccgc
atctggatgt acaactgcca 600cataggtggt tcc 6131530DNAArtificial
sequenceSynthetically constructed single strand DNA oligonucleotide
15acatgcatgc cctgacctgg tcctcaatgc 301630DNAArtificial
sequenceSynthetically constructed single strand DNA oligonucleotide
16cccaagcttg gaaccaccta tgtggcagtt 3017210PRTArtificial
sequenceSynthetically constructed protein sequence derived from
fusion of LOR-1 fragment (1641-2253) to a 5' 6XHis tag 17His His
His His His His Pro Asp Leu Val Leu Asn Ala Glu Met Val 1 5 10 15
Gln Gln Thr Thr Tyr Leu Glu Asp Arg Pro Met Phe Met Leu Gln Cys 20
25 30 Ala Met Glu Glu Asn Cys Leu Ser Ala Ser Ala Ala Gln Thr Asp
Pro 35 40 45 Thr Thr Gly Tyr Arg Arg Leu Leu Arg Phe Ser Ser Gln
Ile His Asn 50 55 60 Asn Gly Gln Ser Asp Phe Arg Pro Lys Asn Gly
Arg His Ala Trp Ile 65 70 75 80 Trp His Asp Cys His Arg His Tyr His
Ser Met Glu Val Phe Thr His 85 90 95 Tyr Asp Leu Leu Asn Leu Asn
Gly Thr Lys Val Ala Glu Gly His Lys 100 105 110 Ala Ser Phe Cys Leu
Glu Asp Thr Glu Cys Glu Gly Asp Ile Gln Lys 115 120 125 Asn Tyr Glu
Cys Ala Asn Phe Gly Asp Gln Gly Ile Thr Met Gly Cys 130 135 140 Trp
Asp Met Tyr Arg His Asp Ile Asp Cys Gln Trp Val Asp Ile Thr 145 150
155 160 Asp Val Pro Pro Gly Asp Tyr Leu Phe Gln Val Val Ile Asn Pro
Asn 165 170 175 Phe Glu Val Ala Glu Ser Asp Tyr Ser Asn Asn Ile Met
Lys Cys Arg 180 185 190 Ser Arg Tyr Asp Gly His Arg Ile Trp Met Tyr
Asn Cys His Ile Gly 195 200 205 Gly Ser 210 18660DNAArtificial
sequenceSynthetically constructed Northern blot probe consisting of
Nucleotides 1-660 of the LOR-1 cDNA 18atggagaggc ctctgtgctc
ccacctctgc agctgcctgg ctatgctggc cctcctgtcc 60cccctgagcc tggcacagta
tgacagctgg ccccattacc ccgagtactt ccagcaaccg 120gctcctgagt
atcaccagcc ccaggccccc gccaacgtgg ccaagattca gctgcgcctg
180gctgggcaga agaggaagca cagcgagggc cgggtggagg tgtactatga
tggccagtgg 240ggcaccgtgt gcgatgacga cttctccatc cacgctgccc
acgtcgtctg ccgggagctg 300ggctatgtgg aggccaagtc ctggactgcc
agctcctcct acggcaaggg agaagggccc 360atctggttag acaatctcca
ctgtactggc aacgaggcga cccttgcagc atgcacctcc 420aatggctggg
gcgtcactga ctgcaagcac acggaggatg tcggtgtggt gtgcagcgac
480aaaaggattc ctgggttcaa atttgacaat tcgttgatca accagataga
gaacctgaat 540atccaggtgg aggacattcg gattcgagcc atcctctcaa
cctaccgcaa gcgcacccca 600gtgatggagg gctacgtgga ggtgaaggag
ggcaagacct ggaagcagat ctgtgacaag 66019530DNAArtificial
sequenceSynthetically constructed Northern blot probe consisting of
nucleotides 1061-1590 of the LOR-2 cDNA 19gagaagctct gagtggcgct
cgcatggggc agggcatggg tgctatccac ctgagtgaag 60ttcgctgctc tggacaggag
ctctccctct ggaagtgccc ccacaagaac atcacagctg 120aggattgttc
acatagccag gatgccgggg tccggtgcaa cctaccttac actggggcag
180agaccaggat ccgactcagt gggggccgca gccaacatga ggggcgagtc
gaggtgcaaa 240tagggggacc tgggcccctt cgctggggcc tcatctgtgg
ggatgactgg gggaccctgg 300aggccatggt ggcctgtagg caactgggtc
tgggctacgc caaccacggc ctgcaggaga 360cctggtactg ggactctggg
aatataacag aggtggtgat gagtggagtg cgctgcacag 420ggactgagct
gtccctggat cagtgtgccc atcatggcac ccacatcacc tgcaagagga
480cagggacccg cttcactgct ggagtcatct gttctgagac tgcatcagat 530
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