U.S. patent application number 10/351284 was filed with the patent office on 2003-11-27 for therapeutic antiangiogenic compositions and methods.
Invention is credited to Folkman, M. Judah, O'Reilly, Michael S..
Application Number | 20030219426 10/351284 |
Document ID | / |
Family ID | 27485527 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030219426 |
Kind Code |
A1 |
O'Reilly, Michael S. ; et
al. |
November 27, 2003 |
Therapeutic antiangiogenic compositions and methods
Abstract
An inhibitor of endothelial cell proliferation, capable of
inhibiting angiogenesis and causing tumor regression, that is
approximately 20 kDa and corresponds to a C-terminal fragment of
collagen type XVIII, and methods of treating angiogenesis-related
disease.
Inventors: |
O'Reilly, Michael S.;
(Wincester, MA) ; Folkman, M. Judah; (Brookline,
MA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
SUITE 2800
ATLANTA
GA
30309
US
|
Family ID: |
27485527 |
Appl. No.: |
10/351284 |
Filed: |
January 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10351284 |
Jan 24, 2003 |
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09174282 |
Oct 16, 1998 |
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6544758 |
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09174282 |
Oct 16, 1998 |
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08740168 |
Oct 22, 1996 |
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5854205 |
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60005835 |
Oct 23, 1995 |
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60023070 |
Aug 2, 1996 |
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60026263 |
Sep 17, 1996 |
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Current U.S.
Class: |
424/94.1 ;
435/184; 435/252.3; 435/254.2; 435/320.1; 435/325; 435/6.14;
435/69.2; 530/388.26; 536/23.2 |
Current CPC
Class: |
Y10S 435/81 20130101;
C07K 14/78 20130101; A61P 17/00 20180101; A61P 35/00 20180101; A61P
27/02 20180101; A61P 7/00 20180101; A61K 38/00 20130101; A61P 9/10
20180101; A61P 1/04 20180101; A61P 29/00 20180101; A61P 1/00
20180101; Y10S 530/84 20130101 |
Class at
Publication: |
424/94.1 ; 435/6;
435/69.2; 435/184; 435/320.1; 435/252.3; 435/254.2; 435/325;
530/388.26; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 038/43; C12N 009/99; C12N 001/18; C12N 001/21; C12P
021/02; C12N 005/06 |
Claims
1. Isolated endostatin.
2. The isolated endostatin of claim 1 comprising, an isolated
protein that is approximately 18 kDa as determined by non-reduced
gel electrophoresis, and approximately 20 kDa as determined by
reduced gel electrophoresis, wherein the protein can be isolated
from the murine hemangioendothelioma EOMA cell line, and wherein
the protein is further characterized by its ability to specifically
inhibit proliferating cultured endothelial cells.
3. The endostatin of claim 1, wherein the N-terminal amino acid
sequence of the protein has substantial sequence homology to Seq ID
No:1.
4. The endostatin of claim 1, wherein the protein has substantial
seuqence homology to a C-terminal peptide fragment of collagen type
XVIII.
5. The endostatin of claim 1 made by a process comprising
recombinantly producing the protein of claim 1 in a recombinant
expression system, and isolating the recombinantly produced protein
in its un-refolded form.
6. The endostatin of claim 5, wherein the recombinant expression
system is E. coli or baculovirus.
7. A compound comprising, an isolated nucleic acid sequence coding
for endostatin protein.
8. The compound of claim 7, wherein the endostatin protein is
approximately 18 kDa as determined by non-reduced gel
electrophoresis, and approximately 20 kDa as determined by reduced
gel electrophoresis, wherein the protein can be isolated from the
murine hemangioendothelioma EOMA, and wherein the protein is
further characterized by its ability to specifically inhibit
proliferating cultured endothelial cells.
9. The compound of claim 7, wherein the N-terminal amino acid
sequence of the protein has substantial sequence homology to Seq ID
No:1.
10. The compound of claim 7, wherein the protein has substantial
seuqence homology to a C-terminal peptide fragment of collagen type
XVIII.
11. A compound comprising, an isolated antibody capable of
specifically binding to endostatin protein.
12. The compound of claim 11, wherein the endostatin protein is
approximately 18 kDa as determined by non-reduced gel
electrophoresis, and approximately 20 kDa as determined by reduced
gel electrophoresis, wherein the protein can be isolated from the
murine hemangioendothelioma EOMA, and wherein the protein is
further characterized by its ability to specifically inhibit
proliferating cultured endothelial cells.
13. The compound of claim 11, wherein the antibody is a monoclonal
antibody.
14. The compound of claim 11, wherein the N-terminal amino acid
sequence of the endostatin protein has substantial sequence
homology to Seq ID No:1.
15. The compound of claim 11, wherein the endostatin protein has
substantial sequence homology to a C-terminal peptide fragment of
collagen type XVIII.
16. An isolated emdostatin made by a process comprising, a.
collecting culture media used to grow murine hemangioendothelioma
cell line EOMA; and b. fractionating the media by heparin column
chromatography, wherein the isolated endostatin is a protein that
is approximately 18 kDa as determined by non-reduced gel
electrophoresis, and approximately 20 kDa as determined by reduced
gel electrophores, and the protein is capable of specifically
inhibiting endothelial cell proliferation in cultured cells.
17. A method of treating an angiogenesis-related disease
comprising, administering to a patient in need of such treatment of
the endostatin of claim 1 in an amount sufficient to inhibit
angiogenesis.
18. The method of claim 17, wherein the endostatin is a
recombinantly produced protein, and wherein the recombinantly
produced protein is administered in its un-refolded form.
19. The method of claim 18, wherein the recombinantly produced
endostatin provides a sustained release of the protein over a
period of at least 8 hours.
20. The method of claim 17, wherein the angiogenesis-related
disease is selected from the group consisting of
angiogenesis-dependent cancers; benign tumors; rheumatoid
arthritis; psoriasis; ocular angiogenesis diseases; Osler-Webber
Syndrome; myocardial angiogenesis; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; wound
granulation; intestinal adhesions, atherosclerosis, scleroderma,
hypertrophic scars, cat scratch disease and Helobacter pylori
ulcers.
21. The method of claim 20, wherein the angiogenesis-related
disease is angiogenesis-dependent cancer.
22. A method of treating a patient with an angiogenesis-dependent
cancer tumor comprising, administering to a patient in need of such
treatment of the endostatin of claim 1 in an amount sufficient to
cause tumor regression.
23. The method of claim 22, wherein the endostatin is a
recombinantly produced protein, and wherein the recombinantly
produced protein is administered in its un-refolded form.
24. The method of claim 23, wherein the recombinantly produced
protein provides a sustained release of the protein for a period of
at least 8 hours.
25. A method of curing a patient with an angiogenesis-dependent
cancer comprising, administering to a patient in need of such a
cure an angiogenesis-dependent cancer curing amount of a
composition comprising, angiostatin combined with the endostatin of
claim 1, wherein the angiostatin and the endostatin are provided in
amounts such that the composition is capable of effectively
inhibiting angiogenesis of angiogenesis-dependent cancers when
administered to patients with angiogenesis-dependent cancers.
26. The method of claim 25, wherein at least one of angiostatin and
endostatin is a recombinantly produced protein, and wherein the
recombinantly produced protein is administered in its un-refolded
form.
27. The method of claim 25, wherein the recombinantly produced
protein provides a sustained release of the protein for a period of
at least 8 hours.
28. A method of birth control comprising, administering to a female
an amount of the endostatin of claim 1 sufficient to prevent embryo
implantation.
29. The method of claim 28, wherein the endostatin is recombinantly
produced protein, and wherein the recombinantly produced protein is
administered in its un-refolded form.
30. The method of claim 29, wherein the recombinantly produced
protein provides a sustained release of the protein for a period of
at least 8 hours.
31. A composition comprising, angiostatin combined with the
endostatin of claim 1, wherein the angiostatin and the endostatin
are provided in amounts such that the composition is capable of
effectively regressing the tumor mass of angiogenesis-dependent
tumors when administered to patients with an angiogenesis-dependent
tumor.
32. A method of making endostatin protein comprising, recombinantly
expressing a protein that is approximately 18 kDa as determined by
non-reduced gel electrophoresis, and approximately 20 kDa as
determined by reduced gel electrophoresis, and which has
substantial sequence homology to endostatin, the protein being
further characterized by its ability to specifically inhibit
proliferating cultured endothelial cells.
33. The method of claim 32, wherein the endostatin protein is
produced in an expression system selected from the group consisting
of bacterial expression systems, yeast expression systems and
insect viral expression systems.
Description
CROSS REFERENCE TO PRIOR RELATED CASES
[0001] This application claims priority to provisional application
Serial No. 60/005,835 filed Oct. 23, 1995; provisional application
Serial No. 60/023,070 filed Aug. 2, 1996; and provisional
application Serial No. 60/026,263 filed Sep. 17, 1996. Each of the
above-referenced applications is incorporated herein in its
entirety.
TECHNICAL FIELD
[0002] This application relates to a novel inhibitor of
angiogenesis useful for treating angiogenesis-related diseases,
such as angiogenesis-dependent cancer. The invention further
relates to a novel composition and method for curing
angiogenesis-dependent cancer. In addition, the present invention
relates to diagnostic assays and kits for endostatin measurement,
to histochemical kits for localization of endostatin, to molecular
probes to monitor endostatin biosynthesis, to antibodies that are
specific for the endostatin, to the development of peptide agonists
and antagonists to the endostatin receptor, and to cytotoxic agents
linked to endostatin peptides.
BACKGROUND OF THE INVENTION
[0003] Several lines of direct evidence now suggest that
angiogenesis is essential for the growth and persistence of solid
tumors and their metastases (Folkman, 1989; Hori et al., 1991; Kim
et al., 1993; Millauer et al., 1994). To stimulate angiogenesis,
tumors upregulate their production of a variety of angiogenic
factors, including the fibroblast growth factors (FGF and BFGF)
(Kandel et al., 1991) and vascular endothelial cell growth
factor/vascular permeability factor (VEGF/VPF). However, many
malignant tumors also generate inhibitors of angiogenesis,
including angiostatin and thrombospondin (Chen et al., 1995; Good
et al., 1990; O'Reilly et al., 1994). It is postulated that the
angiogenic phenotype is the result of a net balance between these
positive and negative regulators of neovascularization (Good et
al., 1990; O'Reilly et al., 1994; Parangi et al., 1996; Rastinejad
et al., 1989). Several other endogenous inhibitors of angiogenesis
have been identified, although not all are associated with the
presence of a tumor. These include, platelet factor 4 (Gupta et
al., 1995; Maione et al., 1990), interferon-alpha,
interferon-inducible protein 10 (Angiolillo et al., 1995; Strieter
et al., 1995), which is induced by interleukin-12 and/or
interferon-gamma (Voest et al., 1995), gro-beta (Cao et al., 1995),
and the 16 kDa N-terminal fragment of prolactin (Clapp et al.,
1993). The only known angiogenesis inhibitor which specifically
inhibits endothelial cell proliferation is angiostatin (O'Reilly et
al. 1994).
[0004] Angiostatin is an approximately 38 kiloDalton (kDa) specific
inhibitor of endothelial cell proliferation. Angiostatin is an
internal fragment of plasminogen containing at least three of the
five kringles of plasminogen Angiostatin has been shown to reduce
tumor weight and to inhibit metastasis in certain tumor models.
(O'Reilly et al., 1994). As it is used hereinafter, the term
"angiostatin" refers to angiostatin as described above; peptide
fragments of angiostatin that have endothelial cell proliferation
inhibiting activity; and analogs of angiostatin that have
substantial sequence homology (as defined herein) to the amino acid
sequence of angiostatin, which have endothelial cell proliferation
inhibiting activity.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a novel protein inhibitor,
and method for its use. The protein is a potent and specific
inhibitor of endothelial proliferation and angiogenesis. Systemic
therapy with the inhibitor causes a nearly complete suppression of
tumor-induced angiogenesis, and it exhibits strong anti-tumor
activity.
[0006] The inhibitory protein has a molecular weight of
approximately 18,000 to approximately 20,000 Daltons (18 to 20 kDa)
and is capable of inhibiting endothelial cell proliferation in
cultured endothelial cells. The protein can be further
characterized by its preferred N-terminal amino acid sequence, the
first twenty (20) of which are as follows:
1 (SEQ ID NO:1) His Thr His Gln Asp Phe Gln Pro Val Leu 1 2 3 4 5 6
7 8 9 10 His Leu Val Ala Leu Asn Thr Pro Leu Ser 11 12 13 14 15 16
17 18 19 20
[0007] A preferred endothelial cell proliferation inhibitor of the
invention is a protein having the above-described characteristics,
and which can be isolated and purified from the murine
hemangioendothelioma cell line EOMA. This inhibitory protein has
been named endostatin.
[0008] The present invention provides methods and compositions for
treating diseases and processes mediated by undesired and
uncontrolled angiogenesis by administering to a human or animal
with the undesired angiogenesis a composition comprising a
substantially purified endostatin or endostatin derivative in a
dosage sufficient to inhibit angiogenesis. The present invention is
particularly useful for treating or for repressing the growth of
tumors. Administration of endostatin to a human or animal with
prevascularized metastasized tumors prevents the growth or
expansion of those tumors.
[0009] The present invention also includes diagnostic methods and
kits for detection and measurement of endostatin in biological
fluids and tissues, and for localization of endostatin in tissues.
The diagnostic method and kit can be in any configuration well
known to those of ordinary skill in the art. The present invention
also includes antibodies specific for the endostatin and antibodies
that inhibit the binding of antibodies specific for the endostatin.
These antibodies can be polyclonal antibodies or monoclonal
antibodies. The antibodies specific for endostatin can be used in
diagnostic kits to detect the presence and quantity of endostatin
which is diagnostic or prognostic for the occurrence or recurrence
of cancer or other disease mediated by angiogenesis. Antibodies
specific for endostatin may also be administered to a human or
animal to passively immunize the human or animal against
endostatin, thereby reducing angiogenic inhibition.
[0010] The present invention also includes diagnostic methods and
kits for detecting the presence and quantity of antibodies that
bind endostatin in body fluids. The diagnostic method and kit can
be in any configuration well known to those of ordinary skill in
the art.
[0011] The present invention also includes endostatin peptide
fragments that can be labeled isotopically or with other molecules
or proteins for use in the detection and visualization of
endostatin binding sites with state of the art techniques,
including, but not limited to, positron emission tomography,
autoradiography, flow cytometry, radioreceptor binding assays, and
immunohistochemistry.
[0012] These endostatin peptides also act as agonists and
antagonists at the endostatin receptor, thereby enhancing or
blocking the biological activity of endostatin. Such peptides are
used in the isolation of the endostatin receptor.
[0013] The present invention also includes endostatin, endostatin
fragments, endostatin antisera, or endostatin receptor agonists and
antagonists linked to cytotoxic agents for therapeutic and research
applications.
[0014] The present invention includes molecular probes for the
ribonucleic acid and deoxyribonucleic acid involved in
transcription and translation of endostatin. These molecular probes
provide means to detect and measure endostatin biosynthesis in
tissues and cells.
[0015] A surprising discovery is that various forms of recombinant
endostatin protein can serve as sustained release anti-angiogenesis
compounds when administered to a tumor-bearing animal. A preferred
form of the sustained release compound is un-refolded recombinantly
produced endostatin.
[0016] Additionally, the present invention encompasses nucleic acid
sequences comprising corresponding nucleotide codons that code for
the above disclosed amino acid sequence and for endostatin and
endothelial cell proliferation inhibiting peptide fragments
thereof.
[0017] The present invention also relates to methods of using the
endostatin protein and peptide fragments, corresponding nucleic
acid sequences, and antibodies that bind specifically to the
inhibitor and its peptides, to diagnose endothelial cell-related
diseases and disorders.
[0018] The invention further encompasses a method for identifying
receptors specific for endostatin, and the receptor molecules
identified and isolated thereby.
[0019] The invention also relates to a method for identifying novel
enzymes capable of releasing endostatin from collagen type XVIII,
and other molecules containing an endostatin amino acid sequence,
and peptides thereof. Such endostatin producing enzymes are also an
aspect of the invention.
[0020] An important medical method is a new form of birth control,
wherein an effective amount of endostatin is administered to a
female such that uterine endometrial vascularization is inhibited
and embryo implantation cannot occur, or be sustained.
[0021] A particularly important aspect of the present invention is
the discovery of a novel and effective method for treating
angiogenesis-related diseases, particularly angiogenesis-dependent
cancer, in patients, and for curing angiogenesis-dependent cancer
in patients. The method unexpectedly provides the medically
important result of inhibition of tumor growth and reduction of
tumor mass. The method relates to the co-administration of the
endostatin of the present invention and another anti-angiogenesis
compound, preferrably angiostatin. Accordingly, the present
invention also includes formulations containing endostatin, and
optionally angiostatin, which are effective for treating or curing
angiogenesis-dependent cancers.
[0022] Accordingly, it is an object of the present invention to
provide a composition comprising an endostatin protein.
[0023] It is another object of the present invention to provide a
method of treating diseases and processes that are mediated by
angiogenesis.
[0024] It is yet another object of the present invention to provide
a diagnostic or prognostic method and kit for detecting the
presence and amount of endostatin in a body fluid or tissue.
[0025] It is yet another object of the present invention to provide
a method and composition for treating diseases and processes that
are mediated by angiogenesis including, but not limited to,
hemangioma, solid tumors, leukemia, metastasis, telangiectasia
psoriasis scleroderma, pyogenic granuloma, myocardial angiogenesis,
plaque neovascularization, corornay collaterals, cerebral
collaterals, arteriovenous malformations, ischemic limb
angiogenesis, corneal diseases, rubeosis, neovascular glaucoma,
diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic
neovascularization, macular degeneration, wound healing, peptic
ulcer, fractures, keloids, vasculogenesis, hematopoiesis,
ovulation, menstruation, and placentation.
[0026] It is another object of the present invention to provide a
composition for treating or repressing the growth of a cancer.
[0027] It is an object of present invention to provide a method for
detecting and quantifying the presence of an antibody specific for
an endostatin in a body fluid.
[0028] Still another object of the present invention is to provide
a composition consisting of antibodies to endostatin that are
selective for specific regions of the endostatin molecule.
[0029] It is another object of the present invention to provide a
method for the detection or prognosis of cancer.
[0030] It is another object of the present invention to provide a
composition for use in visualizing and quantitating sites of
endostatin binding in vivo and in vitro.
[0031] It is yet another object of the present invention to provide
a composition for use in detection and quantification of endostatin
biosynthesis.
[0032] It is yet another object of the present invention to provide
a therapy for cancer that has minimal side effects.
[0033] Still another object of the present invention is to provide
a composition comprising endostatin or an endostatin peptide linked
to a cytotoxic agent for treating or repressing the growth of a
cancer.
[0034] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1: Inhibition of Capillary Endothelial Cell
Proliferation by Conditioned Media from EOMA Cells.
[0036] Conditioned media collected from confluent EOMA cells or
base media was applied to bovine capillary endothelial cells with 1
ng/ml bFGF in a 72 hour proliferation assay. Endothelial cell
proliferation was inhibited by the EOMA conditioned media. Each bar
represents the mean.+-.SEM.
[0037] FIG. 2: Purification of an Inhibitor of Endothelial
Proliferation from EOMA Conditioned Media.
[0038] Conditioned media collected from confluent EOMA cells was
fractionated on a heparin sepharose column. Endothelial
proliferation inhibiting activity eluted at approximately 0.8M
NaCl.
[0039] FIG. 3: Purification of an Inhibitor of Endothelial
Proliferation by Gel Filtration.
[0040] Purified inhibitor from heparin sepharose column
chromatography was applied to a gel filtration column and eluted as
a single peak.
[0041] FIG. 4: Purification of Inhibitor of Endothelial Cell
Proliferation by Reversed Phase Column Chromatography.
[0042] Inhibitor purified by heparin sepharose and gel filtration
chromatography was applied to a reverse phase column. The inhibitor
eluted as a single band from the column at approximately 45% of the
acetonitrile.
[0043] FIG. 5: N-terminal Amino Acid Sequence of An Inhibitor of
Endothelial Cell Proliferation.
[0044] The N-terminal sequence of the purified inhibitor of
endothelial cell proliferation is shown in relation to a schematic
diagram of collagen type 18. The N-terminal sequence revealed
identity of the inhibitor to an approximately 20 kDa C-terminal
fragment (shown in solid shading) for collagen type XVIII. The open
boxes represent the collagenase domains of collagen type XVIII.
[0045] FIG. 6: Treatment of Lewis Lung Carcinoma With Recombinant
Mouse Endostatin Inhibitor.
[0046] Recombinant inhibitor produced in E. coli was administered
to mice seeded with Lewis lung carcinoma that had achieved a tumor
volume of approximately 150 mm.sup.3. The inhibitor was
administered at 20 mg/kg/day. Tumor mass regressed to
non-detectable levels after approximately 12 days of treatment.
[0047] FIG. 7: Systemic Therapy with Recombinant Endostatin
Regresses Lewis Lung Carcinoma Primary Tumors.
[0048] (A) Mice were implanted subcutaneously on the dorsum with
Lewis lung carcinoma cells. Systemic therapy with recombinant mouse
endostatin (20 mg/kg/day) was begun when tumors were approximately
200 mm.sup.3 (1% of body weight). Tumors in the mice treated with
endostatin inhibitor rapidly regressed and were inhibited by
>99% relative to saline-treated controls. Each point represents
mean.+-.SEM for 5 mice. The experiment was repeated with comparable
results.
[0049] (B) Representative treated and untreated tumor-bearing mice
after 11 days of systemic therapy with endostatin. Saline-treated
mice (right) had rapidly growing red tumors with ulcerated
surfaces. Endostatin treated mice (left) had small pale residual
tumors (arrow).
[0050] (C) Residual disease in endostatin treated mice. Three of
the five endostatin treated mice were sacrificed after 16 days of
therapy. Autopsy revealed small white residual tumors at the site
of the original primary implantation (arrows).
[0051] FIG. 8: Treatment of Murine T241 Fibrosarcoma with
Recombinant Mouse Endostatin from E. coli
[0052] Mice were seated with T241 Fibrosarcoma cells. Control mice
were treated with saline. Experimental mice were treated with 20
mg/kg/day of recombinant mouse Endostatin directed from E.
coli.
[0053] FIG. 9: Treatment of Murine B16F10 Melanoma with Recombinant
Mouse Endostatin from E. coli
[0054] Mice were seated with Murine B16F10 melanoma cells. Control
animals were treated with saline. Experimental animals were treated
with 20 mg/kg/day of recombinant mouse Endostatin direct from E.
coli.
[0055] FIG. 10: Treatment of EOMA Hemangioendothelioma with
Recombinant Mouse Endostatin from E. coli
[0056] Mice were seated with EOMA hemangioendothelioma cells.
Control animals were treated with saline. Experimental animals were
treated with 20 mg/kg/day of Recombinant Mouse Endostatin direct
from E. coli.
[0057] FIG. 11: Treatment of Lewis Lung Carcinoma with Recombinant
Mouse or Human Endostatin direct from E. coli.
[0058] Mice were seated with Lewis Lung Carcinoma cells. Control
animals were treated with saline. Experimental animals were treated
with Recombinant Endostatin derived from the mouse sequence or
Recombinant Endostatin direct from the human sequence, wherein both
Endostatin are produced recombinantly in the E. coli. Mouse
Endostatin was administered at either 20 mg/kg/day or 2.5
mg/kg/day, and Human Endostatin was administered at 20
mg/kg/day.
[0059] FIG. 12: Endostatin Results in an Inhibition of Angiogenesis
and an Increase in Apoptosis of Lewis Lung Carcinoma Primary
Tumors.
[0060] Histological sections of tumors from saline versus
endostatin treated mice implanted with Lewis lung carcinomas were
analyzed for proliferation (PCNA), apoptosis (TUNEL), and
angiogenesis (vWF). There was no significant difference in the
proliferative index of tumor cells (A) in treated versus untreated
tumors. In contrast, the apoptotic index of the tumor cells (B)
increased 8-fold (p <0.001) in the endostatin treated mice.
Vessel density (C) was determined by counting the number of
capillary blood vessels per high-power field (HPF) in sections
stained with antibodies against vWF. Angiogenesis was almost
completely suppressed in the residual microscopic tumors of the
endostatin treated mice (p<0.001).
[0061] FIG. 13: Cycle Dormancy Therapy of Lewis Lung Carcinoma with
Recombinant Mouse Endostatin From E. Coli.
[0062] Mice were implanted subcutaneously on the dorsum with Lewis
lung carcinoma cells. Systemic therapy with recombinant mouse
inhibitor (endostatin), administered at a dose of 20 mg/kg/day, was
begun when tumors were approximately 200 mm.sup.3 (1% of body
weight). Tumors in the mice treated with the endostatin inhibitor
rapidly regressed to essentially non-detectable levels after
approximately 15 days of therapy. When treatment was terminated the
tumor volume increased rapidly and was subsequently treatable to
the same non-detectable levels by reinitiation of treatment. The
peaks and valleys in the figure show the cycling effect of
inhibition with endostatin.
[0063] FIG. 14: Combination Therapy with Recombinant Mouse
Angiostatin and Endostatin from E. Coli.
[0064] Mice were implanted subcutaneously on the dorsum with Lewis
lung carcinoma cells. Systemic therapy with a combination of
recombinant mouse endostatin (20 mg/kg/day) and recombinant mouse
angiostatin (20 mg/kg/day) was begun when tumors were approximately
300 mm.sup.3. Tumors in the mice treated with the combination
therapy rapidly regressed to essentially non-detectable level in
about 15 days. Importantly, the regressed tumors remained dormant
and did not increase in size or mass after treatment was stopped.
This is an unexpected result of substantial medical
significance.
DETAILED DESCRIPTION OF THE INVENTION
[0065] Applicants have discovered a new class of protein molecules
that have the ability to inhibit endothelial proliferation when
added to proliferating endothelial cells in vitro. Accordingly,
these protein molecules have been functionally defined as
endostatins, however, it is to be understood that this functional
definition is no way limits the bioactivity of endostatins to
inhibition of endothelial cell growth in vitro or in vivo. Many
other functions of endostatins are likely.
[0066] The term "endostatin" refers to a protein that is preferably
18 kDa to 20 kDa in size as determined by non-reduced and reduced
gel electrophoresis, respectively. The term endostatin also
includes precursor forms of the 18 kDa to 20 kDa protein. The term
endostatin also includes fragments of the 18 kDa to 20 kDa protein
and modified proteins and peptides that have a substantially
similar amino acid sequence, and which are capable inhibiting
proliferation of endothelial cells. For example, silent
substitutions of amino acids, wherein the replacement of an amino
acid with a structurally or chemically similar amino acid does not
significantly alter the structure, conformation or activity of the
protein, is well known in the art. Such silent substitutions are
intended to fall within the scope of the appended claims.
[0067] It will be appreciated that the term "endostatin" includes
shortened proteins or peptides wherein one or more amino acid is
removed from either or both ends of endostatin, or from an internal
region of the protein, yet the resulting molecule retains
endothelial proliferation inhibiting activity. The term
"endostatin" also includes lengthened proteins or peptides wherein
one or more amino acid is added to either or both ends of
endostatin, or to an internal location in the protein, yet the
resulting molecule retains endothelial proliferation inhibiting
activity. Such molecules, for example with tyrosine added in the
first position are useful for labelling such as radioiodination
with .sup.125iodine for use in assays. Labelling with other
radioisotopes may be useful in providing a molecular tool for
destroying the target cell containing endostatin receptors. Other
labelling with molecules such as ricin may provide a mechanism for
destroying cells with endostatin receptors.
[0068] "Substantial sequence homology" means at least approximately
70% homology between amino acid residue sequence in the endostatin
analog sequence and that of endostatin, preferably at least
approximately 80% homology, more preferably at least approximately
90% homology.
[0069] Also included in the definition of the term endostatin are
modifications of the endostatin protein, its subunits and peptide
fragments. Such modifications include substitutions of naturally
occurring amino acids at specific sites with other molecules,
including but not limited to naturally and non-naturally occurring
amino acids. Such substitutions may modify the bioactivity of
endostatin and produce biological or pharmacological agonists or
antagonists. The term endostatin also includes an N terminal
fragment of endostatin consisting of the sequence of the first 20 N
terminal amino acids which is shown in SEQ ID NO:1 and is shown in
Table 1. This sequence of the first 20 N terminal amino acids
corresponds to a C-terminal fragment of newly identified collagen
type XVIII.
2TABLE 1 Amino Acid Residue Abbreviation 1 HIS H 2 THR T 3 HIS H 4
GLN Q 5 ASP D 6 PHE F 7 GLN Q 8 PRO P 9 VAL V 10 LEU L 11 HIS H 12
LEU L 13 VAL V 14 ALA A 15 LEU L 16 ASN N 17 THR T 18 PRO P 19 LEU
L 20 SER S
[0070] Table 1 shows the correspondence of 3 letter and 1 amino
acid designations.
[0071] The N-terminal amino acid sequence of endostatin to an
internal 20 amino acid peptide fragment found in agen alpha 1 type
XVIII starting at amino acid 1105 and mino acid 1124. The
N-terminal amino acid sequence of the so corresponds to an internal
20 amino acid peptide fragment man collagen alpha 1 type XVIII
starting at amino acid 1132 at amino acid 1151.
[0072] Endostatin can be isolated from murine hemangioedothelioma
EOMA. Endostatin may be produced from recombinant sources, from
genetically altered cells implanted into animals, from tumors, and
from cell cultures as well as other sources. It is anticipated that
endostatin is made in cells of the nervous system. Endostatin can
be isolated from body fluids including, but not limited to, serum,
urine and ascites, or synthesized by chemical or biological methods
(e.g. cell culture, recombinant gene expression, peptide synthesis,
and in vitro enzymatic catalysis of precursor molecules to yield
active endostatin). Recombinant techniques include gene
amplification from DNA sources using the polymerase chain reaction
(PCR), and gene amplification from RNA sources using reverse
transcriptase/PCR.
[0073] Endostatin specifically and reversibly inhibits endothelial
cell proliferation. The inhibitor protein molecules of the
invention are useful as a birth control drug, and for treating
angiogenesis-related diseases, particularly angiogenesis-dependent
cancers and tumors. The protein molecules are also useful for
curing angiogenesis-dependent cancers and tumors. The unexpected
and surprising ability of these novel compounds to treat and cure
angiogenesis-dependent cancers and tumors answers a long felt
unfulfilled need in the medical arts, and provides an important
benefit to mankind.
[0074] Important terms that are used herein are defined as follows.
"Cancer" means angiogenesis-dependent cancers and tumors, i.e.
tumors that require for their growth (expansion in volume and/or
mass) an increase in the number and density of the blood vessels
supplying then with blood. "Regression" refers to the reduction of
tumor mass and size.
[0075] The endothelial proliferation inhibiting proteins of the
present invention can be made by automated protein synthesis
methodologies well known to one skilled in the art. Alternatively,
endothelial proliferation inhibiting proteins, or endostatins, of
the present invention may be isolated from larger known proteins,
such as human alpha 1 type XVIII collagen and mouse alpha 1 type
XVIII collagen, proteins that share a common or similar N-terminal
amino acid sequence. Examples of other potential endostatin source
materials having similar N-terminal amino acid sequences include
Bos taurus pregastric esterase, human alpha 1 type 15 collagen,
NAD-dependent formate dehydrogenase (EC 1.2.1.2) derived from
Pseudomonas sp., s11459 hexon protein of bovine adenovirus type 3,
CELF21D12 2 F21d12.3 Caenorhabditis elegans gene product, VAL1 TGMV
AL1 protein derived from tomato golden mosaic virus, s01730 hexon
protein derived from human adenovirus 12, Saccharomyces cerevisiae.
For example, peptides closely related to endostatin may be derived
from BOVMPE 1 pregastric esterase (BOS TAURUS) gene sequence
corresponding to amino acids 502 to 521, and collagen alpha 1 type
15 from humans beginning at amino acid 316 ending at 335.
[0076] Proteins and peptides derived from these and other sources,
including manual or automated protein synthesis, may be quickly and
easily tested for endothelial proliferation inhibiting activity
using a biological activity assay such as the bovine capillary
endothelial cell proliferation assay. Other bioassays for
inhibiting activity include the chick CAM assay, the mouse corneal
assay, and the effect of administering isolated or synthesized
proteins on implanted tumors. The chick CAM assay is described by
O'Reilly, et al. in "Angiogenic Regulation of Metastatic Growth"
Cell, vol. 79 (2), Oct. 21, 1994, pp. 315-328, which is hereby
incorporated by reference in its entirety. Briefly, 3 day old
chicken embryos with intact yolks are separated from the egg and
placed in a petri dish. After 3 days of incubation a
methylcellulose disc containing the protein to be tested is applied
to the CAM of individual embryos. After 48 hours of incubation, the
embryos and CAMs are observed to determine whether endothelial
growth has been inhibited. The mouse corneal assay involves
implanting a growth factor-containing pellet, along with another
pellet containing the suspected endothelial growth inhibitor, in
the cornea of a mouse and observing the pattern of capillaries that
are elaborated in the cornea.
[0077] Applicants' invention also encompasses nucleic acid
sequences that correspond to and code for the endothelial
proliferation-inhibiting protein molecules of the invention, and to
monoclonal and polyclonal antibodies that bind specifically to such
protein molecules. The biologically active protein molecules,
nucleic acid sequences corresponding to the proteins, and
antibodies that bind specifically to the proteins of the present
invention are useful for modulating endothelial processes in vivo,
and for diagnosing and treating endothelial cell-related diseases,
for example by gene therapy.
[0078] Nucleic acid sequences that correspond to, and code for,
endostatin and endostatin analogs can be prepared based upon the
knowledge of the amino acid sequence, and the art recognized
correspondence between codons (sequences of three nucleic acid
bases), and amino acids. Because of the degeneracy of the genetic
code, wherein the third base in a codon may vary yet still code for
the same amino acid, many different possible coding nucleic acid
sequences are derivable for any particular protein or peptide
fragment.
[0079] Nucleic acid sequences are synthesized using automated
systems well known in the art. Either the entire sequence may be
synthesized or a series of smaller oligonucleotides are made and
subsequently ligated together to yield the full length sequence.
Alternatively, the nucleic acid sequence may be derived from a gene
bank using oligonucleotides probes designed based on the N-terminal
amino acid sequence and well known techniques for cloning genetic
material.
[0080] The present invention also includes the detection of
endostatin in body fluids and tissues for the purpose of diagnosis
or prognosis of angiogenesis-mediated diseases such as cancer. The
present invention also includes the detection of endostatin binding
sites and receptors in cells and tissues. The present invention
also includes methods of treating or preventing angiogenic diseases
and processes including, but not limited to, arthritis and tumors
by stimulating the production of endostatin, and/or by
administering substantially purified endostatin, or endostatin
agonists or antagonists, and/or endostatin antisera or antisera
directed against endostatin antisera to a patient. Additional
treatment methods include administration of endostatin, endostatin
fragments, endostatin antisera, or endostatin receptor agonists and
antagonists linked to cytotoxic agents. It is to be understood that
the endostatin can be animal or human in origin. Endostatin can
also be produced synthetically by chemical reaction or by
recombinant techniques in conjunction with expression systems.
Endostatin can also be produced by enzymatically cleaving different
molecules, including endostatin precursors, containing sequence
homology or identity with segments of endostatin to generate
peptides having anti-angiogenesis activity.
[0081] Passive antibody therapy using antibodies that specifically
bind endostatin can be employed to modulate endothelial-dependent
processes such as reproduction, development, and wound healing and
tissue repair. In addition, antisera directed to the Fab regions of
endostatin antibodies can be administered to block the ability of
endogenous endostatin antisera to bind endostatin.
[0082] Antibodies specific for endostatin and endostatin analogs
are made according to techniques and protocols well known in the
art. The antibodies may be either polyclonal or monoclonal. The
antibodies are utilized in well know immunoassay formats, such as
competitive and non-competitive immunoassays, including ELISA,
sandwich immunoassays and radioimmunoassays (RIAs), to determine
the presence or absence of the endothelial proliferation inhibitors
of the present invention in body fluids. Examples of body fluids
include but are not limited to blood, serum, peritoneal fluid,
pleural fluid, cerebrospinal fluid, uterine fluid, saliva, and
mucus.
[0083] The proteins, nucleic acid sequences and antibodies of the
present invention are useful for diagnosing and treating
endothelial cell-related diseases and disorders. A particularly
important endothelial cell process is angiogenesis, the formation
of blood vessels. Angiogenesis-related diseases may be diagnosed
and treated using the endothelial cell proliferation inhibiting
proteins of the present invention. Angiogenesis-related diseases
include, but are not limited to, angiogenesis-dependent cancer,
including, for example, solid tumors, blood born tumors such as
leukemias, and tumor metastases; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis;
Osler-Webber Syndrome; myocardial angiogenesis; plaque
neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; and wound granulation. The endothelial cell
proliferation inhibiting proteins of the present invention are
useful in the treatment of disease of excessive or abnormal
stimulation of endothelial cells. These diseases include, but are
not limited to, intestinal adhesions, atherosclerosis, scleroderma,
and hypertrophic scars, i.e., keloids. They are also useful in the
treatment of diseases that have angiogenesis as a pathologic
consequence such as cat scratch disease (Rochele minalia quintosa)
and ulcers (Helobacter pylori).
[0084] The endothelial cell proliferation inhibiting proteins can
be used as a birth control agent by reducing or preventing uterine
vascularization required for embryo implantation. Thus, the present
invention provides an effective birth control method when an amount
of the inhibitory protein sufficient to prevent embryo implantation
is administered to a female. In one aspect of the birth control
method, an amount of the inhibiting protein sufficient to block
embryo implantation is administered before or after intercourse and
fertilization have occured, thus providing an effective method of
birth control, possible a "morning after" method. While not wanting
to be bound by this statement, it is believed that inhibition of
vascularization of the uterine endometrium interferes with
implantation of the blastocyst. Similar inhibition of
vascularization of the mucosa of the uterine tube interferes with
implantation of the blastocyst, preventing occurrence of a tubal
pregnancy. Administration methods may include, but are not limited
to, pills, injections (intravenous, subcutaneous, intramuscular),
suppositories, vaginal sponges, vaginal tampons, and intrauterine
devices. It is also believed that endostatin administration will
interfere with normal enhanced vascularization of the placenta, and
also with the development of vessels within a successfully
implanted blastocyst and developing embryo and fetus.
[0085] Conversely, blockade of endostatin receptors with endostatin
analogs which act as receptor antagonists may promote
endothelialization and vascularization. Such effects may be
desirable in situations of inadequate vascularization of the
uterine endometrium and associated infertilty, wound repair,
healing of cuts and incisions, treatment of vascular problems in
diabetics, especially retinal and peripheral vessels, promotion of
vascularization in transplanted tissue including muscle and skin,
promotion of vascularization of cardiac muscle especially following
transplantation of a heart or heart tissue and after bypass
surgery, promotion of vascularization of solid and relatively
avascular tumors for enhanced cytotoxin delivery, and enhancement
of blood flow to the nervous system, including but not limited to
the cerebral cortex and spinal cord.
[0086] A surprising discovery is that un-refolded and non-soluble
recombinant endostatin is also a potent anti-angiogenesis compound
which serves as a sustained release depot when administered to a
patient.
[0087] The present invention also relates to methods of using
endostatin and endothelial cell proliferation inhibiting peptide
fragments of endostatin, nucleic acid sequences corresponding to
endostatin and active peptide fragments thereof, and antibodies
that bind specifically to endostatin and its peptides, to diagnose
endothelial cell-related diseases and disorders.
[0088] The invention further encompasses a method for identifying
endostatin-specific receptors, and the receptor molecules
identified and isolated thereby.
[0089] The present invention also provides a method for
quantitation of endostatin receptors.
[0090] A particularly important aspect of the present invention is
the discovery of a novel and effective method for treating and
curing angiogenesis-dependent cancer in patients. It was
unexpectedly found that the co-administration of endostatin and
angiostatin in an amount sufficient to inhibit tumor growth and
cause sustainable regression of tumor mass to microscopic size
cures angiogenesis-dependent cancer. Accordingly, the present
invention also includes formulations effective for treating or
curing angiogenesis-dependent cancers and tumors.
[0091] More particularly, recombinant mouse endostatin, from insect
cells or E. coli, potently inhibits angiogenesis and the growth of
metastases and primary tumors. In a novel method of sustained
release, the E. coli-derived recombinant endostatin was
administered as an un-refolded suspension in an amount sufficient
to inhibit angiogenesis, thereby inhibiting tumor growth. Tumor
mass was reduced when recombinant endostatin was administered in an
amount sufficient to cause regression of the tumor. Primary tumors
of 1-2% of body weight regressed by greater than 150-fold to become
microscopic dormant lesions when treated by endostatin.
Immunohistochemical analysis of the dormant tumors revealed blocked
angiogenesis accompanied by high proliferation of the tumor cells
balanced by a high rate of tumor cell apoptosis. There was no
evidence of toxicity in any of the mice treated with
endostatin.
[0092] It is contemplated as part of the present invention that
endostatin can be isolated from a body fluid such as blood or urine
of patients or the endostatin can be produced by recombinant DNA
methods or synthetic peptide chemical methods that are well known
to those of ordinary skill in the art. Protein purification methods
are well known in the art and a specific example of a method for
purifying endostatin, and assaying for inhibitor activity is
provided in the examples below. Isolation. of human endogenous
endostatin is accomplished using similar techniques.
[0093] One example of a method of producing endostatin using
recombinant DNA techniques entails the steps of (1) identifying and
purifying an endostatin as discussed above, and as more fully
described below, (2) determining the N-terminal amino acid sequence
of the purified inhibitor, (3) synthetically generating a DNA
oligonucleotide probe that corresponds to the N-terminal amino acid
sequence, (4) generating a DNA gene bank from human or other
mammalian DNA, (5) probing the gene bank with the DNA
oligonucleotide probe, (6) selecting clones that hybridize to the
oligonucleotide, (7) isolating the inhibitor gene from the clone,
(8) inserting the gene into an appropriate vector such as an
expression vector, (9) inserting the gene-containing vector into a
microorganism or other expression system capable of expressing the
inhibitor gene, and (10) isolating the recombinantly produced
inhibitor. The above techniques are more fully described in
laboratory manuals such as "Molecular Cloning: A Laboratory Manual"
Second Edition by Sambrook et al., Cold Spring Harbor Press,
1989.
[0094] The gene for endostatin may also be isolated from cells or
tissue (such as tumor cells) that express high levels of endostatin
by (1) isolating messenger RNA from the tissue, (2) using reverse
transcriptase to generate the corresponding DNA sequence and then
(3) using PCR with the appropriate primers to amplify the DNA
sequence coding for the active endostatin amino acid sequence.
[0095] Yet another method of producing endostatin, or biologically
active fragments thereof, is by peptide synthesis. Once a
biologically active fragment of an endostatin is found using the
assay system described more fully below, it can be sequenced, for
example by automated peptide sequencing methods. Alternatively,
once the gene or DNA sequence which codes for endostatin is
isolated, for example by the methods described above, the DNA
sequence can be determined, which in turn provides information
regarding the amino acid sequence. Thus, if the biologically active
fragment is generated by specific methods, such as tryptic digests,
or if the fragment is N-terminal sequenced, the remaining amino
acid sequence can be determined from the corresponding DNA
sequence.
[0096] Once the amino acid sequence of the peptide is known, , for
example the N-terminal 20 amino acids, the fragment can be
synthesized by techniques well known in the art, as exemplified by
"Solid Phase Peptide Synthesis: A Practical Approach" E. Atherton
and R. C. Sheppard, IRL Press, Oxford England. Similarly, multiple
fragments can be synthesized which are subsequently linked together
to form larger fragments. These synthetic peptide fragments can
also be made with amino acid substitutions at specific locations in
order to test for agonistic and antagonistic activity in vitro and
in vivo. Peptide fragments that possess high affinity binding to
tissues can be used to isolate the endostatin receptor on affinity
columns. Isolation and purification of the endostatin receptor is a
fundamental step towards elucidating the mechanism of action of
endostatin. This facilitates development of drugs to modulate the
activity of the endostatin receptor, the final pathway to
biological activity. Isolation of the receptor enables the
construction of nucleotide probes to monitor the location and
synthesis of the receptor, using in situ and solution hybridization
technology.
[0097] Endostatin is effective in treating diseases or processes
that are mediated by, or involve, angiogenesis. The present
invention includes the method of treating an angiogenesis mediated
disease with an effective amount of endostatin or endostatin
agonists and antagonists. The angiogenesis mediated diseases
include, but are not limited to, solid tumors; blood born tumors
such as leukemias; tumor metastasis; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular
angiogenic. diseases, for example, diabetic retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft
rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis;
Osler-Webber Syndrome; myocardial angiogenesis; plaque
neovascularization; telangiectasia; hemophiliac joints;
angiofibroma; and wound granulation. Endostatin is useful in the
treatment of disease of excessive or abnormal stimulation of
endothelial cells. These diseases include, but are not limited to,
intestinal adhesions, atherosclerosis, scleroderma, and
hypertrophic scars, i.e., keloids. Endostatin can be used as a
birth control agent by preventing vascularization required for
blastocyst implantation and for development of the placenta, the
blastcyst, the embryo and the fetus.
[0098] The synthetic peptide fragments of endostatin have a variety
of uses. The peptide that binds to the endostatin receptor with
high specificity and avidity is radiolabeled and employed for
visualization and quantitation of binding sites using
autoradiographic and membrane binding techniques. This application
provides important diagnostic and research tools. Knowledge of the
binding properties of the endostatin receptor facilitates
investigation of the transduction mechanisms linked to the
receptor.
[0099] In addition, labeling these peptides with short lived
isotopes enables visualization of receptor binding sites in vivo
using positron emission tomography or other modern radiographic
techniques in order to locate tumors with endostatin binding
sites.
[0100] Systematic substitution of amino acids within these
synthesized peptides yields high affinity peptide agonists and
antagonists to the endostatin receptor that enhance or diminish
endostatin binding to its receptor. Such agonists are used to
suppress the growth of micrometastases, thereby- limiting the
spread of cancer. Antagonists to endostatin are applied in
situations of inadequate vascularization, to block the inhibitory
effects of angiostatin and possibly promote angiogenesis. This
treatment may have therapeutic effects to promote wound healing in
diabetics.
[0101] Endostatin peptides are employed to develop affinity columns
for isolation of the endostatin receptor from cultured tumor cells.
Isolation and purification of the endostatin receptor is followed
by amino acid sequencing. Next, nucleotide probes are developed for
insertion into vectors for expression of the receptor. These
techniques are well known to those skilled in the art. Transfection
of the.endostatin receptor into tumor cells enhances the
responsiveness of these cells to endogenous or exogenous endostatin
and thereby decreasing the rate of metastatic growth.
[0102] Cytotoxic agents, such as ricin, are linked to endostatin,
and high affinity endostatin peptide fragments, thereby providing a
tool for destruction of cells that bind endostatin. These cells may
be found- in many locations, including but not limited to,
micrometastases and primary tumors. Peptides linked to cytotoxic
agents are infused in a manner designed to maximize delivery to the
desired location. For example, ricin-linked high affinity
endostatin fragments are delivered through a cannula into vessels
supplying the target site or directly into the target. Such agents
are also delivered in a controlled manner through osmotic pumps
coupled to infusion cannulae. A combination of endostatin
antagonists may be co-applied with stimulators of angiogenesis to
increase vascularization of tissue. This therapeutic regimen
provides an effective means of destroying metastatic cancer.
[0103] According to the present invention, endostatin may be used
in combination with other compositions and procedures for the
treatment of diseases. For example, a tumor may be treated
conventionally with surgery, radiation or chemotherapy combined
with endostatin and then endostatin may be subsequently
administered to the patient to extend the dormancy of
micrometastases and to stabilize any residual primary tumor.
[0104] The endostatin of the present invention also can be used to
generate antibodies that are specific for the inhibitor. The
antibodies can be either polyclonal antibodies or monoclonal
antibodies. These antibodies that specifically bind to the
endostatin can be used in diagnostic methods and kits that are well
known to those of ordinary skill in the art to detect or quantify
the endostatin in a body fluid or tissue. Results from these tests
can be used to diagnose or predict the occurrence or recurrence of
a cancer and other angiogenesis mediated diseases.
[0105] The endostatin also can be used in a diagnostic method and
kit to detect and quantify antibodies capable of binding
endostatin. These kits would permit detection of circulating
endostatin-antibodies which indicates the spread of micrometastases
in the presence of endostatin secreted by primary tumors in situ.
Patients that have such circulating anti-endostatin antibodies may
be more likely to develop tumors and cancers, and may be more
likely to have recurrences of cancer after treatments or periods of
remission. The Fab fragments of these anti-endostatin antibodies
may be used as antigens to generate anti-endostatin Fab-fragment
antisera which can be used to neutralize the removal of circulating
endostatin by anti-endostatin antibodies.
[0106] Another aspect of the present invention is a method of
blocking the action of excess endogenous endostatin. This can be
done by passively immunizing a human or animal with antibodies
specific for the undesired endostatin in the system. This treatment
can be important in treating abnormal ovulation, menstruation and
placentation, and vasculogenesis. This provides a useful tool to
examine the effects of endostatin removal on metastatic processes.
The Fab fragment of endostatin antibodies contains the binding site
for endostatin. This fragment is isolated from endostatin
antibodies using techniques known to those skilled in the art. The
Fab fragments of endostatin antisera are used as antigens to
generate production of anti-Fab fragment serum. Infusion of this
antiserum against the Fab fragments of endostatin prevents
endostatin from binding to endostatin antibodies. Therapeutic
benefit is obtained by neutralizing endogenous anti-endostatin
antibodies by blocking the binding of endostatin to the Fab
fragments of anti-endostatin. The net effect of this treatment is
to facilitate the ability of endogenous circulating endostatin to
reach target cells, thereby decreasing the spread of metastases
[0107] It is to be understood that the present invention is
contemplated to include any derivatives of the endostatin that have
endothelial inhibitory activity. The present invention includes the
entire endostatin protein, derivatives of the endostatin protein
and biologically-active fragments of the endostatin protein. These
include proteins with endostatin activity that have amino acid
substitutions or have sugars or other molecules attached to amino
acid functional groups. The present invention also includes genes
that code for endostatin and the endostatin receptor, and to
proteins that are expressed by those genes.
[0108] The proteins and protein fragments with the endostatin
activity described above can be provided as isolated and
substantially purified proteins and protein fragments in
pharmaceutically acceptable formulations using formulation methods
known to those of ordinary skill in the art. These formulations can
be administered by standard routes. In general, the combinations
may be administered by the topical, transdermal, intraperitoneal,
intracranial, intracerebroventricular, intracerebral, intravaginal,
intrauterine, oral, rectal or parenteral (e.g., intravenous,
intraspinal, subcutaneous or intramuscular) route. In addition, the
endostatin may be incorporated into biodegradable polymers allowing
for sustained release of the compound, the polymers being implanted
in the vicinity of where drug delivery is desired, for example, at
the site of a tumor or implanted so that the endostatin is slowly
released systemically. Osmotic minipumps may also be used to
provide controlled delivery of high concentrations of endostatin
through cannulae to the site of interest, such as directly into a
metastatic growth or into the vascular supply to that tumor. The
biodegradable polymers and their use are described, for example, in
detail in Brem et al., J. Neurosurg. 74:441-446 (1991), which is
hereby incorporated by reference in its entirety.
[0109] The dosage of the endostatin of the present invention will
depend on the disease state or condition being treated and other
clinical factors such as weight and condition of the human or
animal and the route of administration of the compound. For
treating humans or animals, between approximately 0.5 mg/kilogram
to 500 mg/kilogram of the endostatin can be administered. A more
preferable range is 1 mg/kilogram to 100 mg/kilogram with the most
preferable range being from 2 mg/kilogram to 50 mg/kilogram.
Depending upon the half-life of the endostatin in the particular
animal or human, the endostatin can be administered between several
times per day to once a week. It is to be understood that the
present invention has application for both human and veterinary
use. The methods of the present invention contemplate single as
well as multiple administrations, given either simultaneously or
over an extended period of time.
[0110] The endostatin formulations include those suitable for oral,
rectal, ophthalmic (including intravitreal or intracameral), nasal,
topical (including buccal and sublingual), intrauterine, vaginal or
parenteral (including subcutaneous, intraperitoneal, intramuscular,
intravenous, intradermal, intracranial, intratracheal, and
epidural) administration. The endostatin formulations may
conveniently be presented in unit dosage form and may be prepared
by conventional pharmaceutical techniques. Such techniques include
the step of bringing into association the active ingredient and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0111] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0112] Preferred unit dosage formulations are those containing a
daily dose or unit, daily sub-dose, as herein above recited, or an
appropriate fraction thereof, of the administered ingredient. It
should be understood that in addition to the ingredients,
particularly mentioned above, the formulations of the present
invention may include other agents conventional in the art having
regard to the type of formulation in question.
[0113] Different peptide fragments of the intact endostatin
molecule can be synthesized for use in several applications
including, but not limited to the following; as antigens for the
development of specific antisera, as agonists and antagonists
active at endostatin binding sites, as peptides to be linked to
cytotoxic agents for targeted killing of cells that bind
endostatin. The amino acid sequences that comprise these peptides
are selected on the basis of their position on the exterior regions
of the molecule and are accessible for binding to antisera. The
amino and carboxyl termini of endostatin, as well as the mid-region
of the molecule are represented separately among the fragments to
be synthesized. The amino terminus distal to the 20th amino acid
and carboxyl termini of endostatin may contain or be modified to
contain tyrosine and lysine residues and are labeled with many
techniques. A tyrosine or lysine is added to fragments that do not
have these residues to facilitate labeling of reactive amino and
hydroxyl groups on the peptide. These peptide sequences are
compared to known sequences using sequence data banks to determine
potential sequence homologies. This information facilitates
elimination of sequences that exhibit a high degree of sequence
homology to other molecules, thereby enhancing the potential for
high specificity in the development of antisera, agonists and
antagonists to endostatin.
[0114] Peptides can be synthesized in a standard microchemical
facility and purity checked with HPLC and mass spectrophotometry.
Methods of peptide synthesis, HPLC purification and mass
spectrophotometry are commonly known to those skilled in these
arts.
[0115] Peptides and endostatin are also produced in recombinant E.
coli, as described below, or in insect or yeast expression systems,
and purified with column chromatography.
[0116] Endostatin and endostatin derived peptides can be coupled to
other molecules using standard methods. The amino terminus distal
to the 20th amino acid and the carboxyl terminus of endostatin may
both contain tyrosine and lysine residues and are isotopically and
nonisotopically labeled with many techniques, for example
radiolabeling using conventional techniques (tyrosine
residues-chloramine T, iodogen, lactoperoxidase; lysine
residues-Bolton-Hunter reagent). These coupling techniques are well
known to those skilled in the art. The coupling technique is chosen
on the basis of the functional groups available on the amino acids
including, but not limited to amino, sulfhydral, carboxyl, amide,
phenol, and imidazole. Various reagents used to effect these
couplings include among others, glutaraldehyde, diazotized
benzidine, carbodiimide, and p-benzoquinone.
[0117] Endostatin peptides are chemically coupled to isotopes,
enzymes, carrier proteins, cytotoxic agents, fluorescent molecules
and other compounds for a variety of applications. The efficiency
of the coupling reaction is determined using different techniques
appropriate for the specific reaction. For example,. radiolabeling
of an endostatin peptide or protein with .sup.125I is accomplished
using chloramine T and Na.sup.125I of high specific activity. The
reaction is terminated with sodium metabisulfite and the mixture is
desalted on disposable columns. The labeled peptide is eluted from
the column and fractions are collected. Aliquots are removed from
each fraction and radioactivity measured in a gamma counter. In
this manner, the unreacted Na.sup.125I is separated from the
labeled endostatin peptide. The peptide fractions with the highest
specific radioactivity are stored for subsequent use such as
analysis of the ability to bind to endostatin antisera.
[0118] Another application of peptide conjugation is for production
of polyclonal antisera. For example, endostatin peptides containing
lysine residues are linked to purified bovine serum albumin using
glutaraldehyde. The efficiency of the reaction is determined by
measuring the incorporation of radiolabeled peptide. Unreacted
glutaraldehyde and peptide are separated by dialysis. The conjugate
is stored for subsequent use.
[0119] Antiserum against endostatin can be generated. After peptide
synthesis and purification, both monoclonal and polyclonal antisera
are raised using established techniques known to those skilled in
the art. For example, polyclonal antisera may be raised in rabbits,
sheep, goats or other animals. Endostatin peptides conjugated to a
carrier molecule such as bovine serum albumin, or endostatin
itself, is combined with an adjuvant mixture, emulsified and
injected subcutaneously at multiple sites on the back, neck,
flanks, and sometimes in the footpads. Booster injections are made
at regular intervals, such as every 2 to 4 weeks. Blood samples are
obtained by venipuncture, for example using the marginal ear veins
after dilation, approximately 7 to 10 days after each injection.
The blood samples are allowed to clot overnight at 4.degree. C. and
are centrifuged at approximately 2400.times.g at 4 C..degree. for
about 30 minutes. The serum is removed, aliquoted, and stored at 4
C..degree. for immediate use or at -20 to -90.degree. C. for
subsequent analysis.
[0120] All serum samples from generation of polyclonal antisera or
media samples from production of monoclonal antisera are analyzed
for determination of titer. Titer is established through several
means, for example, using dot blots and density analysis, and also
with precipitation of radiolabeled peptide-antibody complexes using
protein A, secondary antisera, cold ethanol or charcoal-dextran
followed by activity measurement with a gamma counter. The highest
titer antisera are also purified on affinity columns which are
commercially available. Endostatin peptides are coupled to the gel
in the affinity column. Antiserum samples are passed through the
column and anti-endostatin antibodies remain bound to the column.
These antibodies are subsequently eluted, collected and evaluated
for determination of titer and specificity.
[0121] The highest titer endostatin antisera is tested to establish
the following; a) optimal antiserum dilution for highest specific
binding of the antigen and lowest non-specific binding, b) the
ability to bind increasing amounts of endostatin peptide in a
standard displacement curve, c) potential cross-reactivity with
related peptides and proteins, including endostatin related
species, d) ability to detect endostatin peptides in extracts of
plasma, urine, tissues, and in cell culture media.
[0122] Kits for measurement of endostatin are also contemplated as
part of the present invention. Antisera that possess the highest
titer and specificity and can detect endostatin peptides in
extracts of plasma, urine, tissues, and in cell culture media are
further examined to establish easy to use kits for rapid, reliable,
sensitive, and specific measurement and localization of
angiostatin. These assay kits include but are not limited to the
following techniques; competitive and non-competitive assays,
radioimmunoassay, bioluminescence and chemiluminescence assays,
fluorometric assays, sandwich assays, immunoradiometric assays, dot
blots, enzyme linked assays including ELISA, microtiter plates,
antibody coated strips or dipsticks for rapid monitoring of urine
or blood, and immunocytochemistry. For each kit the range,
sensitivity, precision, reliability, specificity and
reproducibility of the assay are established. Intraassay and
interassay variation is established at 20%, 50% and 80% points on
the standard curves of displacement or activity.
[0123] One example of an assay kit commonly used in research and in
the clinic is a radioimmunoassay (RIA) kit. An endostatin RIA is
illustrated below. After successful radioiodination and
purification of endostatin or an endostatin peptide, the antiserum
possessing the highest titer is added at several dilutions to tubes
containing a relatively constant amount of radioactivity, such as
10,000 cpm, in a suitable buffer system. Other tubes contain buffer
or preimmune serum to determine the non-specific binding. After
incubation at 4.degree. C. for 24 hours, protein A is added and the
tubes are vortexed, incubated at room temperature for 90 minutes,
and centrifuged at approximately 2000-2500.times.g at 4 C. to
precipitate the complexes of antibody bound to labeled antigen.The
supernatant is removed by aspiration and the radioactivity in the
pellets counted in a gamma counter. The antiserum dilution that
binds approximately 10 to 40% of the labeled peptide after
subtraction of the non-specific binding is further
characterized.
[0124] Next, a dilution range (approximately 0.1 pg to 10 ng) of
the endostatin peptide used for development of the antiserum is
evaluated by adding known amounts of the peptide to tubes
containing radiolabeled peptide and antiserum. After an additional
incubation period, for example, 24 to 48 hours, protein A is added
and the tubes centrifuged, supernatant removed and the
radioactivity in the pellet counted. The displacement of the
binding of radiolabeled endostatin peptide by the unlabeled
endostatin peptide (standard) provides a standard curve. Several
concentrations of other endostatin peptide fragments, plasminogen,
endostatin from different species, and homologous peptides are
added to the assay tubes to characterize the specificity of the
endostatin antiserum.
[0125] Extracts of various tissues, including but not limited to,
primary and secondary tumors, Lewis lung carcinoma, cultures of
endostatin producing cells, placenta, uterus, and other tissues
such as brain, liver, and intestine, are prepared using extraction
techniques that have been successfully employed to extract
endostatin. After lyophilization or Speed Vac of the tissue
extracts, assay buffer is added and different aliquots are placed
into the RIA tubes. Extracts of known endostatin producing cells
produce displacement curves that are parallel to the standard
curve, whereas extracts of tissues that do not produce endostatin
do not displace radiolabeled endostatin from the endostatin
antiserum. In addition, extracts of urine, plasma, and
cerebrospinal fluid from animals with Lewis lung carcinoma are
added to the assay tubes in increasing amounts. Parallel
displacement curves indicate the utility of the endostatin assay to
measure endostatin in tissues and body fluids.
[0126] Tissue extracts that contain endostatin are additionally
characterized by subjecting aliquots to reverse phase HPLC. Eluate
fractions are collected, dried in Speed Vac, reconstituted in RIA
buffer and analyzed in the endostatin RIA. The maximal amount of
endostatin immunoreactivity is located in the fractions
corresponding to the elution position of endostatin.
[0127] The assay kit provides instructions, antiserum, endostatin
or endostatin peptide, and possibly radiolabeled endostatin and/or
reagents for precipitation of bound endostatin--endostatin antibody
complexes. The kit is useful for the measurement of endostatin in
biological fluids and tissue extracts of animals and humans with
and without tumors.
[0128] Another kit is used for localization of angiostatin in
tissues and cells. This endostatin immunohistochemistry kit
provides instructions, endostatin antiserum, and possibly blocking
serum and secondary antiserum linked to a fluorescent molecule such
as fluorescein isothiocyanate, or to some other reagent used to
visualize the primary antiserum. Immunohistochemistry techniques
are well known to those skilled in the art. This endostatin
immunohistochemistry kit permits localization of endostatin in
tissue sections and cultured cells using both light and electron
microscopy. It is used for both research and clinical purposes. For
example, tumors are biopsied or collected and tissue sections cut
with a microtome to examine sites of endostatin production. Such
information is useful for diagnostic and possibly therapeutic
purposes in the detection and treatment of cancer.
[0129] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLE 1
[0130] Identification of an Inhibitor of Capillary Endothelial Cell
Proliferation from Hemangioendothelioma Cells
[0131] A murine hemangioendothelioma cell line, EOMA (Obeso et al.,
1990), was evaluated for evidence of the production of inhibitors
of endothelial cell proliferation. Many of the known endogenous
inhibitors of angiogenesis inhibit the in vitro proliferation of
endothelial cells.
[0132] Conditioned Media Collection: Cells of the murine
hemangioendothelioma cell line EOMA were maintained in DMEM
supplemented with 10% bovine calf serum (BCS) and 1%
glutamine-penicillin-streptomycin (GPS) in a 37.degree. C. and 10%
CO.sub.2 incubator. Conditioned media from EOMA cells (i.e. culture
media used to grow EOMA cells) was applied to bovine capillary
endothelial cells, stimulated with bFGF, in a 72 hour proliferation
assay. The conditioned media reversibly inhibited the proliferation
of capillary endothelial cells as compared to controls. The pattern
of inhibition was consistent with the presence of inhibitory and
stimulatory activity of endothelial cell proliferation (FIG.
1).
EXAMPLE 2
[0133] Inhibitory Activity of Endothelial Cell Proliferation is not
Due to Angiostatin
[0134] To determine if the inhibitor of capillary endothelial cell
proliferation produced by the EOMA cells was angiostatin, pooled
conditioned media was applied to a lysine column (lysine conjugated
to Sepharose.TM. chromatography beads). Lysine Sepharose binds
angiostatin and has been used for its purification (O'Reilly et
al., 1996). The endothelial cell inhibitory activity was found only
in the flow-through fraction and not in the bound fraction (data
not shown). The lack of binding of the inhibitory activity to
lysine Sepharose suggested that the novel inhibitor of endothelial
cell proliferation was not angiostatin.
EXAMPLE 3
[0135] Purification of a 20 kDa Protein from the Conditioned Media
of EOMA Cells which Specifically Inhibits Endothelial Cell
Proliferation
[0136] Because several angiogenesis inhibitors have an affinity for
heparin, we applied the flow-through from the lysine Sepharose
column to a heparin Sepharose column. The inhibitory activity bound
heparin with relatively high affinity and was eluted with 0.6-0.8 M
NaCl in 10 mM Tris pH 7.4, as shown in FIG. 2. To further purify
the inhibitory activity, the sample was concentrated and applied to
a gel filtration (Bio-Rad Bio-Gel P-100 fine gel or Pharmacia
Sephacryl S-200HR gel) column (see FIG. 3), followed by several
cycles of reverse-phase HPLC with a C4 column. The inhibitory
activity was eluted from the C4 column with 40-45% acetonitrile in
0.1% trifluoroacetic acid, as exemplified by FIG. 4. After the
final C4 column, the inhibitory activity was associated with a
protein of molecular mass of approximately 20 kDa (reduced) or 18
kDa (non-reduced), by SDS-PAGE, purified to apparent
homogeneity.
[0137] With respect to Examples 2 and 3, lysine Sepharose, heparin
Sepharose, Sephacryl S-200 HR gel (Pharmacia, Uppsala, Sweden),
Bio-Gel P-100 fine polyacrylamide gel (Bio-Rad Laboratories,
Richmond, Calif.), and a SynChropak RP-4 (100.times.4.6 mm) C4
reverse-phase column (Synchrom, Inc., Lafayette, Ind.) were
prepared according to the manufacturers recommendations. A
heparin-Sepharose column (50.times.2.5 cm) was equilibrated with 50
mM NaCI 10 mM Tris-HCI pH 7.4. Pooled conditioned media was applied
and the column was washed with the equilibration buffer. The column
was eluted with a continuous gradient of 50 mM -2 M NaCI in 10 mM
Tris-HCI at pH 7.4 (200 ml total volume) followed by 100 ml of 2 M
NaCI in 10 mM Tris-HCI at pH 7.4. Fractions were collected and an
aliquot of each was applied to capillary endothelial cells.
Fractions which inhibited their proliferation were dialyzed
(MWCO=6,000-8,000) against PBS and concentrated using a 4000 MWCO
Nanospin concentrator (Gelman Sciences, Ann Arbor, Mich.).
[0138] A Bio-Gel P-100 column or a Sephacryl S-200 HR column
(75.times.1.5 cm) was equilibrated with PBS. The sample from
heparin Sepharose chromatography was applied and the column was
fluted with the equilibration buffer. Fractions were collected and
an aliquot of each was applied to endothelial cells. Fractions
which inhibited endothelial proliferation were concentrated and
dialyzed as above.
[0139] A SynChropak RPG (100.times.4.6 mm) column was equilibrated
with H.sub.2O/0.1% trifluoroacetic acid (TFA). HPLC-grade reagents
(Pierce, Rockford, Ill.) were used. The sample from gel filtration
chromatography was applied to the column and the column was fluted
with a gradient of acetonitrile in 0.1% TFA at 0.5 ml/minute and
fractions were collected. An aliquot of each was evaporated by
vacuum centrifugation, resuspended in PBS, and applied to capillary
endothelial cells. Inhibitory activity was further purified to
apparent homogeneity by at least two subsequent cycles on the
SynChropak C4 column.
[0140] To further characterize the 20 kDa inhibitor, we tested it
on several cell lines of endothelial and non-endothelial origin.
For the BCE assay, bovine capillary endothelial cells were obtained
and grown as previously described (Folkman et al., 1979). For the
proliferation assay, cells were washed with PBS and dispersed in a
0.05% solution of trypsin. A cell suspension (25,000 cells/ml) was
made with DMEM+10% BCS+1% GPS, plated onto gelatinized 24-well
culture plates (0.5 mewed), and incubated (37.degree. C., 10%
CO.sub.2) for 24 hours. Themedia was replaced with 0.25 ml of
DMEM+5% BCS+1% GPS and the test sample applied. After 20 minutes of
incubation, media and bFGF were added to obtain a final volume of
0.5 ml of DMEM+5% BCS+1% GPS+1 ng/ml bFGF. After 72 hours, cells
were dispersed in trypsin, resuspended in Hematall (Fisher
Scientific, Pittsburgh, Pa.), and counted by Coulter counter.
[0141] Non-Endothelial Cell Proliferation Assays
[0142] Bovine aortic smooth muscle (SMC), bovine retinal pigment
epithelial (RPE), mink lung epithelial (MLE), Lewis lung carcinoma
(LLC), and EOMA cells and 3T3 fibroblasts were maintained in a 10%
CO.sub.2 and 37.degree. C. incubator. For the proliferation assays,
cells were washed with PBS and were dispersed in a 0.05% solution
of trypsin. Optimal conditions for the cell proliferation assays
were established for each different cell type. Fetal calf serum
(FCS) was used for the RPE, MLE, and LLC cells and BCS was used for
the other cell types. A cell suspension (20,000 cells/ml for SMC,
RPE, MLE; 15,000 cells/ml for 3T3; 10,000 cells/ml for LLC, EOMA)
was made with DMEM+10% bovine serum+1% GPS, plated onto 24-well
culture plates (0.5 ml/well), and incubated (37.degree. C., 10%
CO.sub.2) for 24 hours. The media was replaced with 0.5 ml of
DMEM+5% bovine serum+1% GPS and the test sample applied. After 72
hours, cells were dispersed in trypsin, resuspended in Hematall
(Fisher Scientific, Pittsburgh, Pa.), and counted by Coulter
counter.
[0143] Only endothelial cells were significantly inhibited, as
shown in Table 2.
3TABLE 2 EFFECT OF ENDOSTATIN ON ENDOTHELIAL AND NON-ENDOTHELIAL
CELL PROLIFERATION INHIBITED NON-INHIBITED Bovine capillary Bovine
aortic smooth endothelial cells muscle cells Bovine retinal pigment
epithelial cells 3T3 fibroblasts Mink lung epithelial cells EOMA
hemangioendothelioma cells Lewis Lung carcinoma cells
[0144] The inhibition was first observed at doses of 100 ng/ml with
maximal inhibition observed at doses of 600 ng/ml or greater. No
significant inhibition was seen for cells of non-endothelial origin
at doses 1 log unit higher than those used to inhibit capillary
endothelial cell proliferation (data not shown).
EXAMPLE 4
[0145] Microsequence Analysis of the 20 kDa Protein Reveals
Identity to a Fragment of Collagen XVIII
[0146] The 20 kDa inhibitor of capillary endothelial cell
proliferation from the conditioned media was purified to
homogeneity, as described in the above examples, resolved by
SDS-PAGE, electroblotted onto PVDF (Bio-Rad, Richmond, Calif.),
detected by Ponceau S stain, and excised from the membrane.
N-terminal sequence was determined by automated Edman degradation
on an PE/ABD Model 470A protein sequencer (Foster City, Calif.)
operated with gas-phase delivery of trifluoracetic acid.
[0147] Sequence library searches and alignments were performed
against combined GenBank, Brookhaven Protein, SWISS-PROT, and PIR
databases. Searches were performed at the National Center for
Biotechnology Information through the use of the BLAST network
service.
[0148] Microsequence analysis of the inhibitor revealed identity to
a C-terminal fragment of collagen XVIII. The molecular cloning and
sequence of collagen XVIII was first described by Olsen and his
coworkers and by Rehn and Pihlajaniemi (Oh et al., 1994; Rehn and
Pihlajaniemi, 1994). Collagen XVIII is a novel collagen which
consists of an N-terminal region with 3 splice variants (Muragaki
et al., 1995; Rehn and Pihlajaniemi, 1995), a series of
collagen-like domains with interruptions, and a 35 kDa C-terminal
non-collagenous (NC1) domain. An 18-amino acid N-terminal
microsequence analysis of the purified inhibitor of endothelial
cell proliferation confirms that it is identical to a C-terminal
fragment of this NC1 domain (FIG. 5). We have named this inhibitory
fragment of collagen XVIII "endostatin" and it is included in the
group of molecules that have endostatin activity.
EXAMPLE 5
[0149] Recombinant Mouse Endostatin (Baculovirus or E. coli)
Inhibits Endothelial Cell Proliferation In Vitro and Angiogenesis
in vivo
[0150] The endothelial proliferation cell inhibitor of the present
invention can be recombinantly expressed in any system used to
express proteins. Non-limiting examples of such expressions systems
include bacterial expression systems, yeast expression systems and
insect viral expression systems.
[0151] Recombinant mouse endostatin was expressed using the BacPAK
baculovirus expression system (CLONTECH Laboratories) following the
manufacture's protocol. Briefly, a cDNA fragment encoding the
signal sequence and C-terminal part (endostatin region) of mouse
collagen XVIII was inserted into the pBacPAK8 transfer vector.
BacPAK6 viral DNA (expression vector) and plasmid DNA of the
pBacPAK8-endostatin clone (modified transfer vector) were then
cotransfected into insect Sf21 cells and media containing expressed
mouse endostatin was collected. The BacPAK6 was first digested with
BSU36 enzyme to make it incompetent for independent replication.
The media containing expressed mouse endostatin was applied to a
1.5.times.40 cm heparin Sepharose column which had been
equilibrated with 50 mM NaCl 10 mM Tris pH 7.4. The column was
washed with the equilibration buffer and was then eluted
sequentially with 0.2 M NaCl, 0.4 M NaCl, 0.6 M NaCl, and 1 M NaCl
in 10 mM Tris pH 7.4. All chromatography was performed at 4.degree.
C. The 0.6 M NaCl eluant (which inhibited bovine capillary
endothelial cells in a 72 hour proliferation assay) was dialyzed
(6-8000 MWCO) against PBS and then reapplied to the heparin
Sepharose column. The column was eluted with a gradient of 50 mM
NaCl -1.2 M NaCl in 10 mM Tris pH 7.4. An aliquot of each fraction
was applied to bovine capillary endothelial cells as above and
fractions which inhibited proliferation were pooled, dialyzed
against PBS, and concentrated using a Nanospin Plus (Gelman
Sciences) centrifugal concentrator (MWCO=10,000). SDS-PAGE of the
concentrated sample revealed a discrete band of apparent Mr of 20
kDa.
[0152] Expression and Purification of Recombinant Mouse Endostatin
from E. coli
[0153] The C-terminal part of the cDNA of collagen XVIII was used
to amplify the cDNA of mouse endostatin which was cloned into the
pETKH1 vector (pET11d derivative) (Studier et al., 1990). Induction
resulted in the production of a fusion protein carrying the amino
acid sequence MARRASVGTD (RRAS=protein kinase A recognition
sequence) and 6 histidine residues at the N-terminus followed by
the sequence of mouse endostatin (pTB01#8). The pTB01#8 plasmid was
transformed into BL21:DE3 and the fusion protein was purified on
Ni.sup.+2-NTA-beads as described (QiaExpressionist Handbook,
Qiagen). Briefly, E. coli were grown until an O.D..sub.600 of
0.8-0.9 and expression of the fusion protein was then induced for 3
hours with 1 mM IPTG. The bacteria were pelleted and resuspended in
8 M urea, 10 mM Tris-HCl pH 8.0 containing 10 mM imidazole and
incubated for 1 hour at room temperature. The suspension was
centrifuged for 15 minutes at 20,000 g and the supernatant
incubated with the Ni.sup.+2-NTA beads for 1 hour at room
temperature. The suspension was transferred into a column and
washed with 8 M urea, 0.1 M Na-phosphate, 10 mM Tris-HCl pH 6.25
containing 10 mM imidazole. The protein was eluted with the same
buffer containing 250 mM imidazole. The fractions containing
endostatin were extensively dialyzed against PBS. During dialysis,
the endostatin precipitated. The precipitated endostatin was
resuspended in PBS, the protein concentration was adjusted to 2-4
mg/ml, and the endostatin was stored at -20.degree. C. until use.
For the mouse studies, endostatin was delivered as a suspension in
PBS. For the chick chorioallantoic assay, endostatin was further
dialyzed against water and then lyophilized.
[0154] Recombinant mouse endostatin was produced in both
baculovirus and E. coli expression systems. Using sequential
heparin Sepharose chromatography, recombinant mouse endostatin was
purified to apparent homogeneity from insect cell media.
Ni.sup.+2-NTA-agarose was used to purify the E. coli-derived mouse
endostatin.
[0155] SDS-PAGE revealed a discrete band of approximately-20 kDa or
approximately 22 kDa (reduced) purified to apparent homogeneity for
baculovirus and E. coli-derived recombinant endostatins,
respectively (data not shown). Both were dialyzed against PBS prior
to use. After dialysis, the material from the E. coli system
precipitated and was delivered as a suspension for subsequent in
vivo studies. Recombinant endostatin from baculovirus specifically
inhibited the proliferation of bovine capillary endothelial cells
in a dose-dependent fashion. The inhibition was seen at doses of
100 ng/ml with maximal inhibition observed at doses above 600
ng/ml. No significant inhibition of proliferation of cells of
non-endothelial origin or of the EOMA cells was observed when
endostatin was tested at doses up to 1 log unit higher than those
used to inhibit endothelial cell proliferation.
[0156] The precipitated (un-refolded) material was not testable in
vitro, because of its insolubility. However, a small percentage was
soluble in PBS during dialysis and this fraction was used for the
endothelial cell assays. Furthermore, after refolding, it was
soluble and inhibited endothelial proliferation (data not shown).
When this soluble material was applied to endothelial cells, it was
found to be inhibitory at concentrations comparable to both the
native and baculovirus-derived endostatin.
[0157] To test for the ability of recombinant mouse endostatin to
inhibit in vivo angiogenesis, we used the chick chorioallantoic
membrane (CAM) assay (Folkman, 1985; Nguyen et al., 1994 which are
incorporated herein by reference). Briefly, three day old
fertilized white Leghorn eggs (Spafas, Norwich, Conn.) were
cracked, and embryos with intact yolks were placed in 100.times.20
mm petri dishes (Folkman, 1985). After 3 days of incubation
(37.degree. C. and 3% CO.sub.2), a methylcellulose (Fisher
Scientific, Fair Lawn, N.J.) disc containing endostatin was applied
to the CAM of individual embryos. The discs were made by
desiccation of endostatin in 10 .mu.l of 0.45% methylcellulose (in
H.sub.2O) on teflon rods. After 48 hours of incubation, embryos and
CAMs were observed by means of a stereomicroscope.
[0158] At doses of 10-20 .mu.g/10 .mu.l disc, there was potent
inhibition of in vivo angiogenesis for both the E. coli and the
baculovirus-derived endostatins in all of the tested CAMs
(n=5/group). The E. coli derived-endostatin precipitate gradually
dissolved over 5 days and produced a sustained antiangiogenic
effect on the implanted CAMs. In contrast, the soluble
baculovirus-derived endostatin dissolved within 24 hours and gave a
maximal antiangiogenic effect within a period of 48 hours. There
was no evidence of toxicity in any of the chick embryos tested.
[0159] Human Endostatin was produced recombinanty using similar
methods.
EXAMPLE 6
[0160] Recombinant Mouse Endostatin Inhibits the Growth of
Metastases
[0161] Because tumor growth is angiogenesis dependent, we treated
Lewis lung carcinoma metastases systematically with recombinant
mouse endostatin expressed in the baculovirus system. Animals with
Lewis lung carcinomas of 600-1200 mm.sup.3 tumors were sacrificed
and the skin overlying the tumor was cleaned with betadine and
ethanol. In a laminar flow hood, tumor tissue was excised under
aseptic conditions. A suspension of tumor cells in 0.9% normal
saline was made by passage of viable tumor tissue through a sieve
and a series of sequentially smaller hypodermic needles of diameter
22- to 30-gauge. The final concentration was adjusted to
1.times.107 cells/ml and the suspension was placed on ice. After
the site was cleaned with ethanol, the subcutaneous dorsa of mice
in the proximal midline were injected with 1.times.106 cells in 0.1
ml of saline.
[0162] When tumors were 1500 mm.sup.3 in size, approximately 14
days after implant, the mice underwent surgical removal of the
tumor. The incision was closed with simple interrupted sutures.
From the day of operation, mice received daily intraperitoneal
injections of recombinant (baculovirus) mouse endostatin or saline.
Mice received 0.3 mg/kg/day of endostatin once daily via
subcutaneous injection. When the control mice became sick from
metastatic disease (i.e., after 13 days of treatment), all mice
were sacrificed and autopsied. Lung surface metastases were counted
by means of a stereomicroscope at 4.times.magnification.
[0163] The growth of Lewis lung carcinoma metastases was almost
completely suppressed by the systemic administration of endostatin
at a dose of 0.3 mg/kg/day given subcutaneously (7.+-.3
metastases/mouse, n=4, p<0.001). In contrast, in mice treated
with saline after removal of a Lewis lung carcinoma primary tumor,
lung metastases grew rapidly (77.+-.7 metastases/mouse). Lung
weight, which reflects tumor burden, was 240.+-.25 mg in the
endostatin treated mice versus 760.+-.30 mg in the control mice
(p<0.001). Further, there was no weight loss or evidence of
toxicity in any of the mice treated with endostatin.
EXAMPLE 7
[0164] Recombinant Mouse Endostatin Inhibits the Growth of Primary
Tumors
[0165] The yield of endostatin from the baculovirus system was
lower than that of the E. coli system, i.e. 1-2 mg/liter versus
30-40 mg/liter. We therefore used E. coli-derived endostatin to
study the effect of endostatin therapy on primary tumor growth. We
produced recombinant mouse endostatin from E. coli in sufficient
quantity to treat Lewis lung carcinoma primary tumors. The
endostatin was administered as a suspension of the precipitated
purified protein to mice bearing Lewis lung carcinomas of at least
100-200 mm.sup.3. The protein was purified by conventional means
but was not refolded prior to its administration to the mice. The
injected precipitate was slowly resorbed over 24-48 hours.
[0166] We are unaware of any precedent for the use of an injected
depot of non-refolded recombinant protein as a sustained-release
method in animals. Nevertheless, endostatin gradually resorbed in
vivo and proved to have potent antiangiogenic activity which
resulted in prolonged anti-tumor and antiangiogenic activity.
Therefore, these data suggest a novel general method for the
controlled release of recombinant proteins. Based on this
rationale, we have delivered non-refolded recombinant angiostatin
from E. coli with similar success.
[0167] Accordingly, an aspect of the invention is the
administration of recombinant endostatin or endostatin analogs in
an un- refolded state so as to provide a sustained release depot of
endothelial cell proliferation inhibiting protein over a period of
at least 8 hours, desirably at least 12 hours, more desirably at
least 24 hours or at least 48 hours, depending on the patient and
the disease to be treated. Optionally recombinant and un-refolded
angiostatin is administered to similarly provide a sustained
release depot of protein capable of releasing angiostatin protein
over a period of at least 8 hours, desirably at least 12 hours,
more desirably at least 24 hours or at least 48 hours, depending on
the patient and the disease to be treated.
[0168] Mice were implanted with Lewis lung carcinomas as described
above. Tumors were measured with a dial-caliper and tumor volumes
were determined using the formula
width.sup.2.times.length.times.0.52, and the ratio of treated to
control tumor volume (T/C) was determined for the last time point.
After tumor volume was 100-200 mm.sup.3 (0.5-1% of body weight),
which occurred within 3-7 days, mice were randomized into two
groups. One group received recombinant mouse endostatin (E. coli)
as a suspension in PBS injected subcutaneously at a site distant
from the tumor once daily. The other group received comparable
injections of the vehicle alone. The experiments were terminated
and mice were sacrificed and autopsied when the control mice began
to die.
[0169] The growth of Lewis lung primary tumors was potently
suppressed by systemic therapy with endostatin. Increasing the dose
of endostatin was associated with improved efficacy (data not
shown). At a dose of 10 mg/kg, tumor growth was inhibited by 97% as
compared to control mice treated with vehicle alone. At a dose of
20 mg/kg given once daily, in two separate experiments, there was
an almost complete regression of established primary tumors
(>99% inhibition, p<0.001). These surprising and unexpected
results are shown in FIGS. 6 and 7.
[0170] FIGS. 8, 9, 10 and 11 demonstrate the effectiveness of
recombinant mouse endostatin for inhibiting tumor growth in a
variety of different tumor models. Also demonstrated is the
effectiveness of endostatin derived from human for inhibiting tumor
growth.
[0171] Immunohistochemical analysis (FIG. 12) of the residual small
tumors showed a potent inhibition of angiogenesis in the endostatin
treated tumors. Further, the proliferative index of tumors in the
endostatin and saline treated mice was at the same high level in
both groups while the apoptotic index increased 8-fold after
endostatin therapy. Thus, endostatin therapy results in a similar
pattern of tumor dormancy to the one we have previously described
for angiostatin (Holmgren et al., 1995; O'Reilly et al., 1996).
Further, there was no evidence of drug-related toxicity in any of
the treated mice.
[0172] After discontinuation of endostatin therapy, a tumor
recurred at the primary site within 5-14.days, became vascularized,
and eventually killed the mice (data not shown). Notably, we found
that E. coli-derived recombinant mouse endostatin with a C-terminal
polyhistidine tag, which was expressed. purified and administered
in a comparable fashion to the N-terminal tagged product described
above did not inhibit angiogenesis in the CAM assay and had no
effect on the growth of Lewis lung carcinomas (data not shown).
These data argue strongly that the anti-tumor and antiangiogenic
activity of recombinant endostatin are due to the specific
structure of endostatin and not to a contaminant in the sample.
[0173] FIG. 13 shows the results of cycled treatment of Lewis lung
carcinoma with recombinant mouse endostatin derived from E. coli.
These results clearly show reproducible endostatin-dependent
regression of tumor mass, followed by tumor growth after
termination of endostatin treatment.
[0174] These results show that a murine hemangioendothelioma
produces a novel and specific 20 kDa inhibitor of endothelial cell
proliferation in vitro which is also a potent inhibitor of
angiogenesis and tumor growth in vivo. The N-terminal sequence of
this inhibitor, endostatin, is identical to a C-terminal fragment
of collagen XVIII. Systemic administration of recombinant
endostatin potently inhibits angiogenesis, maintains metastases at
a microscopic size, and regresses primary tumors to less than 1
mm.sup.3, a reduction of over 150-fold. For as long as mice are
treated there is no regrowth of tumors, no evidence of drug
resistance, and no toxicity. It is interesting to note that some
fragments of the C-terminal domain of collagen type XVIII that are
longer than endostatin do not inhibit endothelial cell
proliferation (data not shown).
[0175] Endostatin was discovered by the same strategy employed to
find angiostatin (O'Reilly et al.,1994), i.e., isolation from a
tumor. While it is counter-intuitive that tumors should be a source
of angiogenesis inhibitors, the results reported here seem to
validate this approach.
[0176] This leads to the question of why angiogenesis inhibitors
should be present in tumors that are angiogenic. One possibility is
that an inhibitor could be `left-over` after down-regulation of its
production by a tumor cell undergoing the switch to the angiogenic
phenotype. This appears to be the case for thrombospondin produced
by Li-Fraumeni cells in which the second allele for p53 is mutated
or deleted (Dameron et al., 1994).
[0177] A second possibility is that the proteolytic activity which
accompanies tumor growth, and which is an important component of
capillary blood vessel growth, may also mobilize circulating
angiogenesis inhibitors from precursor proteins which are not
inhibitory themselves. Angiostatin for example, inhibits
angiogenesis and endothelial cell proliferation while plasminogen
does not (O'Reilly et al., 1996; O'Reilly et al., 1994). For
endostatin, a similar pattern is revealed.
[0178] Histology of tumors which regressed under endostatin therapy
showed perivascular cuffing of tumor cells surrounding one or more
microvessels in which angiogenesis was blocked. Tumor cells
displayed high proliferation balanced by high apoptosis, with no
net gain in tumor size. These data are consistent with a model of a
new type of tumor dormancy recently proposed (Holmgren et al.,
1995). Furthermore, endostatin inhibited proliferation of
endothelial cells in vitro, but had no effect on Lewis lung
carcinoma cells, or other cell types including smooth muscle,
epithelium, fibroblasts, and the EOMA cell line from which it was
purified.
[0179] The fact that a specific inhibitor of endothelial cell
proliferation can regress a tumor to a microscopic size and hold it
in a dormant state, despite the fact that the tumor cells are
refractory to the inhibitor from the outset, indicates that the
endothelial population can exert powerful growth regulatory control
over the tumor cells.
[0180] The results with endostatin support the theory (Folkman,
1996) that for therapeutic purposes, it is fruitful to think about
a tumor in terms of two distinct cell populations: a tumor cell
population and an endothelial cell population, each of which can
stimulate growth of the other. Growth of each cell population may
be optimally inhibited by agents which selectively or specifically
target that cell type, i.e., cytotoxic chemotherapy and
antiangiogenic therapy. Furthermore, combined treatment of both
cell populations may be better than treatment of either cell type
alone.
[0181] To test this theory mice seeded with Lewis lung carcinomas,
and bearing tumors which had attained a size of approximately 300
mm.sup.3, were treated with a combination therapy comprising
angiostatin and endostatin, each at a dose of 20 mg/kg/day for 25
days. Tumors regressed to microscopic levels by about day 10 of
treatment. A completely unexpected finding was that tumors remained
regressed and dormant for approximatley three months, even after
all treatment was terminated, as is shown in FIG. 14. Experiments
of longer duration indicate that an initial treatment of tumor with
a combination of angiostatin and endostatin causes a very long term
dormancy, the actual period of which is unknown at this time.
[0182] Such long term dormancy is considered a cure to one skilled
in the art. For example, the NIH guideline for determining when a
treatment is effective as a cancer cure, is that the tumor remain
dormant (i.e. not increasing in size) for ten times the normal
doubling time of the tumor. The dormancy length achieved using a
combination of endostatin and angiostatin far exceeds this
criteria.
[0183] Accordingly, an important aspect of the invention is a
composition comprising a combination of angiostatin and endostatin,
or an endostatin analog, in amounts sufficient to cause long term
dormancy, or cure, of angiogenesis-dependent cancers when
administered to patients with angiogenesis-dependent cancers.
Administration can be systemically, for example by injection, in
which case the dosage is determined depending upon the patient and
the particular cancer, but which generally is at least 0.2
mg/kg/day, desirably at least 2.0 mg/kg/day, more desirably at
least 20 mg/kg/day. Generally, the composition is administered
daily for at least 10 days, desirably at least 20 days, more
desirably at least 25 days. Alternative systemic administration
routes include, orally where the composition is formulated, for
example into coated microbeads, to protect the protein from
inactivating digestive environments; transdermally; and via
pump.
[0184] Alternatively, different dosages and treatment periods can
be used if the composition is administered locally to an
angiogenesis-dependent site, such as a tumor. Such administration
may be, for example, surgical implantation or local injection into,
or near by, the site.
EXAMPLE 8
[0185] Isolation of the Putative Receptor for Endostatin.
[0186] Both endostatin and angiostatin appear to be specific
inhibitors of endothelial cell proliferation. Therefore, it is
likely that endostatin binds to specific structures exclusively
expressed on the surface of endothelial cells. We are not aware of
the existence of any other specific inhibitors of endothelial cell
proliferation.
[0187] Identifying and isolating proteins which specifically bind
to endostatin is accompanied by methods well known in the art, for
example by affinity chromatography and expression cloning.
[0188] Affinity chromatography. Bovine Capillary Endothelial cells
(BCE) are radiolabeled with [35S]-methionine, total cell and
membrane extracts prepared and applied to affinity columns prepared
with endostatin. As a negative control, fibroblast protein extracts
are isolated in a similar way. Bound proteins are eluted from the
column using a NaCl gradient and the different fractions are
analysed using standard SDS-PAGE and autoradiography. This
procedure yields proteins that are tightly bound to the endostatin
column and present only in the endothelial cell derived fractions.
Comparing the gel electrophoretic patterns of the two cell types
reveals expressed proteins unique to the BCE cells. Protein
sequences subsequently are determined and corresponding gene(s)
cloned. A cDNA library of bovine capillary endothelial cells, is
prepared and screened with a degenerative oligo based PCR technique
to locate the cDNA(s) of the endostatin-specific binding
protein(s). Hybridization using degenerative oligonucleotides to
the corresponding cDNA, is also used to identify genes of
endostatin binding proteins. Another approach is to raise
antibodies against the peptide sequences with methods described
earlier in the Detailed Description and immunoscreen the same
library.
[0189] Expression cloning. A cDNA library of BCE cells is prepared.
Poly-A mRNA is isolated from BCE cells whose proliferation has
previously been inhibited by endostatin. These cells express an
endostatin binding protein. The corresponding cDNA library is
transfected into cells allowing high expression of the various
cDNAs. Binding activity of endostatin to cells which express the
receptor protein on the surface is used as a positive selection of
these cells. To select for these cells, purified endostatin is
labeled with biotin and consequently detected using either
streptavidin coupled magnetic-beads or FACS sorting. Alternatively,
an antibody against endostatin is used for screening. After
selection of the positive cells, the corresponding plasmids are
isolated, amplified and transfected again into high expression
cells. After several rounds of positive selection, plasmids are
analysed for identical inserts using endonuclease digestion and
PCR. Using these data, complementation groups are formed, sequenced
and analysed with the BLAST network program. In addition to
computer analysis, individual cDNAs are re-transfected into high
expression cells and tested for endostatin binding activity under
different conditions (e.g., competition with non-labeled
endostatin, time-course of binding, Scatchard analysis, etc. in
other words the use of "classical" receptor characterization
procedures known to those skilled in the art).
EXAMPLE 9
[0190] Determination of the Minimal Region of the Mouse Endostatin
Protein Responsible for its Antiangiogenic Activity.
[0191] Different PCR primers are designed, the corresponding cDNAs
cloned into the E. coli expression system, and the different
endostatin fragments purified to homogeneity. The full length cDNA
is cut from both the N- and C-terminus. As a first screen the
capillary endothelial proliferation assay and the chick embryo
assay are used to determine the residual activity compared to the
full length fragment.
EXAMPLE 10
[0192] Determination of the Putative Enzyme(s) which may Release
Endostatin from Collagen XVIII.
[0193] Collagen XVIII belongs to the non-fibrillar collagen type
family and can be found in three different splicing variants
encoding for proteins with 1315-, 1527-, and 1774 amino acid
residues (Rehn, PNAS 91:4234, 1994). The difference is caused.by
alterations in the N-terminal part of the gene and therefore all
three splicing variants could potentially be the source of
endostatin which itself is a fragment of the non-collagenous domain
11 (NC11). The function of collagen XVIII is not known, but because
its message is substantially expressed in highly vascularized
organs, a role in perivascular matrix assembly and/or structure has
been proposed (Oh, et al., Genomics, 19:494, 1994). A first clue
about the function of collagen XVIII came from the purification of
endostatin as a potent inhibitor of endothelial cell
proliferation.
[0194] From this preliminary data and from our initial observation
that endostatin was purified from conditioned medium of a
hemangioendothelioma (EOMA), we asked whether the enzyme(s) which
release endostatin from collagen XVIII could be identified?
[0195] The last 325 amino acid residues, encoding for the NC11
domain, are expressed in E. coli and the insect cell baculovirus
system, the purified protein is used as a substrate to identify
enzymes that clone this region of collagen XVIII. By PCR, a cDNA
fragment encoding the NC11 domain is cloned into an E. coli
expression vector (pET series) which allows high expression of the
target protein after induction with IPTG. Alternatively, a vector
suitable for insect cell expression is used. The proteins are
tagged with the HIS.sub.6-Tag located on the C-terminus for
purification using Ni.sup.2+-NTA-beads. An Ni.sup.2+-NTA-alkaline
phosphatase conjugate can detect the C-terminus by Western
blotting. Another construct is made which not only has a
HIS.sub.6-Tag on the C-terminus, but will also encode the
hemagglutinin (HA-tag on the N-terminus. This is detected by
Western blotting with an HA-specific monoclonal antibody. The N-
and C-terminus of the protein followed after incubation with EOMA
supernatant and different metalloproteinase extracts.
[0196] Cleavage product is detected by SDS-PAGE analysis or Western
blotting, the protein is re-purified using the Ni.sup.2+-NTA beads,
eluted with imidazole, dialyzed against PBS and tested for
inhibitor activity in the various in vitro and in vivo assays
(e.g., endothelial cell proliferation, chick embryo, and mouse
corneal assay). If the purified cleavage product shows inhibitory
activity, N-terminal amino acid sequencing is performed and
compared to the original starting sequence of endostatin obtained
from the EOMA supernatant. Accordingly, the cleavage procedure can
be scaled up to purify sufficient protein for testing in
tumor-bearing mice, and to compare this activity to that of the
full length NC11 domain.
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Sequence CWU 1
1
3 1 20 PRT Murinae gen. sp. 1 His Thr His Gln Asp Phe Gln Pro Val
Leu His Leu Val Ala Leu Asn 1 5 10 15 Thr Pro Leu Ser 20 2 10 PRT
Artificial Sequence Synthetic 2 Met Ala Arg Arg Ala Ser Val Gly Thr
Asp 1 5 10 3 4 PRT Artificial Sequence Synthetic 3 Arg Arg Ala Ser
1
* * * * *