The present invention is directed to methods of inhibiting tumor
cells by administering an antagonist which inhibits the VEGF/VEGFR-1
autocrine loop of tumor cells. Additional antagonists can be added
to inhibit endothelial paracrine loop by inhibiting other VEGFRs
expressed on endothelial cells, particularly VEGFR-2. Examples of
antagonists include antibodies and small molecules. A preferred
cancer for treatment is breast cancer.
What is claimed is:
1. A method for preventing or reducing the growth of tumor cells
expressing functional VEGF-1 receptors comprising administering
to a mammal an effective amount of a VEGF-1 receptor antagonist
to inhibit autocrine stimulation.
2. The method of claim 1, wherein the mammal is a human.
3. The method of claim 1, wherein the tumor cells are from a cancer
selected from the group consisting of breast cancer, ovarian cancer,
brain cancer, kidney cancer, bladder cancer, adenocarcinoma, malignant
gliomas and leukemias.
4. The method of claim 3, wherein the cancer is breast cancer.
5. The method of claim 3, wherein the cancer has substantially
6. The method of claim 1, wherein the antagonist is a small molecule.
7. The method of claim 1, wherein the antagonist is an antibody.
8. The method of claim 7, wherein the mammal is a human and the
antibody is Mab 6.12, produced by a hybridoma cell line deposited
as ATCC number PTA-3344.
9. The method of claim 1, further comprising administering a second
antagonist directed to VEGFR expressed on edothelial cells, wherein
the VEGFR is selected from the group consisting of VEGFR-2, VEGFR-3
and neuropilin, thereby inhibiting endothelial mediated paracrine
10. The method of claim 9, wherein the second antagonist is a small
11. The method of claim 9, wherein the second antagonist is an
12. The method of claim 9, wherein the second antagonist is directed
13. The method of claim 12, wherein the mammal is a human and the
antagonist is DC101.
14. The method of claim 1, further comprising administering a chemotherapeutic
agent with the antagonist.
15. The method of claim 14, wherein the chmotherapeutic agent is
selected from the group consisting of anthracyclin, methotrexate,
vindesine, neocarzinostatin, cis-platinum, chlorambucil, cytosine
arabinoside, irinotecan, 5-fluorouridine, melphalan, ricin, calicheamicin,
taxol, gemcitibine, fluorouracil, paclitaxel, docetaxel, leucovorin
16. The method of claim 1, further comprising administering radiation.
FIELD OF THE INVENTION
 The present invention is directed to methods of treatment
of tumors in mammals with antagonists of VEGF receptors that are
expressed on tumor cells. The antagonists are preferably neutralizing
antibodies that specifically bind to an extracellular domain of
VEGF receptors that are expressed on tumor cells. In particular,
the present invention is directed to the treatment of breast cancer
via the administration of neutralizing antibodies that specifically
bind to an extracellular domain of human VEGFR-1 in amounts effective
to reduce tumor growth or size.
 Vascular endothelial growth factor (VEGF), placenta-derived
growth factor (PlGF), and their receptors VEGFR-1 (Flt-1), VEGFR-2
(KDR, Flk-1), and VEGFR-3 (Flt-4) have been implicated in vasculogenesis,
angiogenesis, and tumor growth. VEGF is a homodimeric glycoprotein
consisting of two 23 kD) subunits with structural similarity to
PDGF. Four different monomeric isoforms of VEGF exist resulting
from alternative splicing of mRNA. These include two membrane bound
forms (VEGF.sub.206 and VEGF.sub.189) and two soluble forms (VEGF.sub.165
and VEGF.sub.121). In all human tissues except placenta, VEGF.sub.165
is the most abundant isoform.
 VEGF is a strong inducer of vascular permeability, a stimulator
of endothelial cell migration, and an important survival factor
for newly formed blood vessels. VEGF is expressed in embryonic tissues
(Breier et al., Development (Camb.) 114: 521 (1992)), macrophages,
proliferating epidermal keratinocytes during wound healing (Brown
et al., J. Exp. Med., 176: 1375 (1992)), and may be responsible
for tissue edema associated with inflammation (Ferrara et al., Endocr.
Rev. 13: 18 (1992)). In situ hybridization studies have demonstrated
high VEGF expression in a number of human tumor lines including
glioblastoma multiform, heman-gioblastoma, central nervous system
neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al.
(1992) Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res.
53: 5822-5827; Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159;
and Nakamura, S. et al. (1992) AIDS Weekly, 13 (1)). High levels
of VEGF were also observed in hypoxia induced angiogenesis (Shweiki,
D. et al. (1992) Nature 359: 843-845).
 The biological response of VEGF is mediated through its
high affinity VEGF receptors which are selectively expressed on
endothelial cells during embryogenesis (Millauer, B., et al. (1993)
Cell 72: 835-846) and during tumor formation. VEGF receptors typically
are class III receptor-type tyrosine kinases characterized by having
several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal
extracellular receptor ligand-binding domains (Kaipainen et al.,
J. Exp. Med. 178: 2077-2088 (1993)). The other two regions include
a transmembrane region and a carboxy-terminal intracellular catalytic
domain interrupted by an insertion of hydrophilic interkinase sequences
of variable lengths, called the kinase insert domain (Terman et
al., Oncogene 6: 1677-1683 (1991)).
 VEGF receptors include VEGF receptor 1 (VEGFR-1, also called
fins-like tyrosine kinase receptor, or Flt-1), sequenced by Shibuya
M. et al., Oncogene 5, 519-524 (1990); and VEGF receptor 2 (VEGFR-2).
The human form of VEGFR-2 is also called kinase insert domain-containing
receptor (KDR) and is described in PCT/US92/01300, filed Feb. 20,
1992, and in Terman et al., Oncogene 6: 1677-1683 (1991). The murine
form of VEGFR-2 is also called FLK-1 and was sequenced by Matthews
W. et al. Proc. Natl. Acad. Sci. USA, 88: 9026-9030 (1991).
 Release of VEGF by a tumor mass stimulates angiogenesis
in adjacent endothelial cells. When VEGF is expressed by the tumor
mass, endothelial cells closely adjacent to the VEGF+tumor cells
will up-regulate expression of VEGF receptor molecules e.g., VEGFR-1
and VEGFR-2. Upon binding of their ligand, these receptors dimerize
and transduce an intracellular signal through tyrosine phosphorylation.
VEGF plays a crucial role for the vascularization of a wide range
of tumors including breast cancers, ovarian tumors, brain tumors,
kidney and bladder carcinomas, adenocarcinomas, malignant gliomas
and luekemias. Tumors produce ample amounts of VEGF, which stimulates
the proliferation and migration of endothelial cells (ECs), thereby
inducing tumor vascularization by a paracrine mechanism.
 Placenta-derived growth factor (PlGF), another natural specific
ligand for VEGFR-1 (Flt-l), which is produced in large amounts by
villous cytotrophoblast, sincytiotrophoblast and extravillous trophoblast,
is a member of the VEGF family. PlGF is a dimeric secreted factor
which shares close amino acid homology to VEGF. Some of the biological
effects of VEGF and PlGF are also similar, including stimulation
of endothelial cell migration. PlGF and VEGF, thus appear capable
of acting in unison on both myelomonocytic and endothelial lineage
 The administration of neutralizing antibodies and other
molecules that block signaling by VEGF receptors expressed on vascular
endothelial cells is known to reduce tumor growth by blocking angiogenesis
through an endothelial-dependent paracrine loop. One advantage of
blocking the VEGF receptor, as opposed to blocking the VEGF ligand
to inhibit angiogenesis, and thereby inhibit pathological conditions
such as tumor growth, is that fewer antibodies may be needed to
achieve such inhibition. Furthermore, receptor expression levels
may be more constant than those of the environmentally induced ligand.
See, U.S. Pat. Nos. 5,804,301; 5,874,542; 5,861,499; and 5,955,311.
 Certain tumor cells not only produce VEGF, but may also
have acquired the capacity to express functional VEGF receptors
(VEGFR), which results in the generation of an endothelial-independent
autocrine loop to support tumor growth. The present inventors have
recently provided the first demonstration that a VEGF/human VEGFR-2
autocrine loop mediates leukemic cell survival and migration in
vivo. Dias et al., "Autocrine stimulation of VEGFR-2 activates
human leukemic cell growth and migration," J. Clin. Invest.
106: 511-521 (2000). Similarly, VEGF production and VEGFR expression
also have been reported for some solid tumor cell lines in vitro.
(See Tohoku, Sato, J. Exp. Med., 185(3): 173-84 (1998); Nippon,
Sanka Fujinka Gakkai Zasshi,:47(2): 133-40 (1995); and Ferrer, FA,
Urology, 54(3):567-72 (1999)). However whether VEGFRs expressed
on solid tumor cells are functional and convey mitogenic or other
signals has not been demonstrated.
SUMMARY OF THE INVENTION
 The present invention provides a method for treatment of
a tumor in a mammal comprising treating the mammal having such a
tumor with an antagonist of a VEGF receptor that is expressed on
a tumor cell, wherein said VEGF receptor is selected from the group
consisting of human VEGFR-1, VEGFR-2, VEGFR-3, neuropilin, and their
non-human homologs (such as FLK-1); and wherein said antagonist
is administered in an amount effective to reduce tumor growth or
size. Preferably, the antagonist is a neutralizing antibody that
specifically binds to an extracellular domain of a VEGF receptor
that is expressed on a tumor cell, and inhibits autocrine stimulation.
Examples of solid tumors which may be treated with the methods and
antibodies of the present invention include breast carcinoma, lung
carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma, and
lymphoma; examples of liquid tumors include leukemia.
 In a preferred embodiment, the present invention provides
a method for treatment of breast cancer in a mammal comprising treating
the mammal having breast cancer with a neutralizing antibody that
specifically binds to an extracellular domain of human VEGFR-1,
wherein said antibody is administered in an amount effective to
reduce tumor growth or size.
 In another embodiment, a second VEGF receptor antagonist
is also administered. The second antagonist is preferably an antibody
against VEGF receptors expressed on tumor-associated vascular endothelial
cells, resulting in inhibition of endothelial dependent paracrine
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 presents an immunoblot for pErk 1/2 expression in
DU4475 human breast cancer cells treated with growth factors, as
detailed in Example 1.
 FIG. 2 is a chart showing a densiometry analysis of the
blot of FIG. 1.
 FIG. 3 is a chart showing the results of treatment of NOD-SCID
mice inoculated with DU4475 human breast cancer cells with combinations
of antibodies, as detailed in Example 2.
 FIG. 4 presents photographs of tissues from NOD-SCID mice
inoculated with DU4475 human breast cancer cells after treatment
with combinations of antibodies, as detailed in Example 2. The tissues
are stained for morphological evaluation.
 Functional VEGF receptors expressed on tumor cells, and
antibodies that bind to such VEGF receptors, as well as small molecules
that block the activity of such receptors, are useful for treating
tumors by directly inhibiting growth of tumor cells. Therefore,
inhibition of tumor cell growth is not dependent upon blocking angiogenesis.
 The present invention provides methods and compositions
for treating solid tumors, wherein antagonists of VEGF receptors
expressed on the tumor cells are administered to a mammal having
such a tumor. Preferably, the antagonist is a neutralizing antibody
that binds to VEGF receptors expressed on solid tumor cells, and
inhibits autocrine loop. The antagonist may also be a small molecule.
 The present invention provides antibodies for treating tumors,
wherein antibodies bind to and inhibits the activity of VEGF receptors
on the tumor cells.
 Tumors, the growth of which may be reduced using the methods
of the present invention, include tumors that express VEGF receptors.
Examples of tumors include breast carcinoma, lung carcinoma, colorectal
carcinoma, pancreatic carcinoma, glioma, lymphoma, and leukemias.
 The present invention provides methods for identifying antibodies
useful for treating a given tumor type, as well as methods for identifying
antibodies useful for treating a tumor of a specific patient.
 Tumor cells, which may be from established tumor cell lines,
from tissue biopsies, from the blood, or from other appropriate
sources may be assayed to determine whether and which functional
VEGF receptors are expressed thereon. The presence of VEGF receptors
may be detected by imunohistochemical, flow cytometry, ELISA assays,
and other known methods, coupled with the guidance provided herein.
For VEGF receptors found to be present, cells may be tested for
receptor function by exposing them to agonist ligands of VEGF receptors
and determining whether receptor phosphorylation occurs. Methods
of determining receptor phosphorylation are well known in the art
and include, for example, measurement of phosphotyrosine with monoclonal
antibodies or radioactive labels. Other markers of receptor function,
such as cell proliferation and activation of cell signaling pathways
known to be activated by the VEGF receptor of interest, may also
be tested. Appropriate markers for functionality will vary depending
on the VEGF receptor of interest.
 The present invention provides antibodies that are capable
of binding specifically to the extracellular domain of a VEGF receptor
expressed on a tumor cell. VEGF receptors include human VEGFR-1,
VEGFR-2, VEGFR-3, and neuropilin, and their non-human homologs (such
as FLK-1). An extracellular domain of a VEGF receptor as herein
defined includes the ligand-binding domain of the extracellular
portion of the receptor, as well as extracellular portions that
are involved in dimerization and overlapping epitopes. When bound
to the extracellular domain of a VEGF receptor, the antibodies effectively
block receptor activation and/or interfere with receptor dimerization.
As a result of such binding, the antibodies neutralize activation
of the VEGF receptor. Neutralizing a receptor means diminishing
and/or inactivating the intrinsic ability of the receptor to transduce
a signal. A reliable assay for VEGF receptor neutralization is inhibition
of receptor phosphorylation. Methods of determining receptor phosphorylation
are well known in the art and include, for example, measurement
of phosphotyrosine with monoclonal antibodies or radioactive labels.
 In a preferred embodiment, an antibody of the present invention
binds to human VEGFR-1 and blocks VEGF binding and/or PlGF binding
to human VEGFR-1. Mab 6.12 is an example of an antibody that binds
to soluble and cell surface-expressed human VEGFR-1. A hybridoma
cell line producing Mab 6.12, has been deposited as ATCC number
PTA-3344. The deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedure and the regulations thereunder
(Budapest Treaty). This assures maintenance of a viable culture
for 30 years from date of deposit. The organisms will be made available
by ATCC under the terms of the Budapest Treaty, and subject to an
agreement between Applicants and ATCC which assures unrestricted
availability upon issuance of the pertinent U.S. patent. Availability
of the deposited strains is not to be construed as a license to
practice the invention in contravention of the rights granted under
the authority of any government in accordance with its patent laws.
 In addition to the aforementioned antibodies, other anti-VEGF
neutralizing antibodies (e.g., antibodies to VEGFR-1, VEGFR-2, VEGFR-3
and neuropilin) may readily be produced using art-known methods
combined with the guidance provided herein. The antibodies of the
present invention may bind to VEGF receptors with an affinity comparable
to, or greater than, that of the natural ligand.
 Antibodies that are useful in the present invention include
polyclonal and monoclonal antibodies. Both polyclonal and monoclonal
antibodies may be produced by methods known in the art. Methods
for producing monoclonal antibodies include the immunological method
described by Kohler and Milstein in Nature 256, 495-497 (1975) and
Campbell in "Monoclonal Antibody Technology, The Production
and Characterization of Rodent and Human Hybridomas" in Burdon
et al., Eds, Laboratory Techniques in Biochemistry and Molecular
Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985);
as well as by the recombinant DNA method described by Huse et al.
in Science 246, 1275-1281 (1989).
 Chimeric, humanized, and fully human antibodies are also
useful in the present invention. Useful chimeric antibodies include
chimeric antibodies comprising an amino acid sequence of a human
antibody constant region and an amino acid sequence of a non-human
antibody variable region. However, chimeric antibodies comprising
an amino acid sequence of a non-human antibody constant region and
an amino acid sequence of a non-human antibody variable region may
also be useful. The non-human variable region of chimeric antibodies
may be murine. Useful humanized antibodies include humanized antibodies
comprising amino acid sequences of variable framework and constant
regions from a human antibody. The amino acid sequence of the hypervariable
region of humanized antibodies may be murine.
 Chimeric, humanized, or fully human antibodies may be produced
by art-known methods, including phage display. (See, e.g., Jones,
P. T. et al., (1996) Nature 321, 522-525; Riechman, L. et al., (1988)
Nature 332, 323-327; U.S. Pat. No. 5,530,101 to Queen et al.; Kabat,
E. A., et al. (1991) Sequences of Proteins of Immunological Interest.
5th ed. National Center for Biotechnology Information, National
Institutes of Health, Bethesda, Md.; Queen, C. et al., (1989) Proc.
Natl. Acad. Sci. USA 86, 10029-10033; McCafferty et al. (1990) Nature
348, 552-554; Aujame et al. (1997) Human Antibodies 8, 155-168;
and Griffiths et al. (1994) EMBO J. 13, 3245-3260). Human antibodies
may also be produced from transgenic animals (reviewed in Bruggemann
and Taussig (1997) Curr. Opin. Biotechnol. 8, 455-458; see also,
e.g., Wagner et al. (1994) Eur. J. Immunol. 42,2672-2681; Green
et al. (1994) Nature Genet. 7, 13-21).
 Antibodies of the invention also include antibodies that
have been made less immunogenic by replacing surface-exposed residues
to make the antibody appear as self to the immune system. (See,
e.g., Padlan, E. A. (1991) Mol. Immunol. 28, 489-498; Roguska et
al. (1994) Proc. Natl. Acad. Sci. USA 91, 969-973).
 Antibodies useful in the present invention also include
those for which binding characteristics have been improved by direct
mutation or by methods of affinity maturation. (See, e.g., Yang
et al. (1995) J. Mol. Bio. 254, 392-403; Hawkins et al. (1992) J.
Mol. Bio. 226,889-896; Low et al. (1996) J. Mol. Bio. 250, 359-368).
 Functional fragments and equivalents of antibodies are also
useful in the invention, where such fragments and equivalents have
the same binding characteristics as, or that have binding characteristics
comparable to, those of the corresponding whole antibody. Such fragments
may contain one or both Fab fragments or the F(ab')2 fragment. Such
fragments may also contain single-chain fragment variable region
antibodies, i.e., scFv. Fragments may be produced by art-known methods.
(See, e.g. Lamoyi et al, Journal of Immunological Methods 56, 235-243
(1983); and Parham, Journal of Immunology 131, 2895-2902 (1983)).
 In another aspect of the invention, the antibodies can be
chemically or biosynthetically linked to anti-tumor agents or detectable
signal-producing agents. The invention further contemplates antibodies
to which target or reporter moieties are linked.
 In addition to antibodies and their functional equivalents,
other biological antagonists that may be used include proteins,
peptides, or nucleic acid molecules, including antisense oligonucleotides,
which inhibit growth of tumor cells expressing VEGF receptors by
blocking receptor activation, for example.
 Other useful antagonists may be small molecules, which may
be organic or inorganic, and which inhibit growth of tumor cells
expressing VEGF receptors by blocking receptor activation, for example.
Typically such small molecules have molecular weights less than
500, more typically less than 450. Most typically, the small molecules
are organic molecules that usually comprise carbon, hydrogen, and
optionally oxygen and/or sulfur atoms.
 In another embodiment of the invention, a second VEGF receptor
antagonist is administered in addition to an antagonist to a VEGF
receptor expressed on tumor cells, to inhibit endothelial dependent
paracrine loop. If a VEGFR-1 antagonist is used as a first antagonist,
then the second antagonist preferably inhibits another VEGF receptor.
In such a case, the VEGFR-1 antagonist inhibits both autocrine and
paracrine loops associated with VEGFR-1, thus making it unnecessary
to add another VEGFR-1 antagonist. The second antagonist is preferably
a neutralizing antibody and preferably targets a VEGF receptor or
other growth factor receptor expressed on tumor vasculature. Preferably,
the second antagonist inhibits angiogenesis.
 An example of such a second antagonist is an antibody that
binds to human VEGFR-2 (KDR) and blocks VEGF binding to KDR. scFv
p1C11 was produced from a mouse scFv phage display library. (Zhu
et al., 1998). p1C11 blocks VEGF-KDR interaction and inhibits VEGF-stimulated
receptor phosphorylation and mitogenesis of human vascular endothelial
cells (HUVEC). This scFv binds both soluble KDR and cell surface-expressed
KDR on HUVEC, for example, with high affinity (K.sub.d=2.1nM). DC101
is a rat monoclonal antibody that binds to a neutralized mouse VEGFR-2.
A hybridoma cell line producing DC101 was deposited as ATCC Accession
No. ATCC HB 11534 on Jan. 26, 1994. Another example of such antibody
is MF1, an antagonist of murine VEGFR-1, which inhibits endothelial
dependent paracrine and autocrine loop in mice. Yan Wu et al., "Inhibition
of Tumor Growth and Angiogenesis in animal models by a neutralizing
anti-VEGFR 1 monoclonal antibody", ImClone Systems Incorporated,
 When administering an antibody such as Antibody 6.12 to
a human, the Antibody by itself inhibits both autocrine and paracrine
loops; the Antibody inhibits VEGFR-1 regardless of the location
of the receptor on a tumor cell or endothelial cell. With regard
to Example 2, the model involves a human tumor in a mouse, where
the endothelial cells are of murine origin. Antibody 6.12 is specific
for human VEGFR-1, and thus only inhibits the autocrine loop of
the human cancer cells in the mouse model, and not mouse endothelial
cells. The paracrine stimulation of mouse endothelial cells thus
is unaffected by Antibody 6.12 in the model. MF1 is mouse specific,
and inhibits mouse endothelial cells, but not human tumors.
 In yet another aspect of the present invention, a patient
having a tumor that is substantially not vascularized or not yet
vascularized is treated with an antagonist of a VEGF receptor that
is expressed on the tumor cells. An example of such a patient is
one having a tumor that is undergoing metastasis, wherein the metastases
are not yet vascularized. In a preferred embodiment, the patient
has metastatic breast cancer and the antagonist is a neutralizing
antibody against VEGFR-1.
 The antagonists of the present invention may also be used
in combined treatment methods. The antibodies and small molecules
can be administered along with an anti-neoplastic agent such as
a chemotherapeutic agent, a radioisotope, or radiation treatment.
Suitable chemotherapeutic agents are known to those skilled in the
art and include anthracyclines (e.g. daunomycin and doxorubicin),
methotrexate, vindesine, neocarzinostatin, cis-platinum, chlorambucil,
cytosine arabinoside, irinotecan, 5-fluorouridine, melphalan, ricin,
calicheamicin, taxol, gemcitibine, fluorouracil, paclitaxel, docetaxel,
leucovorin and novelbine. The antagonists of the present invention
may be administered in combination with other treatment regimes.
For example, antibodies and/or small molecules of the invention
can be administered with external treatment, e.g., external beam
 It is understood that antibodies and/or small molecules
of the invention, where used in the human body for the purpose of
diagnosis or treatment, will be administered in the form of a composition
additionally comprising a pharmaceutically-acceptable carrier. Suitable
pharmaceutically acceptable carriers include, for example, one or
more of water, saline, phosphate buffered saline, dextrose, glycerol,
ethanol and the like, as well as combinations thereof. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives
or buffers, which enhance the shelf life or effectiveness of the
 Methods of administration to a mammal, including humans,
include but are not limited to oral, intravenous, intraperitoneal,
intracerebrospinal, subcutaneous, intrathecal, intramuscular, inhalation,
or topical administration.
 The compositions of this invention may be in a variety of
forms. These include, for example, solid, semi-solid and liquid
dosage forms, such as tablets, pills, powders, liquid solutions,
dispersions or suspensions, liposomes, suppositories, injectable
and infusible solutions. The preferred form depends on the intended
mode of administration and therapeutic application. The preferred
compositions are in the form of injectable or infusible solutions.
 Effective dosages and scheduling regimens of administration
of antibodies according to the present invention can be determined
by the skilled practitioner using art-known methods, such as clinical
trials and animal studies. Concentrations of the administered substances
will vary depending upon the therapeutic or preventive purpose.
 In embodiments where two antagonists are co-administered,
or where an antagonist is combined with another mode of treatment,
each of the treatments may, if desired, be administered in a dosage
that is smaller or less frequent than the dosage which would be
administered were each treatment administered independently of the
 All citations throughout the specification and the references
cited therein are hereby expressly incorporated by reference.
 The Examples that follow are set forth to aid in understanding
the invention but are not intended to, and should not be construed
to, limit its scope in any way. The Examples do not include detailed
descriptions of conventional methods, such as those employed in
the construction of vectors and plasmids, the insertion of genes
encoding polypepfides into such vectors and plasmids, or the introduction
of plasmids into host cells. Such methods are well known to those
of ordinary skill in the art and are described in numerous publications
including Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Breast Carcinoma Cells Express Functional VEGFR-1 (Flt-1)
 The present experiments show that breast cancer cells express
functional VEGFR-1. Two human cell lines DU-4475 (ER negative) and
MCF-7 (ER positive) were studied extensively. Both cell lines are
VEGFR-1 positive. VEGFR-1 expressed by these breast cancer cells
is functional, as determined by PlGF-induced receptor tyrosine phosphorylation
and activation of the MAP kinase (Erkl/2) pathway. Activation of
the MAP kinase pathway by PlGF or VEGF, ultimately leads to increased
cell proliferation in vitro. Furthermore, DU-4475 and MCF-7 do not
express VEGFR-2, and are therefore growth inhibited only by neutralizing
mAb to VEGFR-1 (6.12--blocks only human VEGFR-1).
 Immunohistochemical Analysis of Human Breast Carcinomas
 Formalin-fixed, paraffin-embedded tissue of 16 human ductal
breast carcinoma biopsies were evaluated for VEGFR-1, human VEGFR-2,
VEGF and von Willebrand factor (VWF) immunoreactivity, following
conventional protocols. The antibodies used were mAb to human VEGFR-1
(FB5); human VEGFR-2 (6.64); VEGF polyclonal antibody, and vWF polyclonal
antibody (Zymed Laboratories Inc., South San Francisco, Calif.,
USA). Secondary peroxidase-labeled antibodies were used at a 1:6000
dilution. The peroxidase reaction was developed with a diaminobenzidine
substract and slides were counterstained with hematoxylin and eosin.
All sections were observed under a light microscope.
 Cell Culture
 The human breast cancer cell lines DU4475, MCF-7, T-47D
and MDA-MB-231 were obtained from ATCC (Manassas, Va., USA). DU4475
cells were grown in suspension, whereas MCF-7, T-47D and MDA-MB-231
cells were grown as subconfluent monolayer cultures in RPMI 1640
(Bio Whittaker Inc., Walkersville, Md., USA) supplemented with 10%
fetal bovine serum, penicillin (100 U/ml), streptomycin (100 .mu.g/ml),
fungizone (0.25 .mu.g/ml) and L-glutamine (0.584 mg/ml) (Gibco BRL,
Rockville, Md., USA). HUVECs were obtained and cultured as previously
described (J Clin Invest. 1973, 52(11): 2745-56). Cells were kept
in a humidified incubator under 5% CO.sub.2 at 37.degree. C.
 RNA Extraction, cDNA Synthesis and RT-PCR
 Total RNA was isolated using Trizol (Gibco BRL, Rockville,
Md., USA), following the manufacturer's instructions. First-strand
cDNA was subsequently synthesized using SuperScript II reverse transcriptase,
according to manufacturer's protocol (Amersham Pharmacia Biotech,
Piscataway, N.J. USA). PCR was performed using Advantage 2 polymerase
mix (Clontech Laboratories Inc., Palo Alto, Calif., USA). Amplification
conditions were as follows: a precycle of 5 minutes at 94.degree.
C., 45 seconds at 63.degree. C., and 45 seconds at 72.degree. C.;
followed by 35 cycles at: 94 minute, 63.degree. C. for 45 seconds,
72.degree. C. for 2 minutes and a 7 minute extension at 72.degree.
C. Primers used for the PCR:
1 VEGFR-1 (forward: ATTTGTGATTTTGGCCTTGC; reverse: CAGGCTCATGAACTTGAAAGC);
human VEGFR-2 (forward: GTGACCAACATGGAGTCGTG; reverse: CCAGAGATTCCATGCCACTT)
VEGF (forward: CGAAGTGGTGAAGTTCATGGATG; reverse: TTCTGTATCAGTCTTTCCTGGTGAG);
PIGF (forward: CGCTGGAGAGGCTGGTGG; reverse: GAACGGATCTTTAGGAGCTG))
and Beta- actin (forward: TCATGTTTGAGACCTTCAA, reverse: GTCTTTGCGGATGTCCACG).
 Oligonucleotide primers designed were used to amplify 3
of the VEGF splicing variants (variants 121, 165, 189).
 Flow Cytometry Analysis
 For identification of VEGFR-1/Flt-1.sup.+and VEGFR-2/KDR.sup.+cells,
DU4475, MCF-7, T-47D and MDA-MB-231 cells were incubated with 2
.mu.l of FITC-labeled high-affinity, mAb to Flt-1 (clone FB5), or
with an unconjugated mAb to KDR (clone 6.64), for 20 minutes. A
secondary PE-labeled Ab (Kirkegaard & Perry Laboratories, Gaithersburg,
Md., USA) was subsequently added to the latter for 20 minutes. The
number of positive cells for VEGFR-1 or human VEGFR-2 was determined
using a Coulter Elite flow cytometer (COULTER, Hialeah, Fla., USA)
and compared to an immunoglobulin G isotype control (FITC; Immunotech,
Marceille, France). Nonviable cells were identified by propidium
iodide (PI) staining.
 Quantification of VEGF and PlGF Levels in Cell Culture Supernatants
 ELISA kits specific for human VEGF.sub.165 or PlGF (R&D
Systems Inc., Minneapolis, Minn., USA) were used to determine VEGF
and PlGF production in human breast cancer cells. DU4475, MCF-7,
T-47D and MDA-MB-231 cell lines were seeded in 6-well plates at
a density of 10.sup.6 cells/well. Cells were cultured in serum-free
conditions, and supernatants were collected after 48 hours. These
were used without further dilution. Each sample was measured in
 Cell Proliferation Assays
 Proliferation of DU4475 cells was determined by counting
the number of viable cells, using the Trypan blue exclusion test,
and by using the BrdU incorporation assay.
 For the trypan blue exclusion test, cells were seeded at
a density of 2.5.times.10.sup.5/well into 12-well plates in serum-free
RPMI. The cultures were treated every 24 hours with: 50 ng/ml PlGF,
20 ng/ml VEGF (R&D Systems Inc., Minneapolis, Minn., USA), 1
.mu.g/ml of the mAb against human VEGFR-1 (clone 6.12) or untreated,
for 48 at 37.degree. C. Viable cells were counted in triplicate
using a hemacytometer. Each experiment was done in triplicate.
 For the BrdU incorporation assay, 5.times.10.sup.3 cells
were plated in 96-well plates for 48 hours, in the following conditions:
serum-free, VEGF (50 ng/mL), PlGF (100 ng/mL), clone 6.12 mAb against
VEGF-1 (1 .mu.g/ml) and co-incubation with 6.12 and PlGF. BrdU was
added to the cultures for the last 24 hours. Incorporated BrdU was
quantified using an ELISA kit (Roche Diagnostics, Mannheim, Germany),
following the manufacturer's protocol.
 VEGFR-1 Phosphorylation Assay
 For receptor phosphorylation assay, DU4475 cells were seeded
in 12 well-plates (5.times.10.sup.5 cells/well) and kept in RPMI
serum-free medium for 18 hours. After replacing the culture medium,
the cells were treated with VEGF (50 ng/ml), PlGF (100 ng/ml) for
10 minutes or co-incubated with mAbs to human VEGFR-1 and human
VEGFR-2 (clone 6.12 and IMC-1C11, respectively), for 1 hour and
PlGF for 10 minutes, at 37.degree. C. After stimulation, total protein
extracts were obtained by lysing cells in cold RIPA buffer (50 mM
Tris, 5 mM EDTA, 1% Triton X-114, 0.4% sodium cacodylate, and 150
mM NaCl), in the presence of protease inhibitors (1 mg/mL aprotinin,
10 mg/mL leupeptin, 1 mM glycerophosphate, 1 mM sodium orthovanadate,
and 1 mM PMSF), for 30 minutes at 4.degree. C. Supernatants from
protein extracts were immunoprecipitated overnight at 4.degree.
C. in the presence of an anti-phosphotyrosine antibody (PY20) and
protein-G agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz,
Calif., USA), to precipitate phosphorylated proteins. The immunoprecipitates
were resuspended in loading buffer, and fractionated under reducing
conditions (in the presence of .beta.-mercaptoethanol) by SDS-PAGE
using 7.5% polyacrylamide gels. Proteins were subsequently electroblotted
onto a nitrocellulose membrane. Blots were blocked in 1% BSA/PBS--1%
Tween-20, for 1 hour at room temperature and then incubated with
primary and secondary antibodies. Mouse monoclonal antibody anti-VEGFR-1
(R&D Systems Inc., Minneapolis, Minn., USA) was used at a concentration
of 1.mu.g/mL, and secondary anti-mouse IgG-HRP (Santa Cruz Biotechnology
Inc., Santa Cruz, Calif., USA) was used at 1:6000. The ECL chemiluminescence
detection system and ECL film (Amersham Pharmacia Biotech, Piscataway,
N.J., USA) were used for the detection of proteins on the nitrocellulose
 MAP Kinase Pathways Activation Through Flt-1
 To evaluate MAPK phosphorylation, DU4475 cells were seeded
in 12 well-plates (5.times.10.sup.5 cells/well) in serum-free RPMI
for 18 hours. The cells were then washed 3 times with cold PBS,
and treated with or without growth factors (VEGF, 50 ng/mL; PlGF,
100 ng/mL) for 10 minutes or preincubated with clone 6.12 for 1
hour and then stimulated with PlGF for 10 minutes. Cells were also
treated with p42/p44 and p38 inhibitors, PD98059 (30 .mu.M) and
SB203580 (20 .mu.M) respectively, for 1 hour and stimulated with
PlGF for 10 minutes. Cell lysis and protein isolation were performed
as described above. Proteins were subjected to a 7.5% SDS-PAGE and
electroblotted onto nitrocellulose membranes. Following transfer,
the membranes were immunoblotted with an antibody against p42/p44
MAP kinases (Thr202/Tyr204) (Santa Cruz Biotechnology Inc., Santa
Cruz, Calif., USA) and p38 MAP kinase (Thr180/Tyr182), at a concentration
of 1 .mu.g/mL, followed by incubation with a secondary anti-mouse
IgG-HRP (1:5000). To ensure equal loading of samples, membranes
were stripped and reprobed with anti-p42/p44 (Santa Cruz Biotechnology
Inc., Santa Cruz, Calif., USA) or anti-p38 antibodies.
 Akt Phosphorylation Assay
 DU4475 cells were seeded in 12 well-plates (5.times.10.sup.5
cells/well) in serum-free RPMI for 18 hours. The cells were then
washed 3 times with cold PBS, treated with or without growth factors
or anti-human VEGFR-1 mAb as indicated above, and also co-incubated
with the PI3-kinase inhibitor wortmannin (30nM) for 1 hour and PlGF
for 10 minutes. Cell lysis, protein isolation, SDS-PAGE and electroblot
into nitrocellulose membranes were performed as described previously.
Levels of Akt phosphorylation (Ser473) were detected using a primary
mouse polyclonal anti-phospho-Akt antibody (Santa Cruz Biotechnology
Inc., Santa Cruz, Calif., USA), at a concentration of 1 .mu.g/mL,
followed by incubation with a secondary anti-mouse IgG-HRP (1:5000).
To confirm equivalent protein loading, membranes were stripped and
reprobed with anti-Akt antibodies (Santa Cruz Biotechnology Inc.,
Santa Cruz, Calif., USA).
 Analysis of Apoptosis in Breast Cancer Cell Lines
 DU4475 cells were seeded in 12 well-plates (5.times.10.sup.5
cells/well), and kept for 48 hours under the following conditions:
serum-free RPMI 1640, RPMI with 10% FCS, clone 6.12 (2 .mu.g/mL),
clone 6.12 (10 .mu.g/mL) and 4% paraformaldehyde (positive control).
Cells were harvested and stained by fluorescein isothiocyanate-conjugated
annexin V and by PI, following the manufacturer's instructions (Immunotech,
 Results were analyzed using a Coulter Elite flow cytometer
(COULTER, Hialeah, Fla., USA). Cells which were double positive
for FITC-labeled annexin V, and PI were considered apoptotic.
 In vivo Effects of VEGFR-1 mAbs in the Growth of Established
DU4475 Breast Tumors
 To evaluate the effect of VEGFR-1 Abs against fully established
tumors, DU4475 human breast tumor cells (1.times.10.sup.6) were
injected subcutaneous into athymic nude mice (Jackson Labs, Bar
Harbor, Me., USA). Mice were divided in groups of 16 animals each
and tumors were allowed to grow up to approximately 20, 120 and
400 mm.sup.3 in size. Treated animals received intraperitoneal injections
of 1000 .mu.g of: anti-mouse VEGFR-1 mAb (mF1), anti-human VEGFR
-1 mAb (6.12), or the combination of both, every 3 days. The control
group was injected with PBS. Tumors were measured twice a week for
42 days. Tumor tissues were taken for histological examination on
days 14, 30 and at the end of the experiment after antibodies treatment.
mAb to VEGFR-1 Blocks Breast Cancer Gowth in vivo
 Subcutaneous inoculation into NOD-SCID mice of DU-4475 human
breast carcinoma cells resulted in the generation of large solid,
highly vascularized tumors in vivo, which could be detected and
measured after 4-5 days.
 Treatment of DU-4475 tumor-bearing mice with neutralizing
mAb against murine VEGFR-1 (clone MF1) or mAb against murine VEGFR-2
(DC101), 400 .mu.p every three days, to block host-derived angiogenesis,
delayed the growth of this breast carcinoma, an effect that was
particularly clear between days 14 and 21 post-inoculation. However,
this treatment alone was not sufficient to completely block tumor
growth, and 21 days after implantation the tumors in MF1 or DC101-treated
mice grew to the same size as control (untreated) mice.
 Treatment of tumor-bearing mice with neutralizing mAb to
human VEGFR-1 (6.12) (400 .mu.g every three days) resulted in a
dramatic delay in tumor growth, which was sustained for up to 28
days post- inoculation. Notably, DU-4475-bearing mice did not respond
to IMC-IC11 (anti-human VEGFR-2) treatment, confirming these breast
tumor cells express only functional VEGFR-1 (Flt-1). However, despite
a significant delay in tumor growth, tumors from mice treated with
the mAb 6.12 still had viable tumor areas after 21 days. These tumors
eventually grew to 1 cm.sup.3 after 36 days and started invading
the surrounding skin, at which point the mice were sacrificed.
 Co-administration of 6.12 (targeting the VEGF/human VEGFR-1
autocrine loop) with MF1 or DC101 (targeting the endothelial-dependent
paracrine loop) to DU-4475 bearing mice resulted in a synergistic
inhibition of tumor growth. Mice treated simultaneously with 6.12+DC101
(400 .mu.g of each every three days) or 6.12+MF1 (400 .mu.g of each
every three days) showed a significant and sustained delay in tumor
growth, producing fully necrotic and regressing tumors after 21-28
days, which in the case of the 6.12/DC101 (mAb against human VEGFR-1/mAb
against murine VEGFR-2) combination could no longer be measured
after 36 days. Therefore, a sustained delay in tumor growth was
produced only in mice treated with mAbs against both paracrine and
autocrine VEGF/NVEGF receptor signaling pathways.
 In vivo Experiments with the DU4475 Breast Cancer Cell Line
 Non-obese diabetic immunocompromised (NOD-SCID) mice (Jackson
Labs, Bar Harbor, Me., USA) were used in all experiments. DU4475
cells (1.times.10.sup.6/mouse) were injected subcutaneously into
21 NOD-SCID mice, and 4 days after injection mice were divided into
7 groups of three mice each. Intraperitoneal treatments started
4 days after cell inoculation. Six of the groups were treated three
times a week with the neutralizing mAb: 400 .mu.g of anti-human
Flt-1 (clone 6.12), 400 .mu.g of anti-murine VEGFR-1 (mF1), 400
.mu.g of anti-human VEGFR-2 (IMC1-C11), 800 .mu.g of anti-murine
VEGFR-2 (DC101). The control group was untreated.
 Tumors were measured every 3-4 days for 35 days. When tumors
reached approximately 1000 mm.sup.3, mice were sacrificed. Tumors
were excised, fixed in 2% paraformaldehyde, stored in 70% ethanol
and processed for immunohistochemical analysis, following conventional
protocols (see above). Paraffin blocks were cut to 5-.mu.m sections
and stained with hematoxylin and eosin (H&E), for morphology