U.S. patent application number 10/369369 was filed with the patent office on 2003-08-21 for methods for inhibiting brain tumor growth.
This patent application is currently assigned to Childrens Hospital Los Angeles. Invention is credited to Laug, Walter E..
Application Number | 20030157098 10/369369 |
Document ID | / |
Family ID | 26815994 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157098 |
Kind Code |
A1 |
Laug, Walter E. |
August 21, 2003 |
Methods for inhibiting brain tumor growth
Abstract
The present invention describes methods for inhibition of tumor
growth in the brain, using antagonists of integrins such as
.alpha..sub.v.beta..sub- .3 and .alpha..sub.v.beta..sub.5.
Antagonists of the present invention can inhibit angiogenesis in
brain tumor tissue. They can also inhibit vitronectin and
tenascin-mediated cell adhesion and migration in brain tumor cells.
They can further induce direct brain tumor cell death.
Inventors: |
Laug, Walter E.; (La
Crescenta, CA) |
Correspondence
Address: |
OLSON & HIERL, LTD.
36th Floor
20 North Wacker Drive
Chicago
IL
60606
US
|
Assignee: |
Childrens Hospital Los
Angeles
|
Family ID: |
26815994 |
Appl. No.: |
10/369369 |
Filed: |
February 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10369369 |
Feb 18, 2003 |
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09489391 |
Jan 21, 2000 |
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6521593 |
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60118126 |
Feb 1, 1999 |
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Current U.S.
Class: |
424/143.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 38/39 20130101; A61P 35/00 20180101; A61P 43/00 20180101; C07K
16/2848 20130101; C07K 16/2839 20130101 |
Class at
Publication: |
424/143.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of inhibiting tumor growth in the brain of a host,
comprising administering to the host in need of such an inhibition
a therapeutically effective amount of an integrin antagonist which
is selected from the group consisting of an antibody against
.alpha..sub.v.beta..sub.3, an antibody against
.alpha..sub.v.beta..sub.5 and a combination of antibodies
respectively against .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5.
2. The method of claim 1, wherein the antagonist is a monoclonal
antibody immunospecific for .alpha..sub.v.beta..sub.3.
3. The method of claim 2, wherein the monoclonal antibody has the
immunoreaction characteristics of a monoclonal antibody designated
LM-609.
4. The method of claim 1, wherein the combination of the antibodies
respectively against .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 is a combination of a monoclonal antibody
immunospecific to .alpha..sub.v.beta..sub.3 and a monoclonal
antibody immunospecific to .alpha..sub.v.beta..sub.5.
5. The method of claim 4, wherein the monoclonal antibody
immunospecific to .alpha..sub.v.beta..sub.3 has the immunoreaction
characteristics of a monoclonal antibody designated LM-609 and the
monoclonal antibody immunospecific to .alpha..sub.v.beta..sub.5 has
the immunoreaction characteristics of a monoclonal antibody
designated P1-F6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 09/489,391 filed on Jan. 21, 2000, now U.S. Pat. No.
6,521,593, which application claims the benefit of U.S. Provisional
Patent Application Serial No. 60/118,126 filed on Feb. 1, 1999.
BACKGROUND OF THE INVENTION
AREA OF THE ART
[0002] The invention relates generally to inhibition of tumor
growth and specifically to inhibition of brain tumor growth.
DESCRIPTION OF THE PRIOR ART
[0003] Throughout this application various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end of this application, preceding
the claims.
[0004] Brain tumors, like other solid tumors, require a perpetually
increasing blood supply to maintain continuous growth beyond 1-2
mm.sup.3 (1,2). This is accomplished through angiogenesis, a
process which occurs in response to endothelial growth factors
released by tumor cells. Angiogenesis involves the induction of
endothelial cell proliferation from quiescent microvasculature,
migration of neoendothelium toward the tumor bed and, finally,
maturation into a new capillary network (3). Brain tumors are the
most angiogenic of all human neoplasias. The principal angiogenic
factors demonstrated by either in situ hybridization or specific
antibodies in tissue sections of patients with glioblastoma and
medulloblastoma, the most common malignant brain tumors, are
vascular endothelial growth factor (VEGF) and basic fibroblast
growth factor (bFGF) (4-7). In fact, VEGF expression and
microvessel density in glial tumors directly correlate with the
degree of malignant characteristics and overall outcome (8-9).
[0005] Recent evidence suggests that angiogenesis is regulated by
the activation of endothelial cell integrins, a family of
transmembrane receptors which direct cell adhesion to extracellular
matrix (ECM) proteins by binding to the amino acid sequence
Arg-Gly-Asp (RGD)(10). In response to bFGF and VEGF, endothelial
cells upregulate the expression of integrins
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5,
respectively (11-13). Glioblastomas and their associated vascular
endothelium have been found to express the integrins
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 (14,15).
Integrin-mediated adhesion results in the propagation of
intracellular signals which promote cell survival, proliferation,
motility and capillary sprouting (16,17). Failure of these
integrins to bind ligand results in endothelial cell apoptosis
(18,19). The matrix glycoprotein, vitronectin, serves as ligand for
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 and is
produced at the leading invasive edge by malignant gliomas (15,20).
Together, these findings suggest a complex paracrine interaction
between tumor cells, brain ECM and endothelial cell integrins for
the continued angiogenesis and growth of malignant brain
tumors.
[0006] Studies using the anti-.alpha..sub.v.beta..sub.3 antibody,
LM-609, or an RGD cyclic peptide antagonist of
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5, which
prevents integrin-ECM interactions, have demonstrated an
anti-angiogenesis response in the chicken chorioallantoic membrane
(CAM) and a mouse chimera model (21-23). Other agents which act
upon alternative sites of the angiogenesis pathway, such as
antibodies to VEGF or its tyrosine kinase receptor fit, have also
been effective in inhibiting angiogenesis (24,25).
[0007] Prior studies have shown that the attachment of breast
carcinoma, melanoma and HT29-D4 colonic adenocarcinoma cells to
vitronectin is dependent on .alpha..sub.v,
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5,
respectively (51-53). Vitronectin, which is produced by tumor and
endothelial cells, is recognized by .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 and is an ECM protein found at sites of
tumor invasion and neovascularization (54-55). Thus, in addition to
supporting endothelial cell survival through .alpha..sub.v
ligation, and hence angiogenesis, vitronectin expression may
further enhance the adhesion of tumor and endothelial cells which
express .alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5
integrins, thereby promoting their invasion. In one study using a
SCID mouse/human chimeric model for breast cancer, tumor invasion
was considerably reduced following the administration of the
anti-.alpha..sub.v.beta..sub.3 antibody LM-609, suggesting a direct
effect upon the tumor cell biology through a
.alpha..sub.v.beta..sub.3 blockade (56).
[0008] Brain tumors, because of their highly invasive nature and
degree of angiogenesis, afford an excellent model with which to
further study the importance of integrins in tumor progression.
Multiple studies have shown that microvessel density correlates
with outcome and malignant grade in astrocytomas (57-59).
Angiogenesis inhibitors, such as TNP-470, thrombospondin-1 and
platelet factor 4, have been introduced into experimental brain
tumors and have shown an inhibition of tumor growth (60-62).
However, to date, no study has examined the effect of integrin
antagonism on brain tumor invasion and angiogenesis. Therefore, a
need exists to study the effect and thus provide a novel method for
treating brain tumors.
SUMMARY OF THE INVENTION
[0009] The present invention is based on the surprise discovery
that targeted antagonism of integrins, specifically
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5, can
substantially inhibit brain tumorigenesis in vivo. It is also based
on the discovery that the microenvironment of the brain tumor is
critical to the tumor behavior and in determining its
responsiveness to such biologically directed therapies. The
invention is further based on the discovery that integrin
antagonism can have an anti-tumorigenic effect independent of
anti-antiogenesis, which may act synergistically to retard tumor
growth. For example, it is a discovery of the present invention
that integrin antagonism may induce direct brain tumor cell
death.
[0010] Accordingly, one aspect of the present invention provides a
method of inhibiting tumor growth in the brain of a host. The
method comprises administering to the host in need of such an
inhibition a therapeutically effective amount of an antagonist of
an integrin.
[0011] In one embodiment of the present invention, the integrin may
be .alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5. The
antagonist may be a polypeptide antagonist of .alpha..sub.v, an
antibody against .alpha..sub.v.beta..sub.3, an antibody against
.alpha..sub.v.beta..sub.5 or a combination of antibodies
respectively against .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5.
[0012] Another aspect of the present invention provides a method
for inhibiting angiogenesis in a tumor tissue located in the brain
of a host. The method comprises administering to the host a
composition comprising an angiogenesis-inhibiting amount of an
antagonist of an .alpha..sub.v integrin.
[0013] In one embodiment of the present invention, the intergrin is
.alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5. The
antagonist is a polypeptide antagonist of .alpha..sub.v, an
antibody against .alpha..sub.v.beta..sub.3 an antibody against
.alpha..sub.v.beta..sub.5 or a combination of antibodies
respectively against .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5.
[0014] A further aspect of the present invention provides a method
of inhibiting ECM-dependent cell adhesion of brain tumor cells
growing in the brain of a host. The method comprises administering
to the host a therapeutic effective amount of an antagonist of an
.alpha..sub.v integrin, i.e., integrins .alpha..sub.v.beta..sub.3
or .alpha..sub.v.beta..sub.5. In one embodiment of the present
invention, the antagonist is a polypeptide antagonist of
.alpha..sub.v or a combination of antibodies respectively against
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5.
[0015] Yet another aspect of the present invention provides a
method of inhibiting vitronectin-dependent cell migration in brain
tumor cells growing in the brain of a host. The method comprises
administering to the host a therapeutically effective amount of an
antagonist to .alpha..sub.v.
[0016] In one embodiment of the present invention, the antagonist
is a polypeptide antagonist of .alpha..sub.v or an antibody against
.alpha..sub.v.beta..sub.3.
[0017] A further aspect of the present invention provides a method
of inducing apoptosis in tumor cells growing in the brain of a
host. The method comprises administering to the host a
therapeutically effective amount of an antagonist of an
integrin.
[0018] In one embodiment of the present invention, the integrin may
be .alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5. The
antagonist may be a polypeptide antagonist of .alpha..sub.v.
[0019] The methods of the present invention are well suited for use
in treating brain tumors in vivo. The present invention provides a
novel therapeutic approach to treat brain tumors.
DESCRIPTION OF THE FIGURES
[0020] The above-mentioned and other features of this invention and
the manner of obtaining them will become more apparent, and will be
best understood, by reference to the following description, taken
in conjunction with the accompanying drawings. These drawings
depict only a typical embodiment of the invention and do not
therefore limit its scope. They serve to add specificity and
detail, in which:
[0021] FIGS. 1a and 1b show the inhibition of angiogenesis (a) and
tumor growth on the CAM (b) by .alpha..sub.v-antagonist.
[0022] FIG. 2 shows the tumor size (A) and mouse survival (B) after
intracerebral injection of DAOY and U87MG cells.
[0023] FIGS. 3a and 3b show histopathology of orthotopically
injected brain tumor cells DAOY (a) and U87MG (b), daily treated
with the inactive (A) or active peptide (B-D). Large intracerebral
tumors (arrowheads) are visible in the control animals (A), whereas
no tumors (B) or only microscopic residual tumors (arrowheads) are
detected in the .alpha..sub.v-antagonist-treated animals (C and
D).
[0024] FIG. 4 shows the survival of mice after orthotopical brain
tumor implantation, receiving either the active or inactive
peptide.
[0025] FIG. 5 shows the effect of .alpha..sub.v-antagonist on
orthotopically (brain) and heterotopically (subcutis) implanted
DAOY cells.
[0026] FIGS. 6a-6d show the integrin profile (a), and effect of
.alpha.v-antagonist on adhesion to (b), migration on vitronectin
(c) and cell viability on vitronectin (d) for brain tumor and brain
capillary endothelial cells.
[0027] FIG. 7 shows the effect of cyclic pentapeptide on tumor cell
adhesion to ECM proteins.
[0028] FIG. 8 shows the effect of inhibition of adhesion, resulting
in cell death (apoptosis) in both brain tumor cells and brain
capillary cells. This effect is restricted to the ECM vitronectin
and tenascin.
[0029] FIG. 9 shows the immunohistochemistry of U87 brain tumors
xenotransplanted into the forebrain of nude mice and treated with
an active (anti-.alpha.v) or control peptide.
DETAILED DESCRIPTION OF THE INVENTION
[0030] One aspect of the present invention provides a method of
inhibiting tumor growth in the brain of a host, comprising
administering to the host in need of such an inhibition a
therapeutically effective amount of an antagonist of an
integrin.
[0031] The method of the present invention may be used to treat any
tumors that grow in the brain, as long as the growth of tumors in
the brain requires the interaction of the integrin with its ligand.
Examples of such a tumor include, but are not limited to,
glioblastoma, medulloblastoma (astrocytoma, other primitive
neuroectoderma and brain stem glioma cancers). For the purpose of
the present invention, preferably, the tumor growth is located
intracerebrally in the brain of a host. The host may be any mammal,
including, but not limited to, rat and human. The tumor growth is
inhibited if the growth is impaired by the treatment.
[0032] For the purpose of the present invention, an integrin is any
member of a specific family of homologous heterodimeric
transmembrane receptors. The receptors direct cell adhesion by
binding to the amino acid sequence Arg-Gly-Asp (RGD). The receptors
may be expressed on both tumor and normal cells. The characteristic
of integrins are well known and well characterized in the art and
are described in detail in the cited references (1, 2), the
relevant content of which is incorporated herein by reference.
Examples of integrins include, but are not limited to, the
.alpha..sub.v family, such as .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.v.beta..sub.1,
.alpha..sub.v.beta..sub.6 .alpha..sub.v.beta..sub.8 and the
like.
[0033] An antagonist of an integrin is a molecule that blocks or
inhibits the physiologic or pharmacologic activity of the integrin
by inhibiting the binding activity of the integrin, a receptor, to
its ligand, various matrix proteins, including, but not limited to,
vitronectin, tenascin, fibronectin and collagen I. In one
embodiment of the present invention, an antagonist of an integrin
may be an antagonist of integrin .alpha..sub.v.beta..sub.3 or
integrin .alpha..sub.v.beta..sub.5. Preferred integrin antagonists
can be either a monoclonal antibody, a fragment of the monoclonal
antibody, or a peptide.
[0034] For example, an antagonist of .alpha..sub.v.beta..sub.3 may
be any factor that inhibits the binding of
.alpha..sub.v.beta..sub.3 to one of its multiple ligands, namely
vitronectin or tenascin. Examples of antagonists of
.alpha..sub.v.beta..sub.3 are described in the PCT publications WO
96/37492 and WO 97/45137, the relevant content of which is
incorporated herein by reference.
[0035] Likewise, an antagonist of .alpha..sub.v.beta..sub.5 may be
any factor that inhibits the binding of .alpha..sub.v.beta..sub.5
to its ligand, namely vitronectin. Examples of antagonists of
.alpha..sub.v.beta..sub.5 are described in the PCT publication WO
97/06791, the relevant content of which is incorporated herein by
reference.
[0036] In one embodiment of the present invention, the antagonist
is a polypeptide antagonist of .alpha..sub.v, namely, a polypeptide
antagonist of .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5. Preferably, the polypeptide is an
Arg-Gly-Asp (RGD)-containing polypeptide. In one embodiment, the
polypeptide antagonist is an RGD cyclic pentapeptide antagonist of
.alpha..sub.v.
[0037] In accordance with another embodiment of the present
invention, the antagonist may be a monoclonal antibody
immunospecific for .alpha..sub.v.beta..sub.3 or
.alpha..sub.v.beta..sub.5. Alternatively, it may be a combination
of antibodies respectively against .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5. In one embodiment, a monoclonal antibody
immunospecific for .alpha..sub.v.beta..sub.3 has the immunoreaction
characteristics of a monoclonal antibody designated LM-609. In
another embodiment of the present invention, a monoclonal antibody
immunospecific for .alpha..sub.v.beta..sub.5 has the immunoreaction
characteristics of a monoclonal antibody designated P1-F6. Both the
LM-609 antibody and P1-F6 antibody are well known in the industry
and are commercially available through Chemicon, Temecula,
Calif.
[0038] For the purpose of the present invention, the antagonists
may be used alone or in combination with each other in a method of
the present invention for inhibiting tumor growth in the brain.
[0039] The therapeutically effective amount is an amount of
antagonist sufficient to produce a measurable inhibition of a tumor
growth in the brain of a host being treated. Inhibition of tumor
growth can be determined by microscopic measurement after staining,
as described herein, by mouse brain MRI scanning, or by 3H-Thymidin
Incorporation, which methods are well known to one skilled in the
art.
[0040] Insofar as an integrin antagonist can take the form of an
RGD-containing peptide, an anti-.alpha..sub.v.beta..sub.3
monoclonal antibody or fragment thereof, an
anti-.alpha..sub.v.beta..sub.5 monoclonal antibody or a fragment
thereof, or a combination of the monoclonal antibodies of
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5, it is to
be appreciated that the potency, and therefore the expression, of a
"therapeutically effective amount" can vary. However, as shown by
the present assay methods, one skilled in the art can readily
assess the potency of a candidate antagonist of this invention.
[0041] The potency of an antagonist can be measured by a variety of
means including, but not limited to, inhibition of angiogenesis in
the CAM assay, in the in vivo brain tumor assay, and by measuring
inhibition of the binding of natural ligands to integrins such as
.alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5, all as
described herein, and like assays.
[0042] The dosage ranges for the administration of the antagonist
depend upon the form of the antagonist and its potency to a
particular integrin. One skilled in the art can find out the proper
dosage for a particular antagonist in view of the disclosure of the
present invention without undue experimentation. The dosage should
be large enough to produce the desired effect in which tumor growth
in the brain is inhibited. The dosage should not be so large as to
cause adverse side effects, such as brain edema or the rapid
release of cytokines from brain tumors inducing kachexia, for
example, when an integrin antagonist of the present invention is
administered in the form of a polypeptide. The dosage per kg body
weight can vary from 1 to 20 mg, in one or more dose
administrations daily, for one or several days or indefinitely.
When an integrin antagonist of the present invention is
administered in the form of a monoclonal antibody, the dosage can
vary from 1 to 20 mg/kg, in one dose administrations once to twice
weekly for an indefinite time.
[0043] The polypeptide or monoclonal antibodies of the present
invention can be administered parenterally by injection or by
gradual infusion over time. Although the tissue to be treated is
most often by intraperitoneal or subcutaneous (antibody)
administration, the antagonists of the present invention may also
be administered intraocularly, intravenously, intramuscularly,
intracavity, transdermally, and can be delivered by peristaltic
means.
[0044] The compositions are administered in a manner compatible
with the dosage formulation and in a therapeutically effective
amount. The quantity to be administered and timing of
administration depend on the subject to be treated, capacity of the
subject's system to utilize the active ingredient, and degree of
therapeutic effect desired. Precise amounts of the active
ingredient required to be administered depend on the judgement of
the practitioner and are peculiar to each individual. However,
suitable dosage ranges for systemic application are disclosed
herein and depend on the route of administration. Suitable regimes
for administration are also variable but are typified by an initial
administration, followed by repeated doses at one or more hour
intervals by a subsequent injection or other administration.
[0045] In accordance with one embodiment of the present invention,
the present invention also provides a pharmaceutical composition
useful for practicing the therapeutic methods described herein. The
compositions contain an antagonist of the present invention in
combination with a pharmaceutically acceptable carrier. As used
herein, the terms "pharmaceutically acceptable," "physiologically
tolerable" and grammatical variations thereof, as they refer to
compositions, carriers, dilutents and reagents, are used
interchangeably and represent that the materials are capable of
administration to or upon a mammal without the production of
undesirable physiological effects.
[0046] Preparations for parental administration of a peptide or an
antibody of the invention include sterile aqueous or non-aqueous
solutions, suspension, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parental vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present, such as, for example, antimicrobials, antioxidants,
chelating agents, and inert gases and the like.
[0047] Another aspect of the present invention provides a method
for inhibiting angiogenesis in a tumor tissue located in the brain
of a host. The method comprises administering to the host a
composition comprising an angiogenesis-inhibiting amount of an
antagonist of an integrin.
[0048] As discussed in the background section, angiogenesis is the
formation of a neovascular network from pre-existing host vessels
and is required for tumor growth beyond 1-2 mm.sup.3. For the
purpose of the present invention, angiogenesis is inhibited if
angiogenesis and the disease symptoms mediated by angiogenesis are
ameliorated.
[0049] In one embodiment of the present invention, the tumor tissue
is located intracerebrally in the brain of a host. The host may be
any mammal. Examples of the host include, but are not limited to,
mouse, rat and human.
[0050] The dosage ranges for the administration of the antagonist
depend upon the form of the antagonist and its potency to a
particular integrin. One skilled in the art can find out the proper
dosage for a particular antagonist in view of the disclosure of the
present invention without undue experimentation. The dosage should
be large enough to produce the desired effect in which angiogenesis
and the disease symptoms mediated by angiogenesis are ameliorated.
The dosage should not be so large as to cause adverse side effects,
such as brain edema due to rapid tumor lysis with cytokine release
or hemorrhage.
[0051] In one embodiment of the present invention, an antagonist of
an integrin may be an antagonist of integrin
.alpha..sub.v.beta..sub.3 or integrin .alpha..sub.v.beta..sub.5.
Preferred integrin antagonists can be either a monoclonal antibody
or a peptide. In one embodiment of the present invention, the
antagonist is a polypeptide antagonist of .alpha..sub.v, namely, a
polypeptide antagonist of .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5. Preferably, the polypeptide is an
Arg-Gly-Asp (RGD)-contained polypeptide. In one embodiment, the
polypeptide antagonist is an RGD cyclic pentapeptide antogonist of
.alpha..sub.v.
[0052] In accordance with another embodiment of the present
invention, the antagonist may be an antibody against
.alpha..sub.v.beta..sub.3 and a combination of antibodies
respectively against .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5.
[0053] The therapeutically effective amount is an amount of
antagonist sufficient to produce a measurable inhibition of
angiogenesis in the tisuue being treated, i.e., an
angiogenesis-inhibiting amount. Inhibition of angiogenesis can be
measured in situ by immunohistochemistry, as described herein, or
by other methods known to one skilled in the art.
[0054] In accordance with one embodiment of the present invention,
the present invention also provides a pharmaceutical composition
useful for practicing the therapeutic methods described herein. The
compositions contain an antagonist of the present invention in
combination with a pharmaceutically acceptable carrier.
[0055] A further aspect of the present invention provides a method
of inhibiting ECM-dependent cell adhesion in brain tumor cells
growing in the brain of a host, comprising administering to the
host a therapeutically effective amount of an antagonist to
integrins .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5.
[0056] For the purpose of the present invention, ECM-dependent cell
adhesion includes any cell adhesion that is ECM-depedent and that
is mediated by .alpha..sub.v-integrins. Examples of such adhesion
include, but are not limited to, vitronectin-dependent cell
adhesion and tenascin-dependent cell adhesion. It is a discovery of
the present invention that human brain tumors may produce
vitronectin and tenascin, and those ECM play an important role in
tumor cell adhesion and migration by interacting with integrins.
For the purpose of the present invention, the inhibition is
achieved if tumor cell adhesion to ECM is reduced.
[0057] The phrase "therapeutically effective amount" as used herein
indicates an amount of antagonist that is sufficient so that
ECM-mediated tumor cell adhesion is reduced. Cell adhesion can be
measured by the methods described herein and by the methods
commonly known in the art.
[0058] In accordance with one embodiment of the present invention,
the antagonist may be a polypeptide antagonist of .alpha..sub.v or
a combination of antibodies respectively against
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5. For
example, the antagonist may be an RGD cyclic pentapeptide
antagonist of .alpha..sub.v or a combination of monoclonal
antibodies designated LM-609 and P1-F6.
[0059] Another aspect of the present invention provides a method of
inhibiting vitronectin-dependent cell migration in brain tumor
cells growing in the brain of a host, comprising administering to
the host a therapeutically effective amount of an antagonist to
.alpha..sub.v.beta..sub.3.
[0060] For the purpose of the present invention, the inhibition is
achieved if tumor cell migration is reduced.
[0061] The phrase "therapeutically effective amount" as used herein
indicates an amount of antagonist that is sufficient so that
vitronectin-mediated tumor cell migration is reduced. Cell
migration can be measured by the methods described herein and by
the methods commonly known in the art.
[0062] In accordance with one embodiment of the present invention,
the antagonist may be a polypeptide antagonist of .alpha..sub.v or
a monoclonal antibody immunoreactive against
.alpha..sub.v.beta..sub.3. For example, the antagonist may be an
RGD cyclic pentapeptide antogonist of .alpha..sub.v or a monoclonal
antibody designated LM-609.
[0063] The present invention also provides a method of inducing
apoptosis in tumor cells growing in the brain of a host. The method
comprises administering to the host a therapeutically effective
amount of an antagonist of an integrin.
[0064] For the purpose of the present invention, brain tumor cell
apoptosis is induced if an increased amount of tumor cell apoptosis
is observed in brain after the administration of the antagonist.
The therapeutically effective amount is an amount of antagonist
sufficient to produce a measurable tumor cell apoptosis in the
brain of a host being treated. Tumor cell apoptosis in brain may be
measured by methods described herein or commonly known in the
art.
[0065] In accordance with one embodiment of the present invention,
the integrin may be .alpha..sub.v, .alpha..sub.v.beta..sub.3 or
.alpha..sub.v.beta..sub.5. The antagonist may be a polypeptide
antagonist of .alpha..sub.v.
EXAMPLES
Materials and Methods
[0066] Materials: The active cyclic RGD pentapeptide EMD 121974,
cyclo(Arg-Gly-Asp-D-Phe-[N-Me]-Val), and the inactive control
peptide cRAD (EMD 135981) were provided by A. Jonczyk, Ph.D., Merck
KgaA, Darmstadt, Germany. The monoclonal antibodies LM609 and P1F6
have been described (13). The brain tumor cell lines DAOY and U87MG
were purchased from ATCC, Rockville, Md. Primary cultures of human
brain capillary endothelial cells were provided by M. Stins, Ph.D.,
Childrens Hospital, Los Angeles (49). Human vitronectin was
purchased from Promega, Madison, Wis.
[0067] FACS analysis: A FACScan cytometer (Becton-Dickinson, San
Jose) was used. Conditions were as previously described (43).
Primary antibodies were LM 609 and P1F6 at 1:100, and secondary Ab
was FITC labeled goat anti-mouse IgG at 1:250. Apoptosis
determination was as per the manufacturer's instruction (Clontech,
Palo Alto, Calif., Apo Alert Annexin V-FITC Apoptosis kit).
[0068] Adhesion: Assay conditions were as previously described
(15). Non-tissue culture treated wells incubated overnight at
4.degree. C. with vitronectin (1-10 .mu.g/ml PBS), washed and
blocked for 30 min. with heat denatured 1% BSA in PBS, followed by
washing with PBS. Control and test cells, pre-incubated at
37.degree. C. with the test substance (20 .mu.g/ml) in adhesion
buffer for 30 min. were plated into wells (5.times.10.sup.4
cells/well) and incubated for 1 hr at 37.degree. C. After gentle
washing of the wells with adhesion buffer, adhering cells were
fixed and stained with crystal violet, the dye solubilized in
methanol and the OD determined at 600.degree. A.
[0069] Migration: Transwell polycarbonate filters (8 .mu.m pore
size) were incubated for 30 min. at 37.degree. C. with vitronectin
in PBS (1 .mu.g/ml on the upper side and 10 .mu.g on the bottom
side), blocked for 30 min. with 1% heat denatured BSA and washed
with PBS. Test cells (5.times.10.sup.5/well) were added in adhesion
buffer containing the test substances indicated (20 .mu.g/ml) and
incubated for 4 hrs at 37.degree. C. with the lower chamber
containing adhesion buffer. Cells in the upper chamber were removed
with a cotton swab and cells on the bottom part of the filter were
fixed and stained with crystal violet and the number of migrated
cells determined by counting.
[0070] Apoptosis: Adhesion conditions were as above, except that 12
well plates covered with vitronectin and 5.times.10.sup.5
cells/well were plated in adhesion buffer. After 30 min. of
incubation at 37.degree. C., the adhesion buffer was replaced with
buffer containing 20 .mu.g/ml of active cRGD or inactive cRAD
peptide and incubated further for 4 hrs. Attached cells were
trypsinized and combined with the detached cells in the supernatant
and then examined for the presence of apoptotic cells using the Apo
Alert Annexin V-FITC apoptosis kit (Clontech).
[0071] CAM assay: Egg supplier and preparation have been described
earlier (23). Tumor cells were plated in 50 .mu.l PBS at
4.times.10.sup.6 cells/egg for DAOY and 3.5.times.10.sup.6
cells/egg for U87MG. Cells were grown to tumors for 7 days, then
harvested under sterile conditions, trimmed to similar sizes and
repeated onto the CAM of 10-day-old embryos. The following day the
active or inactive peptide (100 .mu.g/egg) was injected into a CAM
vein. Tumors were photographed in situ after 7 days of growth, then
harvested, weighed and fixed in 4% buffered formalin and embedded
in paraffin. After serial sectioning, slides were stained with
hematoxilin and eosin.
[0072] Brain tumor model: Details of the model have been described
by us (31). Tumor cells (10.sup.6/10 .mu.l PBS) were injected
intracerebrally at the coordinates mentioned. Intraperitoneal
treatment with the active cRGD or the inactive cRAD peptide (100
.mu.g/50 .mu.l/mouse) was initiated on day 3 after implantation for
U87MG and on day 10 for DAOY cells and repeated daily until
cachexia and/or moribund status occurred. The animals were then
sacrificed by CO.sub.2 anesthesia, the brains removed and either
snap frozen in liquid nitrogen or fixed in buffered formalin,
embedded in paraffin and the cut sections stained with
Hematoxylin-Eosin.
[0073] Subcutaneous tumor growth: For subcutaneous tumor growth,
the cells (10.sup.6 mouse) were injected s.c. below the right
shoulder pad immediately following intracerebral injection.
[0074] Animal studies: Animal studies were done according to the
NIH guidelines and approved by the local animal care committee.
Experiments
Angiogenesis on CAM
[0075] To assess the effect of .alpha.v antagonism on brain tumor
associated angiogenesis, DAOY and U87MG human brain tumor cells
were growrron chick chorioallantoic membranes (CAMs). The tumors
were allowed to grow for 7 days before they were removed and
reimplanted onto fresh CAMs. 24 hours after transfer, 100 .mu.g of
either the active cRGD peptide or control peptide (cRAD) was
injected into a CAM vein. Tumors were grown for an additional 6
days and then weighed and analyzed for vascularization. Under
stereomicroscope examination, tumors receiving the control peptide
exhibited extensive angiogenesis, while tumors treated with the
.alpha.v-antagonist showed significant suppression of angiogenesis
(FIG. 1a). Tumor growth was also inhibited by the
.alpha.v-antagonist. In contrast to control tumors, which increased
80% in weight, .alpha.v-antagonist treated tumors decreased 30% in
weight (FIG. 1b). These data suggest that the inhibition of tumor
growth by .alpha.v-antagonism resulted as a consequence of
disrupted tumor-associated angiogenesis.
[0076] FIGS. 1a and 1b show the inhibition of angiogenesis (a) and
tumor growth on the CAM (b) by an .alpha..sub.v-antagonist. Brain
tumors grown on CAMs were harvested and cut to similar sizes and
placed on fresh CAMs of 10-day-old chicken eggs. The following day,
100 .mu.g of active .alpha..sub.v-inhibitor or control peptide were
injected into the chicken veins and grown for another 6 days. Eggs
which received the control peptide showed significant angiogenesis
(a) (DAOY=A and U87MG=C), while significant suppression of
neovascularization was observed in the eggs receiving the active
peptide (DAOY=B and U87MG=D). Tumors placed on the CAM were weighed
at the beginning and end of the experiment (b). The weight of the
tumors treated with the control peptide increased by 80% for both
tumor types (left, n=8), whereas it decreased by about 30% when the
active peptide was administered (right, n=8).
Orthotopic Tumor Model
[0077] DAOY and U87MG cells (10.sup.6 cells/mouse) were
stereotactically injected into the right frontal cortex of nu/nu
mice in order to establish a system that recapitulates the brain
microenvironment. This method allowed for a highly reproducible
model of human brain tumorigenesis and enabled the measurement of
tumor growth and mouse survival over time prior to the introduction
of .alpha.v-antagonism (FIGS. 2a and 2b). FIG. 2 shows the tumor
size (A) and mouse survival (B) after intracerebral injection of
DAOY and U87MG cells. U87MG cells show a rapid growth and reach a
plateau of about 6 mm in 6 weeks, whereas the DAOY cells grow
slower and reach a diameter of 5.5 mm in 9 weeks (A). All animals
are dead by week 9 (B). For each time, point n=5 or 6.
[0078] In order to test the effect of the cRGD peptide in this
model, tumors were first grown for 7 days (DAOY) or 3 days (U87MG)
before the daily i.p. administration of the .alpha.v-antagonist
(100 .mu.g/mouse) or its inactive control for an additional 3
weeks. At the time of sacrifice (4 weeks total time of tumor
growth), control mice had grossly visible brain tumors averaging 3
mm (DAOY) and 5.5 mm (U87MG) in size. Treated mice had either no
tumor or only scant residual tumor cells found in microscopic
clumps along the ventricular and dural surfaces (FIGS. 3a and 3b).
FIGS. 3a and 3b show the histopathology of orthotopically injected
brain tumor cells DAOY (a) and U87MG (b), daily treated with the
inactive (A) or active peptide (B-D). Large intracerebral tumors
(arrowheads) are visible in the control animals (A), whereas no
tumors (B) or only microscopic residual tumors (arrowheads) are
detected in the .alpha..sub.v-antagonist-treated animals (C and
D).
[0079] The treated mice also maintained their baseline weight of 21
g; however, the DAOY and U87MG control mice weighed 16 g and 15 g,
respectively, representing a loss of 5-6 g from baseline weight
(see Table 1). The mean time to the onset of neurologic symptoms in
the control group was 2.5 weeks for U87MG and 4.5 weeks for DAOY
mice, while treated animals showed no evidence of neurological
symptoms at any time.
1 TABLE 1 Tumor size (mm) Cell Line Brain Subcutis Mouse weight
(gr) DAOY Control peptide 3.0 .+-. 0.71 18 .+-. 1.41 16 .+-. 1.3
Active peptide NM 17 .+-. 0.84 21 .+-. 0.89 U87MG Control peptide
5.5 .+-. 0.55 20 .+-. 1.0 15 .+-. 0.45 Active peptide NM 19 .+-.
0.77 20 .+-. 0.71 NM = Not measurable
[0080] Additional animals were tested for survival. Survival was
measured as the time point in which mice required sacrifice, due to
a moribund state. All control animals were sacrificed prior to 6
weeks tumor growth and the majority (>50%) in both control
groups were sacrificed by weeks 3-4 (FIG. 4). FIG. 4 shows the
survival of mice after orthotopical brain tumor implantation,
receiving either the active or inactive peptide; i.p. treatment
(100 .mu.g/mouse/day) was initiated on day 3 for U87MG and day 7
for DAOY cells. N for DAOY and U87MG control is 16 mice each and 32
mice for treated DAOY and 24 for treated U87MG. FIG. 4 shows that
none of the treated animals required sacrifice and, presently, the
treated DAOY mice have survived up to 24 weeks and U87MG mice 15
weeks since tumor cell injection. None of the treated animals have
developed any neurological signs and all continue to grow
normally.
Heterotopic Tumor Model
[0081] To determine the influence of the microenvironment on tumor
growth subjected to .alpha.v-antagonism, we injected the same human
brain tumor cells subcutaneously (10.sup.6 cells/nude mouse) before
initiating treatment with the active and control peptides (100
.mu.g/mouse daily i.p.) at 3 (U87MG) and 7 days after tumor cell
injection (DAOY). Under these conditions, there was no inhibition
of tumor growth by the active peptide, and after 6 weeks, the
tumors from the treated and untreated groups appeared identical on
both the macroscopic and microscopic level, each demonstrating the
presence of extensive angiogenesis (data not shown). This indicates
that the intrinsic properties of the brain tumor cells tested are
not sufficient to render them sensitive to the activity of the cRGD
peptide antagonist, but rather require conditions unique to the
brain microenvironment.
[0082] To further investigate this finding, we simultaneously
injected the DAOY and U87MG cells intracranially and subcutaneously
before initiating treatment with the peptides as outlined under the
orthotopic model. FIG. 5 shows the effect of
.alpha..sub.v-antagonist on orthotopically (brain) and
heterotopically (subcutis) implanted DAOY cells. Tumor cells
(10.sup.6) were injected into the brain and the subcutis and
treatment with the inactive (a) or active peptide initiated on day
7 (b). Pictures were taken at week 4. Animals receiving either the
inactive (a) or active (b) peptide showed a similar size of
subcutaneous tumors (A) and vascularization (B). In both
conditions, the s.c. tumors grew into the underlying muscle layer
(arrowheads) (C). In contrast, the control animals developed large
brain tumors (arrowheads), while the mice receiving the active
peptide showed absence or microscopic residual disease (D).
Therefore, FIG. 5 shows that the subcutaneous tumors in the control
and treated groups both grew to an average size of 18 mm.sup.3 with
evidence of extensive vascularization (FIG. 5). The control mice
also developed large brain tumors similar in size to that
previously observed in the orthotopic tumor model, as well as
clinical evidence of neurologic compromise (Table 1). In contrast,
mice receiving the active peptide survived without evidence of
neurologic symptoms, and necropsy revealed the absence of
intracerebral tumor or only residual microscopic tumor cell
clusters (FIG. 5). However, this group had progressive growth of
their subcutaneous tumors, which necessitated sacrifice after 10
weeks. This confirmed that tumors growing intracerebrally are
responsive to the treatment with the cyclic RGD peptide, while the
same tumors growing subcutaneously are not.
Integrin Expression
[0083] Vitronectin is a matrix protein which could influence the
different biological responses observed in the brain and
subcutaneous compartments. It is abundant in extraneural sites, but
is not normally produced by glial and neuronal tissue (15, 20).
However, malignant glioma cells synthesize vitronectin, which in
turn may be crucial for allowing their attachment and spread (15).
Alternatively, vitronectin may promote endothelial cell adhesion
and migration toward the tumor bed and thus enhance angiogenesis.
Since cell attachment to vitronectin is mediated by the integrins
.alpha.v.beta.3 and .alpha.v.beta.5, we performed FACS analysis to
determine whether the human DAOY medulloblastoma, U87MG
glioblastoma and primary cultures of human brain endothelial cells
(HBEC) express the integrins .alpha.v.beta.3 and .alpha.v.beta.5
(FIG. 6a).
[0084] FIGS. 6a-6d show the integrin profile (a), effect of
.alpha..sub.v-antagonist on adhesion to (b), migration on (c) and
cell viability on vitronectin (d) for brain tumor and brain
capillary endothelial cells. Both DAOY and U87MG tumor cells and
brain capillary cells express the integrins
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 (a).
Adhesion to vitronectin was significantly inhibited by the active
peptide, as well as by the combination of antibodies to
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 while
neither the control peptide nor the specific
anti-.alpha..sub.v.beta..sub.3 nor anti-.alpha..sub.v.beta..sub.5
antibody alone had any significant effect. Migration of the cells
on a vitronectin gradient was abolished by
.alpha..sub.v.beta..sub.3 antibodies and the .alpha..sub.v
antagonist, while neither the control peptide nor antibodies to
.alpha..sub.v.beta..sub.5 had any inhibitory effect. Cell viability
was tested by letting the cells attach to vitronectin for 1 hour,
and then exposing them for 4 hours to the control peptide or
.alpha..sub.v-antagonist (20 .mu.g/ml) (d). Adherent and floating
cells were combined and examined by FACS for apoptosis, using FITC
labeled anti-annexin V and propidium iodide. While little apoptosis
was observed in the cultures exposed to the control peptide (d,
left), both brain capillary endothelial cells and DAOY cells showed
significant numbers of apoptotic cells (d, right).
[0085] FIGS. 6a-6d show that the U87MG cells express high levels of
.alpha.v.beta.3 relative to DAOY cells, but the latter express
higher amounts of .alpha.v.beta.5. HBEC cells grown in
VEGF-containing medium express high amounts of .alpha.v.beta.5,
while 50% of the cells express the .alpha.v.beta.3 integrin in
short-term culture. Cells were also tested by FACS analysis,
following 2 days growth in culture on plates coated with
vitronectin. Expression of .alpha.v.beta.3 and .alpha.v.beta.5 did
not change under these conditions (data not shown).
Vitronectin Adhesion
[0086] To determine if .alpha.v.beta.3 and .alpha.v.beta.5 regulate
DAOY, U87MG and HBEC adhesion to vitronecin, the cells were plated
in vitronectin-coated wells and allowed to adhere in the presence
or absence of either blocking antibodies to .alpha.v.beta.3
(LM-609) and .alpha.v.beta.5 (P1-F6) or the cRGD peptide. P1-F6
minimally inhibited the attachment of DAOY cells, while LM-609 had
no effect on the attachment of any of the cells. Significant
inhibition of adhesion of all cells was observed by incubating
P1-F6 and LM-609 together, or cRGD alone (FIG. 6b). These data
indicate that blocking either integrin separately is insufficient
and that dual blockade of .alpha.v.beta.3 and .alpha.v.beta.5 is
required to significantly disrupt the vitronectin-dependent
adhesion demonstrated by the cells tested.
Vitronectin Migration
[0087] To address whether .alpha.v.beta.3 and .alpha.v.beta.5
modulate migration on vitronectin, independent of adhestion, we
tested the same cells on vitronectin-coated membranes in Boyden
chambers while in the presence of blocking antibodies or cRGD.
P1-F6 did not effect migration; however, LM-609 and cRGD
significantly inhibited the migration of all three cell types (FIG.
6c). Since LM-609, in the absence of P1-F6, was incapable of
blocking adhesion, this anti-migratory effect must be mediated
through a distinct pathway which is independent of forces
responsible for adhesion. Furthermore, LM-609 blocked migration
equally well as cRGD, suggesting that the .alpha.v.beta.3-mediated
cell signaling is a critical component in the determination of the
migration of the brain tumor and brain endothelial cells
tested.
Apoptosis
[0088] High-grade astrocytomas produce vitronectin at the leading
invasive edge, suggesting that this protein plays an important role
in brain tumor cell attachment, migration and possibly cell
survival (15). We have previously shown that pre-incubation of
brain tumor or capillary cells with the cRGD peptide, or the
combination of anti-.alpha.v.beta.3 and anti-.alpha.v.beta.5
antibodies, prevents cell adhesion to vitronectin. Endothelial and
melanoma cell apoptosis has been demonstrated, following similar
inhibition of integrin-dependent attachment to matrix proteins
(26-28). It was therefore of interest to determine whether the
.alpha.v-antagonists could induce apoptosis of these cells after
their detachment from vitronectin. DAOY cells and primary HBEC were
seeded in adhesion buffer on vitronectin-covered wells (1 .mu.g/ml)
and allowed to attach for 30 min. at 37.degree. C. The buffer was
then replaced with buffer containing cRGD or cRAD peptide (control)
at 20 .mu.g/ml, and further incubated for 4 hrs. Attached cells
were combined after trypsinization with floating cells and the
number of apoptotic cells was determined by FACS, using
anti-annexin V-FITC antibody and propidium iodide (Clontech Apo
alert Annexin V-FITC detection apoptosis kit). Cells exposed to the
control peptide cRAD showed little apoptosis, while both DAOY and
HBEC cRGD-treated cells demonstrated significant apoptosis (FIG.
6d). These data indicate that cRGD peptide not only detaches these
cells from vitronectin, but also induces their subsequent
apoptosis.
Effect of Pentapeptide on Brain Tumor Cell Adhesion to Different
ECM Substrates
[0089] FIG. 7 shows the effect of cyclic pentapeptide on tumor cell
adhesion to ECM proteins. Non tissue culture dishes were incubated
1 hour at 37.degree. C. with vitronectin, tenascin, fibronectin or
collagen I (10 .mu.g/ml), then washed with PBS. After the wash,
5.times.10.sup.5 cells/well were plated and incubated for 16 hours
at 37.degree. C. The cultures were then washed and an adhesion
buffer containing 20 .mu.g/ml of pentapeptide or control peptide
were added and incubated for an additional 2-24 hours. The cultures
were then washed twice with adhesion buffer and stained with
Crystal violet and the OD 600 determined. The more adherent cells
are present, the higher the OD. Data represent adherent cells after
8-hour incubation. U87=glioblastoma and DAOY=medulloblastoma.
[0090] As illustrated in FIG. 7, the tumor cells detached from
vitronectin and tenascin, whose adherence is mediated by
.alpha.v-integrins, but not from collagen and fibronectin, which
interact with non .alpha.v- integrins. Similar data were obtained
with brain capillary cells (not shown).
The Effect of Cell Detachment on Cell Survival
[0091] FIG. 8 shows apoptosis of brain tumor and capillary cells
grown on ECM and exposed to the pentapeptide. Test conditions were
as in FIG. 7, except that detached cells and adherent cells were
combined after trypsinisation. The cells were then washed,
suspended in 50 .mu.l PBS and centrifuged in glass cover slides
(cytospin). After fixation with 4% paraformaldehyde, the cells were
stained for apoptosis using the Boehringer Apoptosis Kit. Results
are expressed in a percentage of apoptotic cells versus total
number of cells. Experiment was done after 24 hours of incubation
with the peptides.
[0092] The effect of cell detachment on cell survival is shown in
FIG. 8. As shown, cells exposed to the active peptide for 24 hours
and grown on vitronectin or tenascin showed increased number of
dead cells when compared to control cells. In contrast, the
pentapeptide did not alter the survival of cells grown on collagen
I or fibronectin. Decreased cell survival was observed in both
types of tumor cells and brain capillary cells. Similar data were
obtained when the cells were stained for an early marker of cell
death, expressed on the cell surface (Annexin-V, data not shown).
Thus, the active pentapeptide induces cell detachment and death in
both brain tumor and capillary cells adherent to vitronectin and
tenascin.
Production of ECM Proteins in Human Brain Tumor
[0093] As outlined above, brain tumors produce vitronectin and
tenascin in humans and it is thought that these substrates improve
survival of tumor cells and enhance their invasion. We tested in
our brain tumor model for the production of these proteins.
[0094] FIG. 9 shows immunohistochemistry of U87 brain tumors
xenotransplanted into the forebrain of nude mice and treated with
active (anti-.alpha.v) or control peptide. Mice were injected with
the tumor cells (10.sup.6/cells/mouse) and treatment with the
active or inactive peptide (100 .mu.g/mouse/day) initiated on day 7
after injection. Tumors were removed after 2 weeks of treatment,
fixed in buffered formalin, embedded in paraffin and 5 .mu.m
sections obtained. Sections were examined for CD31 expression
(marker for capillary cells) using a monoclonal rat anti-mouse
antibody (Pharmingen), monoclonal mouse anti-human vitronectin
antibody (Sigma), mouse anti-human tenascin antibody (Neo-Marker)
and the Boehringer Apoptosis Kit to test for dead cells. CD
31=capillary marker, VN=vitronectin, TN=tenascin and
APO=apoptosis.
[0095] As shown in FIG. 9, the brain tumors indeed produced
vitronectin and tenascin, and their origin from tumor cells was
proven with the use of antibodies specific for human proteins. The
administration of the active pentapeptide had no influence on the
synthesis of these proteins. Furthermore, we were also able to
demonstrate that the brain tumor cells produce these proteins in
tissue culture (data not shown). Thus, our mouse model mimics the
situation of human brain tumors.
[0096] The anti-angiogenic effect of the pentapeptide is shown at
the top of FIG. 9, where we stained tissue sections for a specific
marker for capillary cells, namely CD 31. While tumors treated with
the control peptide had multiple vessels, which stained positive
for this marker, there were very few such vessels in tumors treated
with the active pentapeptide (p<0.005), indicating suppression
of capillary growth. The bottom of FIG. 9 shows staining for
apoptotic (dead) cells. The method is similar to that described in
FIG. 8. Tumors treated with the active pentapeptide had
significantly more dead cells than those treated with the control
peptide (p<0.02). Pictures were taken for the glioblastoma U87,
but similar data were obtained with the medulloblastoma DAOY (not
shown).
Relationship of Cell Death with Tumor Cell Apoptosis
[0097] In order to demonstrate that this increased cell death is
due to direct tumor cell apoptosis and not a consequence of the
suppression of capillary growth, we implanted into mouse brains
melanoma cells which either do (M21 L .alpha.v pos.) or do not
(M21L .alpha.v neg.) express .alpha.v-integrins.alpha. If mice
transplanted with .alpha.v positive cells and treated with the
active pentapeptide survive longer than mice treated with the
control peptide, then direct tumor cell apoptosis must be
responsible.
[0098] Table 2 shows the survival of mice with .alpha.v.beta.3
negative and positive tumors treated with RGDFV. Tumor cells
(10.sup.6) were injected into the forebrain of 6-week-old nude mice
and treatment with the active or control peptide (100
.mu.g/mouse/day) was initiated on day 3. Mice were sacrificed when
becoming cachectic and the presence of tumor was verified by
autopsy.
2 TABLE 2 Survival (days) Cell Line EMD 121974 (active) EMD
135981(control) M21L.alpha.v neg 15.7 .+-. 3.8 15.3 .+-. 3.3
M21L4.alpha.v pos 36.5 .+-. 2.9 17.3 .+-. 1.9
[0099] As shown in Table 2, mice transplanted with
.alpha.v-negative melanoma cells survived 15 days, independent of
treatment received. In contrast, the life span of mice with
.alpha.v-positive tumors increased to 36 days when treated with the
active pentapeptide, compared to 17 days when treated with the
control peptide. This indicates that direct tumor cell apoptosis
must be responsible for tumor cell death.
[0100] In conclusion, the above-discussed data support the
hypothesis that the cyclic pentapeptide induces direct tumor cell
death.
DISCUSSION
[0101] Brain tumors are highly angiogenic and are dependent on
neovascularization for their continued growth. Anti-angiogenesis is
thus a potentially important therapeutic strategy aimed against
these malignancies. Integrins .alpha.v.beta.3 and .alpha.v.beta.5
are candidate anti-angiogenic targets, since their expression on
endothelium is activated during angiogenesis (12,13). Their role in
neovascularization was previously confirmed by studies which showed
that angiogenesis induction by bFGF and TNF-alpha in the CAM could
be disrupted by the presence of either the .alpha.v.beta.3-blocking
antibody LM-609 or an RGD cyclic peptide antagonist of
.alpha.v.beta.3 and .alpha.v.beta.5 (21,23). In each case, the
anti-angiogenesis effect is thought to occur through the induction
of endothelial cell apoptosis as a result of preventing essential
.alpha.v-matrix protein binding interactions (28-30). In this
study, we used a similarly specific RGD cyclic peptide antagonist
of .alpha.v.beta.3 and .alpha.v.beta.5 (EMD 121974) and
demonstrated its ability to inhibit CAM angiogenesis induced by two
human brain tumor cell lines, DAOY and U87MG. cRGD treatment not
only suppressed CAM angiogenesis, but subsequently led to tumor
necrosis and tumor involution.
[0102] To address whether similar responses could be achieved in a
model that recapitulates the brain microenvironment, brain tumor
cells were stereotactically injected into the nude mouse forebrain.
U87MG glioma and DAOY medulloblastoma cells were chosen to study
because of their responsiveness to the peptide in the CAM assay and
their extensive invasiveness and angiogenesis previously
demonstrated in vivo (31,32). In this orthotopic model, cRGD again
significantly inhibited U87MG and DAOY brain tumor growth. Control
mice succumbed to tumor progression and were found to have highly
invasive tumors larger than 3 mm.sup.3 on average. The cRGD-treated
mice survived without evidence of morbidity, and viable tumors
could not be detected, apart from scant residual cells along the
site of implantation or in small clusters along the ventricular and
dural surfaces. All residual tumor foci in the treated group were
smaller than 1 mm.sup.3 and hence did not acquire a host
angiogenesis response. Alternative inhibitors of angiogenesis, such
as thrombospondin-1, TNP-470 and platelet factor 4, have also been
shown to inhibit experimental brain tumor growth, although most of
these results were limited to subcutaneous xenografts or required
the direct delivery of drug to the tumor bed by stereotactic
injection (32-34). A peptide antagonist of .alpha.v.beta.3 in a
SCID mouse/rat Leydig cell subcutaneous tumor model was shown to
inhibit tumor growth by 80%, following its intraperitoneal
administration (35). Angiostatin, an anti-angiogenic agent with an
as yet unclear mechanism of action, also demonstrated considerable
growth inhibition of intracranial C6 and 9L rat glioma xenografts
(36). In contrast to our study, treatment with angiostatin (1
mg/mouse/day) or the peptidomimetic antagonist (2 mg/mouse/day) was
initiated immediately after tumor cell implantation. Our study
revealed near ablation of established tumor xenografts and is the
first to show the inhibition of intracranial brain tumor growth and
angiogenesis through integrin antagonism.
[0103] Interestingly, DAOY and U87MG tumors grown simultaneously
under the skin showed little or no effect to cRGD treatment in our
study. This underscores the importance of the extracellular
environment in regulating host-tumor-cell integrin responses.
Angiostatin was found to equally inhibit the growth of s.c. and
i.c. injected brain tumor cells, indicating that this compound
suppresses angiogenesis through a different mechanism than the RGD
peptide (36). One possible explanation for the difference in cRGD
growth inhibition is that the endothelial cells of the subcutis may
be inherently different from those of the CNS microvasculature,
rendering angiogenesis less susceptible to integrin antagonism.
This is supported by the recent finding that in .alpha.v-knockout
mice, only the brain and intestinal capillary cells are abnormal,
while the remainder of the circulatory system is intact (37).
Alternatively, matrix proteins, which serve as ligands for
endothelial cell integrins, may be preferentially expressed by
tumor cells, depending on an orthotopic or heterotopic location.
For instance, human glioblastoma cells implanted subcutaneously
were not found to produce vitronectin, however, their placement in
the cerebral microenvironment induced their expression of the
vitronectin gene (15). Normal brain tissue may likewise alter its
expression of ECM proteins in response to invading glioma cells
(38). Unlike extraneural sites, vitronectin is normally absent in
the brain, and thus small changes in the concentration of this
protein within the CNS may profoundly modify cell responses.
Studies indicate that endothelial cell locomotion is dependent on
the concentration of vitronectin and the distance between points of
cell-matrix contacts which allow for cell spread (39,40).
[0104] The anti-tumorigenic effects of the peptide in our study are
in general greater than those observed with other anti-angiogenic
agents. Since many tumors also express .alpha.v.beta.3 and
.alpha.v.beta.5 integrins (including U87MG and DAOY), the effect of
integrin antagonists may not be limited to the host endothelium,
although the importance of specific integrins in tumor cell
responses is less clear (41). Prior studies have shown that the
attachment of glioma, breast carcinoma, melanoma and HT29-D4
colonic adenocarcinoma cells to vitronectin is dependent on
.alpha.v integrins (42-45). Vitronectin is produced by tumor and
stromal cells and is most abundant at sites of tumor invasion and
neovascularization, including malignant brain tumors (46,47). Thus
vitronectin, in addition to supporting endothelial cell survival,
may also be critical for enhancing the adhesion and invasion of
tumor cells, which express .alpha.v.beta.3 and .alpha.v.beta.5. In
a SCID mouse/human chimeric model for breast cancer, tumor invasion
was markedly reduced following the administration of the
anti-.alpha.v.beta.3 antibody LM-609, suggesting a direct effect of
.alpha.v.beta.3 blockade upon the tumor independent of angiogenesis
(48).
[0105] In order to address this question, we examined cellular
adhesion and migration on vitronectin in the presence of
.alpha.v-antagonists, using DAOY, U87MG and isolated primary human
brain endothelial cells (HBEC) (49). The anti-.alpha.v.beta.3
antibody LM-609 inhibited U87MG, DAOY and HBEC migration on
vitronectin, and in combination with the anti-.alpha.v.beta.5
antibody P1-F6 inhibited the adhesion of these cells to
vitronectin. cRGD alone significantly inhibited the adhesion to and
migration on vitronectin by all three cell types. In a similar
study, melanoma cell invasion was enhanced by vitronectin and
blocked by RGD peptides (50). Finally, cRGD treatment resulted in
increased apoptosis not only in HBEC cells, but also in DAOY and
U87MG tumor cells as well. These results suggest that cRGD may act
to inhibit tumor invasion and proliferation directly, in addition
to its anti-angiogenic function.
[0106] Our data further demonstrates that cyclic pentapeptide may
inhibit the adhesion of brain tumor cells to different ECM
substrates such as vitronectin or tenascin. The effect of such an
inhibition of adhesion resulted in cell death (apoptosis) in both
brain tumor cells and brain capillary cells. This effect is
restricted to the ECM vitronectin and tenascin. We have also
demonstrated that the increased cell death is due to direct tumor
cell apoptosis and not a consequence of the suppression of
capillary growth. In other words, it is a discovery of the present
invention that the cyclic pentapeptide induces direct tumor cell
death.
[0107] In summary, this study provides evidence that targeted
antagonism of integrins, specifically .alpha.v.beta.3 and
.alpha.v.beta.5, can substantially inhibit brain tumorigenesis in
vivo and may thus represent an important novel therapeutic approach
to brain tumors. Our results also suggest that the microenvironment
is critical to the tumor behavior and in determining its
responsiveness to such biologically directed therapies. Finally, we
show that integrin anatagonism can have an anti-tumorigenic effect
independent of anti-angiogenesis, which may act synergistically to
retard tumor growth.
[0108] The foregoing is meant to illustrate, but not to limit, the
scope of the invention. Indeed, those of ordinary skill in the art
can readily envision and produce further embodiments, based on the
teachings herein, without undue experimentation.
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