Note: Descriptions are shown in the official language in which they were submitted.
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THERAPEUTIC ANTIANGIOGENIC ENDOSTATIN COMPOSITIONS AND
METHODS OF USE
Technical Field
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.
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Background of the Invention
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).
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.
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Summary of the Invention
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.
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:
His Thr His Gin 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
(SEQ ID NO:1)
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.
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.
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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 diseases 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.
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.
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.
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.
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.
The present invention includes molecular probes for the
ribonucleic acid and deoxyribonucleic acid involved in transcription and
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translation of endostatin. These molecular probes provide means to
detect and measure endostatin biosynthesis in tissues and cells.
A surprising discovery is that various forms of recombinant
endostatin protein can serve as sustained release anti-angiogenesis
5 compounds when administered to a tumor-bearing animal. A preferred
form of the sustained release compound is un-refolded recombinantly
produced endostatin.
Additionally, the present invention encompass.es 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.
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.
The invention further encompasses a method for identifying
receptors specific for endostatin, and the receptor molecules identified
and isolated thereby.
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.
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.
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
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present invention also includes formulations containing endostatin and
optionally
angiostatin, which are effective for treating or curing angiogenesis-dependent
cancers.
Accordingly, the present invention seeks to provide a composition comprising
an endostatin protein.
Further, the present invention seeks to provide a method of treating disease
and processes that are mediated by angiogenesis.
Still further the present invention seeks to provide a diagnostic or
prognostic
method and kit for detecting the presence and amount of endostatin in a body
fluid
or tissue.
Yet further the present seeks 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, coronary collaterals, cerebral collaterals, arteriovenous
malformations, ischemic limb angiogenesis, comeal 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.
Further still, the present invention seeks to provide a composition for
treating
or repressing the growth of a cancer.
Further still, the present invention seeks to provide a method for detecting
and
quantifying the presence of an antibody specific for an endostatin in a body
fluid.
Moreover the present invention seeks to provide a composition consisting of
antibodies to endostatin that are selective for specific regions of the
endostatin
molecule.
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An aspect of the present invention provides isolated endostatin protein having
a molecular weight of approximately 18 kDa as determined by non-reducing gel
electrophoresis, or approximately 20 kDa as determined by reducing gel
electrophoresis. The protein has an amino acid sequence (SEQ IDN: 1) of a
fragment
of a collagen type XVIII that begins at any of the amino acids from amino acid
1105
to amino acid 1124, also amino acid 1135 to amino acid 1151 or a collagen type
XV,
that begins at any of the amino acids from 316 to amino acid 335. The
endostatin
protein binds to a heparin affinity column and does not bind to a lysine
column and
is further characterized by its ability to specifically inhibit angiogenesis.
A further aspect of the present invention provides use of a method for
expressing an endostatin protein comprising providing a cell and transfecting
a vector
into the cell, the vector having the nucleic acid sequence.
Another aspect of the present invention provides for the use of the endostatin
protein for the manufacture of a medicament for the treatment of an
angiogenesis
related disease.
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Yet fiarther the present invention seeks to provide a method for the detection
or
prognosis of cancer.
Still further, the present invention seeks to provide a composition for use in
visualizing and quantitating sites of endostatin binding in vivo and in vitro.
Further still, the present invention seeks to provide a composition for use in
detection and quantification of endostatin biosynthesis.
Yet fijrther, the present invention seeks to provide a therapy for cancer that
has
minimal side effects.
Additionally, the present invention seeks to provide a composition comprising
endostatin or an endostatin peptide linked to a cytotoxic agent for treating
or repressing the
growth of a cancer.
These: and other aspects, 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
Figure 1: Inhibition of Capillary Endothelial Cell Proliferation by
Conditioned Media from
EOMA Cells.
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.
Figure 2: Purification qf an Inhibitor of Endothelial Proliferation from EOMA
Conditioned
Media.
Conditioned media collected from confluent EOMA cells was fractionated on a
heparin Sepharose"" column. Endothelial proliferation inhibiting activity
eluted at
approximately 0.8M: NaCI.
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Figure 3: Purification of an Inhibitor of Endothelial Proliferation by
Gel Filtration.
Purified inhibitor from heparin sepharose column
chromatography was applied to a gel filtration column and eluted as a
single peak.
Figure 4: Purification of Inhibitor of Endothelial Cell Proliferation by
Reversed Phase Column Chromatography.
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.
Figure 5: N-terminal Amino Acid Sequence of An Inhibitor of
Endothelial Cell Proliferation.
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.
Figure 6: Treatment of Lewis Lung Carcinoma With Recombinant Mouse
Endostatin Inhibitor.
Recombinant inhibitor produced in E. coli was administered
to mice seeded with Lewis lung carcinoma that had achieved a tumor
volume of approximately 150 mm3. The inhibitor was administered at
20 mg/kg/day. Tumor mass regressed to non-detectable levels after
approximately 12 days of treatment.
Figure 7A-C: Systemic Therapy with Recombinant Endostatin Regresses
Lewis Lung Carcinoma Primary Tumors.
Figure 7A. 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
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tumors were approximately 200 mm3 (1% 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.
Figure 7B. 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).
Figure 7C. 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).
Figure 8: Treatment of Murine 7241 Fibrosarcoma with Recombinant
Mouse Endostatin from E. coli
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.
Figure 9: Treatment of Murine B16F1O Melanoma with Recombinant
Mouse Endostatin from E. coli
Mice were seated with Murine B 16F10 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.
Figure 10: Treatment of EOMA Hemangioendothelioma with
Recombinant Mouse Endostatin from E. coli
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.
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Figure 11: Treatment of Lewis Lung Carcinoma with Recombinant
Mouse or Human Endostatin direct from E. coli.
Mice were seated with Lewis Lung Carcinoma cells.
Control animals were treated with saline. Experimental animals were
5 treated with Recombinant Endostatin derived from the mouse sequence
or Recombinant Endostatin direct from the human sequence, wherein
both Endostatin were 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.
Figure 12A-C: Endostatin Results in an Inhibition of Angiogenesis and
an Increase in Apoptosis of Lewis Lung Carcinoma Primary Tumors.
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 (Figure 12A) in treated versus untreated tumors. In contrast, the
apoptotic index of the tumor cells (Figure 12B) increased 8-fold (p <
0.001) in the endostatin treated mice. Vessel density (Figure 12C) 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).
Figure 13: Cycle Dormancy Therapy of Lewis Lung Carcinoma with
Recombinant Mouse Endostatin From E. Coli.
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 mm3 (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-
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detectable levels by reinitiation of treatment. The peaks and valleys in
the figure show the cycling effect of inhibition with endostatin.
Figure 14: Combination Therapy with Recombinant Mouse Angiostatin
and Endostatin from E. Coli.
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 mm3. 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
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.
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
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activity of the protein, is well known in the art. Such silent substitutions
are intended to fall within the scope of the appended claims.
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 labeling such as radioiodination with
12siodine for use in assays. Labeling with other radioisotopes may be
useful in providing a molecular tool for destroying the target cell
containing endostatin receptors. Other labeling with molecules such as
ricin may provide a mechanism for destroying cells with endostatin
receptors.
"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.
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.
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Table 1 shows the correspondence of 3 letter and 1 letter
amino acid designations.
TABLE 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
LEU L
11 HIS H
12 LEU L
13 VAL V
14 ALA A
LEU L
16 ASN N
17 THR T
18 PRO P
19 LEU L
SER S
The N-terminal amino acid sequence of endostatin
corresponds to an internal 20 amino acid peptide fragment found in
mouse collagen alpha 1 type XVIII starting at amino acid 1105 and
10 ending at amino acid 1124. The N-terminal amino acid sequence of the
inhibitor also corresponds to an internal 20 amino acid peptide fragment
found in human collagen alpha 1 type XVIII starting at amino acid 1132
and ending at amino acid 1151.
Endostatin can be isolated from murine
15 hemangioendothelioma 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.
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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.
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.
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 them
with blood. "Regression" refers to the reduction of tumor mass and size.
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.,
s 11459 hexon protein of bovine adenovirus type 3, CELF21 D 12 2
F21dl2.3 Caenorhabditis elegans gene product, VAL1 TGMV ALl
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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
5 beginning at amino acid 316 ending at 335.
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,
10 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), October 21, 1994, pp. 315 -
328, which
may be referred to for further details. 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
15 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.
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.
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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.
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.
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
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segments of endostatin to generate peptides having anti-angiogenesis
activity.
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. .
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.
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
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18
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 (Helobacterpylori).
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
occurred, thus providing an effective method of birth control, possibly 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.
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
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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.
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.
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.
The invention further encompasses a method for identifying
endostatin-specific receptors, and the receptor molecules identified and
isolated thereby.
The present invention also provides a method for
quantitation of endostatin receptors.
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.
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
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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
5 apoptosis. There was no evidence of toxicity in any of the mice treated
with endostatin.
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
10 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
15 accomplished using similar techniques.
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
20 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.
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
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21
with the appropriate primers to amplify the DNA sequence coding for
the active endostatin amino acid sequence.
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.
Once the amino acid sequence of the peptide is known, for
example the N-termina120 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.
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.
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The angiogenesis mediated diseases include, but are not limited to, solid
tumors; blood born tumors such as leukernias; 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.
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.
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.
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
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possibly promote angiogenesis. This treatment may have therapeutic
effects to promote wound healing in diabetics.
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.
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.
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.
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
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24
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.
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.
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.
CA 02235393 2000-08-29
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
5 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.
The proteins and protein fragments with the endostatin activity described
above
can be provided as isolated and substantially purified proteins and protein
fragments in
10 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,
15 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
20 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 may be referred to for further details.
The dosage of the endostatin of the present invention will
depend on the disease state or condition being treated and other clinical
25 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
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mg/kilogram to 100 mg/kilogram with the most preferable range being
from 2mg/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.
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.
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.
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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.
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.
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.
Peptides and endostatin are also produced in recombinant
E. coli, as described below, or in insect or yeast expression systems, and
purified with colunm chromatography.
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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.
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 125I is accomplished using chloramine T and
NaluI 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 Na125I 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.
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.
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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 4C and are centrifuged at approximately
2400 X g at 4C for about 30 minutes. The serum is removed, aliquoted,
and stored at 4C for immediate use or at -20 to -90C for subsequent
analysis.
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 deternunation of titer
and specificity.
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,
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SUBSTITUTE SHEETS
including endostatin related species, d) ability to detect endostatin
peptides in extracts of plasma, urine, tissues, and in cell culture media.
Kits for measurement of endostatin are also contemplated
as part of the present invention. Antisera that possess the highest titer
5 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
10 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,
15 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.
One example of an assay kit commonly used in research
and in the clinic is a radioimmunoassay (RIA) kit. An endostatin RIA is
20 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-
25 specific binding. After incubation at 4C 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 X g at 4C to
precipitate the complexes of antibody bound to labeled antigen. The
supernatant is removed by aspiration and the radioactivity in the pellets
30 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.
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
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31
radiolabeled peptide and antiserum. After an additional incubation
period, for example, :?4 to 48 hours, protein A is added and the tubes
centrifuged, supernatant removed and the radioactivity in the pellet
counted. T'he 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 antiserunl.
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 beer.i successfully employed to extract endostatin. After
lyophilization or Spe(.-.d 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 tc) measure endostatin in tissues and body fluids.
Tissue extracts that contain endostatin are additionally
characterized by subjecting aliquots to reverse phase HPLC. Eluate
fractions are collected, dried in Speed Va~Mreconstituted 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 endostatiii.
The assay kit provides instructions, antiserum, endostatin or
endostatin peptide, and possibly radiolabeled endostatin and/or reagents
for precipitation of hound endostatin - endostatin antibody complexes.
The kit is useful for the measurernent of endostatin in biological fluids
and tissue extracts of animals arid humans with and without tumors.
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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 irnmunohistochemistry 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.
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
Identification of an Inhibitor of Capillary Endothelial Cell Proliferation
from Hemangioendothelioma Cells
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.
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 C and 10% C02
incubator. Conditioned media from EOMA cells (i.e. culture media used
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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 (Figure 1).
Example 2
Inhibitory Activity of Endothelial Cell Proliferation is not due to
Angiostatin
io 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
SepharoseTM chromatography beads). Lysine Sepharose binds
angiostatin and has been used for its purification (O'Reilly et al., 1996).
IL 5 The endotlielial 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 irihibitor of endothelial cell proliferation was not angiostatin.
Example 3
20 Purification of a 20 kDa Protein from the Conditioned Media of EOMA
Cells whicl~~ Specifically inhibits Endothelial Cell Proliferation
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
25 with relatively high affinity and was eluted with 0.6-0.8 M NaCI in 10
mM Tris pH 7.4, as shown in Figure 2. To further purify the inhibitory
activity, the sam le was concentrated and applied to a el filtration
(Bio-Rad Bio-Gel 100 fine gel or Pharmacia Sephacryl~~200HR gel)
column (see Figure 3), followed by several cycles of reverse-phase
3o 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 Figure 4. After the final C4 column, the inhibitory
activity was associated with a protein of molecular mass of
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approximately 20 kDa (reduced) or 18 kDa (non-reduced), by
SDS-PAGE, purified to apparent homogeneity.
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, CA), and a SynChropak-TPRP-4 (100 x 4.6 mm) C4
reverse-phase columrl (Synchrom, Inc., Lafayette, IN) were prepared
according to the manufacturers recommendations. A heparin-Sepharose
column (50 x 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 NanospinMoncentrator (Gelman
Sciences, Anll Arbor, MA).
A Bio-Gel P-100 column or a Sephacryl S-200 HR column
(75 x 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.
A SynChropak RPG (100 x 4.6mm) column was
equilibrated with H20l0.1 %a trifluoroacetic acid (TFA). HPLC-grade
reagents (Pierce, 'Rockford, IL) 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.
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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,
5 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 C, 10% C02) for 24 hours. The media was
replaced with 0.25 nll of DMEM + 5% BCS + 1% GPS and the test
10 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 Hematal.frFisher Scientific, Pittsburgh, PA), and counted
by Coulter counter.
15 Non-Endothelial Cell Proliferation Assays
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% C02 and 3 7 C incubator. For the proliferation assays, cells
20 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 ce:ll suspensiori (20,000 cells/ml for SMC, RPE, MLE; 15,000
25 cells/ml for 3T3; 10,000 cells/mi for LLC, EOMA) was made with
DMEM + 10% bovine serum + 1% GPS, plated onto 24-well culture
plates (0.5 rnl/well), and incubated (37 C, 10% C02) 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
30 trypsin, resuspended in Hematall (Fisher Scientific, Pittsburgh, PA), and
counted by Coulter counter.
Only endothelial cells were significantly inhibited, as
shown in Table 2.
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TABLE 2
EFFECT OF ENDOSTATIN ON ENDOTHELIAL
AND NON-ENDOTHELIAL CELL PROLIFERATION
INHIBITED NON-INHIBITED
Bovine capillary Bovine aortic smooth
endothelial cells niuscle cells
~ Bovine retinal pigment
epithelial cells
3T3 fibroblasts
Mink lung epithelial cells
FOMA
heman ioendothelioma cells
Lewis Lung carcinoma cells
The inhibi'tion 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 l. log unit
higher than those used to inhibit capillary endothelial cell proliferation
(data not shown).
Example 4
Microsequence Analysis of the 20 kDa Protein Reveals Identity to a
Fragment of Collagen.XVIII
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, CA), detected by
PonceauTN stain, and excised from the membrane. N-terminal sequence
was detennined by automated Edman degradation on an PE/ABD Model
470A pratein sequencer (Foster City, CA) operated with gas-phase
delivery of trifluoracetic acid.
Sequence library searches and alignments were performed
against combined (.TenBanffMBrookhaven Protein, SWISS-PROT, and
PIR databases. Searches were performed at the National Center for
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Biotechnology Information through the use of the BLAST network
service.
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 Relln and Pihlajaniemi (Oh et al., 1994; Rehn and
Pihlajanieini, 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 (NC 1) 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 dornain (Figure 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
Recombinant Mouse Endostatin (Baculovirus or E. coli) Inhibits
Endothelial Cell Proliferation In Vitro and Angiogenesis In Vivo
The endothelial proliferation cell inhibitor of the present
invention can be recorribinantly 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.
Recombinant mouse endostatin was expressed using the
BacPAWbaculovirus 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
pBacPAKg-endostatin clone (modified transfer vector) were then
cotransfected into insect Sf21 cells and media containing expressed
mouse endost.atin 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 x 40
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cm heparin Sepharose column which had been equilibrated with 50 mM
NaC1 10 rnM Tris pH 7.4. The column was washed with the
equilibration buffer aryd was then- eluted sequentially with 0.2 M NaCI,
0.4 M NaC1, 0.6 M NaCI, and 1 M NaCI in 10 mM Tris pH 7.4. All
chromatography was performed at 4 ' C. The 0.6 M NaC1 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 NaCI - 1.2 M NaCI 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.
Expression and Purif a`cation of Recombinant Mouse Endostatin from E.
coli
The C-terminal part of the cDNA of collagen XVIII was
used to amplify the cDNA of mouse endostatin which was cloned into
the pETKHl vector (pETl ld derivative) (Studier et al., 1990). Induction
2o resulted in. the prodluction of a fusion protein carrying the amino acid
sequence MARRASVGTD (SEQ ID NO:2) (RRAS = protein kinase A
recognition sequence) and 6 histidine residues at the N-terminus
followed by the sequence of mouse endostatin (pTB01#8). The
pTBO1#8 plasmid was transformed into BL21:DE3 and the fusion
protein was purified on Ni+2-NTA-beads as described (QiaExpressionisT M
Handbook, Qiagen). Briefly, E. coli were grown until an O.D.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.-HCI pI-1 8.0 containing 10 mM imidazole and
incubated for 1 hour at roorn temperature. The suspension was
centrifuged for 15 nlinutes at 20,000 g and the supernatant incubated
with the Ni+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 T'ris-HCI pH 6.25 containing 10 mM
imidazole. The protein was eluted with the same
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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 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.
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+2-NTA-agarose was
used to purify the E. coli-derived mouse endostatin.
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.
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.
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To test for the ability of recombinant mouse endostatin to inhibit in vivo
angiogenesis, we used the chick chorioallantoic tnembrane (CAM) assay
(Folkman, 1985;
Nguyen et al, 1994 which may be referred to for further details). Briefly,
three day old
fertilized white Leghorn eggs (Spafas, Norwich, CT) were cracked and embryos
with intact
5 yolks were placed in 100 x 20 mm petri dishes (Folkman, 1985). After 3 days
of incubation
(37 C and 3% C02), 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 l of 0.45% methylcellulose (in H20) on teflon rods. After
48 hours of
incubation, embryos and CAMs were observed by means of a stereomicroscope.
10 At doses of 10 - 20 g/10 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 anti-angiogenic effect on the implanted CAMs. In
contrast, the
soluble baculovirus-derived endostatin dissolved within 24 hours and gave a
maximal anti-
15 angiogenic effect wirhin a period oiF 48 hours. There was no evidence of
toxicity in any of the
chick embryos tested.
Human endostatin was produced recombinantly using similar methods.
Example 6
Recombinant Mouse Endostatin Inhibits the Growth of Metastases
20 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 - 1.200 mm3
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
25 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
x 10' cells/ml and the suspension 'was placed on ice. After the site was
cleaned with ethanol,
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the subcutaneous dorsa of mice in the proximal midline were injected
with 1 x 106 cells in 0.1 ml of saline.
When tumors were 1500 mm3 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 4x
magnification.
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
Recombinant Mouse Endostatin Inhibits the Growth of Primary Tumors
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 mm3. 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.
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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.
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.
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 width2 x length x 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 mm3 (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.
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
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43
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 Figures 6 and 7.
Figures 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.
Immunohistochemical analysis (Figure 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.
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 irecombinant
endostatin are due to the specific structure of endostatin and not to a
contaminant in the sample.
Figure 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.
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
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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 mm3, 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).
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.
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).
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.
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).
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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.
5 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.
10 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
15 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.
To test this theory mice seeded with Lewis lung
20 carcinomas, and bearing tumors which had attained a size of
approximately 300 mm3, 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
25 regressed and dormant for approximately three months, even after all
treatment was terminated, as is shown in Figure 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.
30 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
35 of endostatin and angiostatin far exceeds this criteria.
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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.
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
Isolation of the putative receptor for endostatin.
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.
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.
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
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47
using a NaC1 gradient and the different fractions are analyzed 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.
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
analyzed for identical inserts using endonuclease digestion and PCR.
Using these data, complementation groups are formed, sequenced and
analyzed 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).
CA 02235393 2000-08-29
SUBSTITUTE SHEETS
48
Example 9
Determination of the minimal region of the mouse endostatin protein
responsible for its antiangiogenic activity.
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
Determination of the putative enzyme(s) which may release endostatin
from collagen XVIII.
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 (NC 11). 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.
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.
The last 325 amino acid residues, encoding for the NC 11
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
NC 11 domain is cloned into an E. coli expression vector (pET series)
CA 02235393 2003-04-29
49
which allows high expression of t:he target protein after induction with IPTG.
Alternatively,
a vector suitable for insect cell expression is used. The proteins are tagged
with the HIS6
-Tag located on the C-terminus for purification using Nil+-NTA-beads. An Ni2+-
NTA-
alkaline phosphatase conjugate can detect the C-terminus by Western blotting.
Another
construct is made which not only has an HIS6-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.
Cleavage product is detected by SDS-PAGE analysis or Western blotting, the
protein is re-purified using the NiZ+-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-termirial amino acid sequencing is performed and
cornpared 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 LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: The Children's Medical Center Corporation
(B) STREET: 300 Longwood Avenue
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02115
(G) TELEPHONE: (617) 735-7050
(H) TELEFAX: (617) 232-7485
(ii) TITLE OF INVENTION: Therapeutic Antiangiogenic Compositions and
Methods
(iii) NUMBER OF SEQUENCES: 1
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Murine
(F) TISSUE TYPE: Collagen
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 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
