Disclosed are methods for the detection, diagnosis and prediction
of tamoxifen-resistant breast cancer. Genetic and antibody probes
and methods useful in determining the presence and monitoring the
progression of breast cancer are also described. The methods involve
determining polypeptide or mRNA expression of the genes encoding
the angiogenic agents or receptors TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR. Also described are procedures for combination
therapies utilizing antiangiogenic agents or gene therapy directed
towards TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or
bFGFR, in combination with tamoxifen treatment of breast cancer.
1. A method for detecting tamoxifen-resistant breast cancer cells,
comprising: a) obtaining a sample suspected of containing tamoxifen-resistant
breast cancer cells; b) contacting said sample with an antibody
that specifically binds to a polypeptide selected from the group
consisting of tyrosine protein kinase receptor (TIE-2), endothelin-1
receptor (EDNRA), transforming growth factor .beta.3 (TGF.beta.3),
transforming growth factor receptor .beta.III (TGFR.beta.III), vascular
permeability factor receptor (VEGFR1), vascular endothelin growth
factor (VEGF) and basic fibroblast growth factor receptor (bFGFR),
under conditions effective to bind said antibody and form a complex;
c) measuring the amount of said polypeptide present in said sample
by quantitating the amount of said complex; and d) comparing the
amount of polypeptide present in said sample with the amount of
polypeptide in estrogen-stimulated, tamoxifen-sensitive and tamoxifen-resistant
breast cancer cells, wherein an increase in the amount of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGF or VEGFR1 polypeptide or
a decrease in the amount of bFGFR polypeptide in said sample compared
with the amount in estrogen-stimulated or tamoxifen-sensitive breast
cancer cells indicates the presence of tamoxifen-resistant breast
2. The method of claim 1, further comprising: a) measuring the
amounts of two or more polypeptides selected from the group consisting
of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF and bFGFR;
and b) for each polypeptide, comparing the amount of said polypeptide
present in said sample with the amount of the same polypeptide present
in estrogen-stimulated, tamoxifen-sensitive and tamoxifen-resistant
breast cancer cells.
3. The method of claims 1 or 2, further comprising providing a
diagnosis of tamoxifen-sensitive or tamoxifen-resistant breast cancer.
4. The method of claims 1 or 2, further comprising providing a
prediction of the existence or development of tamoxifen-resistant
5. A method of determining survival for an individual with breast
cancer, comprising determining the levels of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR polypeptide in a breast cancer
tissue sample from said individual, wherein the presence of elevated
levels of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGF or VEGFR1
polypeptide or decreased levels of bFGFR polypeptide in said tissue
sample relative to estrogen-stimulated or tamoxifen sensitive breast
cancer samples is associated with a decreased survival of the individual.
6. A method for detecting tamoxifen-resistant breast cancer cells,
comprising: a) isolating a nucleic acid from a sample suspected
of containing tamoxifen-resistant breast cancer cells; b) contacting
said nucleic acid with a pair of primers effective to amplify the
nucleic acid sequences of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR; c) amplifying said nucleic acid using said
primers to form an amplification product; d) quantitating the amount
of said amplification product formed; and e) comparing the amount
of said amplification product formed from said sample with the amount
of amplification product formed under identical conditions from
estrogen-stimulated, tamoxifen-sensitive and tamoxifen-resistant
breast cancer cells, wherein a difference in the amount of said
amplification product formed from said sample compared with the
amount formed from estrogen-stimulated or tamoxifen-sensitive breast
cancer cells indicates the presence of tamoxifen-resistant breast
7. The method of claim 6, further comprising: a) measuring the
amount of two or more amplification products using primers effective
to amplify the nucleic acid sequences of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR; and b) for each amplification
product, comparing the amount of amplification product formed from
said sample with the amount of amplification product formed from
estrogen-stimulated, tamoxifen-sensitive and tamoxifen-resistant
breast cancer cells.
8. The method of claim 6 or 7, further comprising providing a diagnosis
of tamoxifen-sensitive or tamoxifen-resistant breast cancer.
9. The method of claim 6 or 7, further comprising providing a prediction
for likelihood of development of tamoxifen-resistant breast cancer
and subsequent patient survival.
10. A method of determining survival for an individual with breast
cancer, comprising determining the levels of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR amplification product formed
from a breast cancer tissue sample from said individual, wherein
the presence of elevated levels of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGF or VEGFR1 amplification product or decreased levels of bFGFR
amplification product formed from said tissue sample compared with
estrogen-stimulated or tamoxifen-sensitive breast cancer cells is
associated with a decreased survival of the individual.
11. A method for altering the phenotype of a breast cancer cell
comprising contacting the cell with (i) a gene selected from the
group consisting of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF and bFGFR and (ii) a promoter active in said cancer cell, wherein
said promoter is operably linked to the region encoding said gene,
under conditions effective for the uptake and expression of said
gene by said breast cancer cell.
12. A method for treating breast cancer, comprising: a) providing
an effective amount of an antiangiogenic agent; and b) providing
an effective amount of tamoxifen.
13. The method of claim 12, wherein the antiangiogenic agent is
selected from the group consisting of AGM-1470 (TNP-470), platelet
factor 4 and angiostatin.
14. A method for treating breast cancer, comprising: a) providing
an effective amount of an antisense construct containing a gene
selected from the group consisting of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGF and VEGFR1 under conditions allowing for the
uptake and expression of said construct by said breast cancer; and
b) providing an effective amount of tamoxifen.
15. A method for treating breast cancer, comprising: a) providing
an effective amount of an expression construct containing a gene
encoding bFGFR under conditions allowing for the uptake and expression
of said construct by said breast cancer; and b) providing an effective
amount of tamoxifen.
16. A kit comprising: a) one or more antibodies that specifically
bind to TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or
bFGFR polypeptide; and b) a container for each of said antibodies.
17. A kit comprising: a) one or more pairs of primers effective
to amplify the nucleic acid sequences of messenger RNAs encoded
by TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR;
and b) a container for each of said primers.
18. A method of detecting markers for tamoxifen-resistant breast
cancer, comprising: a) isolating nucleic acids from samples of estrogen-stimulated,
tamoxifen-sensitive and tamoxifen-resistant breast cancers; b) converting
messenger RNAs to cDNAs; c) screening the cDNA species with a human
cDNA expression array; and d) identifying cDNA species that are
differentially expressed in tamoxifen resistant breast cancers versus
estrogen-stimulated or tamoxifen sensitive breast cancers, wherein
differential expression indicates a marker for tamoxifen-resistant
19. A pharmaceutical composition comprising two or more nucleic
acids selected from the group consisting of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF and bFGFR.
20. The composition of claim 19, wherein said nucleic acids are
in the form of vectors.
21. A pharmaceutical composition comprising two or more polypeptides
selected from the group consisting of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF and bFGFR.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention relates generally to methods of detecting
antiestrogen resistant human breast cancer and the use of polypeptides
and nucleic acids encoding angiogenic factors or angiogenic receptors
for such methods. More particularly, certain methods utilizing differential
expression of genes encoding tyrosine protein kinase receptor (TIE-2),
endothelin-1 receptor (EDNRA), transforming growth factor .beta.3
(TGF.beta.3), transforming growth factor receptor .beta.III (TGFR.beta.III),
vascular permeability factor receptor (VEGFR1), vascular endothelin
growth factor (VEGF) and basic fibroblast growth factor receptor
(bFGFR) are described that may provide the basis for predictive
and diagnostic evaluations of human breast cancer patients.
 2. Description of Related Art
 Breast cancer is the leading cause of death for women between
30-50 years of age in the United States. Pathological breast cancer
staging (tumor size, nodal status) is still the most reliable method
for predicting outcome. In contrast to other forms of cancer, only
a few tumor markers have been identified for breast cancer (e.g.,
estrogen receptor, progesterone receptor, S-phase, P53, Erb-2, cathepsin
D) (see, e.g. Slamon et al., 1987).
 Mutational analysis of important tumor suppressor genes
such as p53 (Elledge, 1994) and BRCA1 (Miki et al., 1994) has recently
been introduced as a diagnostic and prognostic test for breast cancer.
Mutations in the breast cancer susceptibility genes BRCA1 (chromosome
17q21) and BRCA2 (chromosome 13q13) are associated with familial
breast cancer, accounting for about 5% of total breast cancer cases,
but have not been found in sporadic breast cancer (Stratton and
Wooster, 1996). To date, none of these markers has proven to be
reliable enough to be used for routine screening for breast cancer
in the clinic. Therefore, there is an urgent need for better prognostic
markers in breast cancer diagnosis, measured either by "traditional"
methods (e.g., immunohistochemistry, Western blot), or genetic test.
 Tamoxifen is the most commonly prescribed drug for breast
cancer in the world (Johnston, 1997). Tamoxifen is thought to inhibit
breast cancer growth by competitively blocking the estrogen receptor
(ER), thereby inhibiting estrogen-induced growth (Osborne and Fuqua,
1994). Over the past two decades its role has expanded from primary
treatment for advanced metastatic disease to established adjuvant
therapy following surgery for primary disease (Johnston, 1997).
Tamoxifen prolongs both disease-free and overall survival in breast
cancer patients (Johnston, 1997). But, while tamoxifen is effective
in many breast cancer patients, eventually all patients develop
tamoxifen resistance (Johnston, 1997). Thus, the widespread use
of tamoxifen in clinical practice has resulted in a significant
increase in the number of patients presenting at recurrence with
tamoxifen-resistant disease (Johnston, 1997). The mechanisms for
tamoxifen resistance are largely unknown and their identification
could have profound clinical implications for alternative treatment
strategies (Osborne and Fuqua, 1994; Johnston, 1997).
 Previous studies in the areas of tamoxifen resistance and
breast cancer progression have focused on alterations in the estrogen
receptor (Osborne and Fuqua, 1994; lemieux and Fuqua, 1996; Zhang
et al., 1997a), changes in ER accessory proteins (Osborne &
Fuqua, 1994), clonal selection of ER negative tumor cells (Johnston,
1997), apoptosis factors (Johnston, 1997), AP-1 (Schiff et al.,
1998), SRC-1 (Berns et al., 1998) and growth factor receptors (Johnston,
1997). It has been reported that overexpression of single growth
factor genes such as cyclin D1 (Neuman et al., 1997), protein kinase
A (Fujimoto and Katzenellenbogen, 1994) and transforming growth
factor .beta. (Thompson et al., 1991) can influence a cell's response
to tamoxifen treatment. Despite this extensive work, the precise
mechanisms underlying acquired tamoxifen resistance remain poorly
 Breast cancer is a heterogeneous disease and the development
of tamoxifen resistance is probably multifactorial (Osborne and
Fuqua, 1994). Thus, complex changes in patterns of gene expression
may accompany the resistant phenotype. The present invention satisfies
a long-standing need in the field by identifying changes in gene
expression that are associated with the development of tamoxifen
 Those genes identified herein as differentially expressed
during the development of tamoxifen resistance generally fall into
the categories of angiogenic factors or angiogenic receptors. An
association between angiogenesis and tumor growth has been reported
and anticancer therapies based upon antiangiogenic agents have been
explored (Folkman, 1995a; Lin et al., 1998). However, the present
application is the first report of an association between the development
of tamoxifen resistance and the differential expression of angiogenic
factors or receptors in human cancer.
SUMMARY OF THE INVENTION
 The present invention addresses deficiencies in the art
by identifying specific gene products whose expression levels serve
as markers for tamoxifen-resistant breast cancer. More particularly,
differential expression of the genes encoding tyrosine protein kinase
receptor (TIE-2, GenBank Accession No. L06139), endothelin-1 receptor
(EDNRA, GenBank Accession No. L06622), transforming growth factor
.beta.3 (TGF.beta.3, GenBank Accession No. J03241), transforming
growth factor receptor .beta.3 (TGFR.beta.III, GenBank Accession
No. L07594), vascular permeability factor receptor (VEGFR1, GenBank
Accession No. U01134), vascular endothelin growth factor (VEGF,
GenBank Accession Nos. M32977) and basic fibroblast growth factor
receptor (bFGFR, GenBank Accession No. M60485) are reported herein
to be associated with tamoxifen-resistant breast cancer. This surprising
result is the first report of an association between the development
of tamoxifen-resistant tumors and changes in expression of angiogenic
factors or receptors. These results provide the basis for methods
directed toward detection of expression levels of TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF and bFGFR in breast tissue
samples which will have utility for diagnosis and prediction of
tamoxifen-resistant breast cancer.
 One aspect of the present invention encompasses antibodies
specific for TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
and bFGFR and immunological methods for detection and measurement
of these proteins in tissue samples. Such methods may include the
use of Western blots, immunohistochemistry (IHC), ELISA, and other
well known techniques for antibody assay of protein expression.
Another aspect concerns the use of such antibodies for methods of
breast cancer cell detection, diagnosis and prediction, by comparing
the levels of for TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF and bFGFR polypeptide in suspected tamoxifen-resistant cancer
cells with levels present in groups of known estrogen stimulated,
tamoxifen-sensitive and tamoxifen-resistant breast cancer cells.
 One embodiment of the invention encompasses a kit for use
in the detection and measurement of these proteins in tissue samples,
comprising antibodies specific for TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR. Additional components of kits for immunologic
detection of disease-state associated antigens are well known in
the art, and may include components such as molecular weight marker
proteins, secondary antibodies, reagents for staining or otherwise
detecting bound antibodies, control samples containing known amounts
of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF and bFGFR
protein or peptide, and negative controls lacking these proteins.
 The invention also comprises nucleic acid segments that
are either identical to or complementary with the cDNA sequences
of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF and bFGFR.
Such nucleic acid segments are expected to have utility not only
as probes or primers for the genetic analysis of breast tumor samples
but also, for example, as components of expression vectors or antisense
vectors for transformation of tamoxifen-resistant breast cancer
cells that differentially express these proteins. Such vectors may
have utility in the treatment of tamoxifen-resistant breast cancer.
 An additional embodiment encompasses genetic analysis of
tissue samples to obtain information relating to tumor progression
and tamoxifen-resistance. Such analyses typically employ PCRT amplification,
using primers specific for the human cDNA sequences of TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF and bFGFR, followed by quantitative
analysis of the amplification products. Quantitative analysis of
amplification products or of the mRNA species themselves may be
performed by any standard means, including Southern blots, slot-blots,
and Northern blots. In a preferred embodiment, the mRNA species
present in a tissue sample are converted to cDNA prior to amplification,
using reverse transcriptase. One example of such a protocol is the
well known procedure of RT-PCR.TM.. Tumors with differentially expressed
levels of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
and bFGFR are recognized as associated with a poorer five-year survival
rate for breast cancer patients. One may therefore assess potential
survival rates in such patients by assaying the levels of these
mRNA or protein species.
 Yet another aspect of the present invention encompasses
host cells or vectors comprising a nucleic acid encoding TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR. Such cells
or vectors are expected to have utility in the therapeutic treatment
of breast cancer. Insertion of a vector comprising an antisense
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III or VEGFR1, or an expression
cassette for VEGF or bFGFR into tumor cells from breast cancers
may result in suppression of tumor growth and colony formation.
Thus, an embodiment of the present invention comprises a method
for altering the phenotype of a tumor cell by contacting the cell
with a nucleic acid encoding antisense or expression cassettes for
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR,
operably linked to a functional promoter, under conditions permitting
uptake and expression of the nucleic acid by the tumor cell.
 A further embodiment of the present invention concerns the
use of antiangiogenic agents or gene therapy as an adjunct to tamoxifen
treatment, or to convert tamoxifen resistant tumors into tamoxifen
sensitive tumors. Antiantiogenic gene therapy may be accomplished,
for example, by the methods of Lin et al., (1998), incorporated
herein by reference in its entirety. Alternatively, antiangiogenic
agents, such as AGM-1470 (TNP-470), platelet factor 4 and angiostatin
may be used as tamoxifen adjuncts or for conversion of tamoxifen-resistant
to tamoxifen-sensitive tumors (Folkman, 1995b). Additional antiangiogenic
agents that may be used in the practice of the present invention
are identified in Augustin (1998), incorporated herein by reference
in its entirety. Antiangiogenic therapy may be combined with traditional
forms of chemotherapy or radiation therapy (Folkman, 1995a), targeted
specifically against tamoxifen-resistant breast tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 shows a scatterplot matrix of expression data for
588 genes, collected from estrogen-stimulated (ES), tamoxifen-sensitive
(TS) and tamoxifen-resistant (TR) breast cancers. Data were collected
as described in the EXAMPLES section.
 FIG. 2A shows a scatterplot of log-transformed expression
data for TS and TR tumors, showing the line of identity (solid line)
and 99% prediction region (dashed line). Genes that are overexpressed
in TR tumors compared to TS tumors are indicated by open circles
and underexpressed genes are indicated by solid triangles.
 FIG. 2B shows a scatterplot of first and second principal
components from the same data as shown in FIG. 2A.
 FIG. 3 illustrates a scatterplot of second and third principal
components from PCA (principal component analysis) of log-transformed
gene expression data from ES, TS and TR tumors, back transformed
to show approximate fold alterations. Axis labels describe the qualitative
interpretation of PCA coefficients. Genes inside the 99% prediction
ellipse (indicated by solid line) are shown as open circles, genes
outside the ellipse are shown as closed circles.
 FIG. 4 shows a Western blot analysis with erk-2 and HSF-1
antibodies in ES, TS and TR tumors. Molecular weight marker positions
are indicated on the right side.
 FIG. 5 illustrates the fold change in expression in estrogen-stimulated
(E2), tamoxifen-sensitive (TS) and tamoxifen-resistant (TR) breast
cancers for the TGFR.beta.III, VEGR1, TGF.beta.3, EDNRA and TIE-2
genes. Data were collected as described in the EXAMPLES section.
 FIG. 6 describes the fold change in expression in estrogen-stimulated
(E2), tamoxifen-sensitive (TS) and tamoxifen-resistant (TR) breast
cancers for the VEGF and bFGFR genes, as described in the legend
to FIG. 5.
 FIG. 7 shows a Western blot analysis using a commercial
antibody (Santa Cruz, Inc., Santa Cruz, Calif.) to the TIE-2 receptor
protein. Five tumors of each group (E2, TS and TR) were examined.
Only the TR tumors exhibited detectable expression of a high molecular
weight (220 kDa) form of TIE-2 (putative TIE-2 related protein).
 FIG. 8 shows a Western blot analysis using a commercial
antibody (Santa Cruz, Inc., Santa Cruz, Calif.) to the TIE-2 receptor
protein. Human vascular endothelial cells (HuVec) and one tumor
of each mouse breast cancer group (E2, TS and TR) were examined.
HuVec cells express a TIE-2 protein of approximately 140 kDa, compared
to the 220 kDa TIE-2 related protein expressed in TR tumors.
 FIG. 9 shows a Western blot analysis using an antibody to
the VEGF protein. Five tumors of each group (E2, TS and TR) were
examined. VEGF monomer and dimers were relatively overexpressed
in the TR tumors.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 This application concerns, at least in part, isolated proteins
and nucleic acids encoded by tyrosine protein kinase receptor (TIE-2,
GenBank Accession No. L06139), endothelin-1 receptor (EDNRA, GenBank
Accession No. L06622), transforming growth factor .beta.3 (TGF.beta.3,
GenBank Accession No. J03241), transforming growth factor receptor
.beta.III (TGFR.beta.III, GenBank Accession No. L07594), vascular
permeability factor receptor (VEGFR1, GenBank Accession No. U01134),
vascular endothelin growth factor (VEGF, GenBank Accession Nos.
M32977) and basic fibroblast growth factor receptor (bFGFR, GenBank
Accession No. M60485) as well as methods of detection, diagnosis,
prediction and therapeutic treatment of tamoxifen-resistant breast
cancer directed towards such proteins and nucleic acids.
 In referring to the function of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR or "wild-type" activity,
it is meant that the molecule in question has the ability to inhibit
angiogenesis, or to prevent metastasis or invasive tumor growth.
Molecules possessing this activity may be identified using assays
familiar to those of skill in the art. For example, transfer of
genes encoding TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR, or variants thereof, into cells that do not have
a functional product, and hence exhibit impaired growth control,
will identify, by virtue of growth suppression, those molecules
having TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or
 The term "TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR gene" refers to any DNA sequence that
is substantially identical to a DNA sequence encoding a TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR protein as defined
above. Allowing for the degeneracy of the genetic code, sequences
that have at least about 50%, usually at least about 60%, more usually
about 70%, most usually about 80%, preferably at least about 90%,
and most preferably about 95% of nucleotides that are identical
to the cDNA sequences of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR are "as set forth in" those sequences.
Sequences that are substantially identical or "essentially
the same" as the cDNA sequences of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR also may be functionally defined
as sequences that are capable of hybridizing to a nucleic acid segment
containing the complement of the cDNA sequences of TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR under conditions
of relatively high stringency. Such conditions are typically relatively
low salt and/or high temperature conditions, such as provided by
about 0.02 M to about 0.15 M NaCl at temperatures of about 50.degree.
C. to about 70.degree. C. Such selective conditions tolerate little,
if any, mismatch between the complementary stands and the template
or target strand. "TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR gene" is also intended to include RNA,
or antisense sequences compatible with the cDNA sequences. Any such
gene sequences may also comprise associated control sequences.
 The term "substantially identical," when used
to define either an amino acid sequence or a nucleic acid sequence,
means that a particular subject sequence, for example, a mutant
sequence, varies from the sequence of the natural TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR genes by one or
more substitutions, deletions, or additions, the net effect of which
is to retain at least some biological activity of the protein or
 Alternatively, DNA analog sequences are "substantially
identical" to specific DNA sequences disclosed herein if: (a)
the DNA analog sequence is derived from coding regions of the natural
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR gene;
or (b) the DNA analog sequence is capable of hybridization of DNA
sequences of (a) under moderately stringent conditions and which
encode biologically active TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR; or (c) DNA sequences which are degenerative
as a result of the genetic code to the DNA analog sequences defined
in (a) or (b).
 The present invention also relates to fragments of the polypeptides
that may or may not retain the angiogenic (or other) activity of
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR.
Fragments including the N-terminus of the molecule may be generated
by genetic engineering of translation stop sites within the coding
region (discussed below). Alternatively, treatment of the protein
molecule with proteolytic enzymes, known as proteases, can produce
a variety of N-terminal, C-terminal and internal fragments. Examples
of fragments may include contiguous residues of the TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR amino acid sequences
of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95,
100, 200, 300, 400, or more amino acids in length. These fragments
may be purified according to known methods, such as precipitation
(e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity
chromatography (including immunoaffinity chromatography), or various
size separations (e.g., sedimentation, gel electrophoresis, gel
 Substantially identical analog proteins will be greater
than about 80% similar to the corresponding sequence of the native
protein. Sequences having lesser degrees of similarity but comparable
biological activity are considered to be equivalents. In determining
nucleic acid sequences, all subject nucleic acid sequences capable
of encoding substantially similar amino acid sequences are considered
to be substantially similar to a reference nucleic acid sequence,
regardless of differences in codon sequence.
 Purification of Proteins
 It may be desirable to purify TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR or variants thereof. Protein
purification techniques are well known to those of skill in the
art. These techniques involve, at one level, the crude fractionation
of the cellular milieu to polypeptide and non-polypeptide fractions.
Having separated the polypeptide from other proteins, the polypeptide
of interest may be further purified using chromatographic and electrophoretic
techniques to achieve partial or complete purification (or purification
to homogeneity). Analytical methods particularly suited to the preparation
of a pure peptide are ion-exchange chromatography, gel exclusion
chromatography, polyacrylamide gel electrophoresis, affinity chromatography,
immunoaffinity chromatography and isoelectric focusing. A particularly
efficient method of purifying peptides is fast protein liquid chromatography
(FPLC) or even HPLC.
 Certain aspects of the present invention concern the purification,
and in particular embodiments, the substantial purification, of
an encoded protein or peptide. The term "purified protein or
peptide" as used herein, is intended to refer to a composition,
isolatable from other components, wherein the protein or peptide
is purified to any degree relative to its naturally-obtainable state.
A purified protein or peptide, therefore, also refers to a protein
or peptide free from the environment in which it may naturally occur.
 Generally, "purified" will refer to a protein
or peptide composition that has been subjected to fractionation
to remove various other components, and which composition substantially
retains its expressed biological activity. Where the term "substantially
purified" is used, this designation will refer to a composition
in which the protein or peptide forms the major component of the
composition, such as constituting about 50%, about 60%, about 70%,
about 80%, about 90%, about 95%, or more of the proteins in the
 Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or assessing
the amount of polypeptides within a fraction by SDS/PAGE analysis.
A preferred method for assessing the purity of a fraction is to
calculate the specific activity of the fraction, to compare it to
the specific activity of the initial extract, and to thus calculate
the degree of purity therein, assessed by a "-fold purification
number." The actual units used to represent the amount of activity
will, of course, be dependent upon the particular assay technique
chosen to follow the purification, and whether or not the expressed
protein or peptide exhibits a detectable activity.
 Various techniques suitable for use in protein purification
will be well known to those of skill in the art. These include,
for example, precipitation with ammonium sulphate, PEG, antibodies
and the like, or by heat denaturation, followed by: centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of these and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be changed,
or that certain steps may be omitted, and still result in a suitable
method for the preparation of a substantially purified protein or
 There is no general requirement that the protein or peptide
always be provided in their most purified state. Indeed, it is contemplated
that less substantially purified products will have utility in certain
embodiments. Partial purification may be accomplished by using fewer
purification steps in combination, or by utilizing different forms
of the same general purification scheme. For example, it is appreciated
that a cation-exchange column chromatography performed utilizing
an HPLC apparatus will generally result in a greater "-fold"
purification than the same technique utilizing a low pressure chromatography
system. Methods exhibiting a lower degree of relative purification
may have advantages in total recovery of protein product, or in
maintaining the activity of an expressed protein.
 It is known that the migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE (Capaldi
et al., 1977). It will, therefore, be appreciated that under differing
electrophoresis conditions, the apparent molecular weights of purified
or partially purified expression products may vary.
 High Performance Liquid Chromatography (HPLC) is characterized
by a very rapid separation with extraordinary resolution of peaks.
This is achieved by the use of very fine particles and high pressure
to maintain an adequate flow rate. Separation can be accomplished
in a matter of min, or at most an h. Moreover, only a very small
volume of the sample is needed because the particles are so small
and close-packed that the void volume is a very small fraction of
the bed volume. Also, the concentration of the sample need not be
very great because the bands are so narrow that there is very little
dilution of the sample.
 Gel chromatography, or molecular sieve chromatography, is
a special type of partition chromatography that is based on molecular
size. The theory behind gel chromatography is that the column, which
is prepared with tiny particles of an inert substance that contain
small pores, separates larger molecules from smaller molecules as
they pass through or around the pores, depending on their size.
As long as the material of which the particles are made does not
adsorb the molecules, the sole factor determining rate of flow is
the size of the pores. Hence, molecules are eluted from the column
in decreasing size, so long as the shape is relatively constant.
Gel chromatography is unsurpassed for separating molecules of different
size because separation is independent of all other factors such
as pH, ionic strength, temperature, etc. Thus the elution volume
is related in a simple matter to molecular weight.
 Affinity chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule to which it can specifically bind to. This is a receptor-ligand
type of interaction. The column material is synthesized by covalently
coupling one of the binding partners to an insoluble matrix. The
column material is then able to specifically adsorb the substance
from the solution. Elution occurs by changing the conditions to
those in which binding will not occur (e.g., altered pH, ionic strength,
 The matrix should be a substance that itself does not adsorb
molecules to any significant extent and that has a broad range of
chemical, physical and thermal stability. The ligand should be coupled
in such a way as to not affect its binding properties. The ligand
should also provide relatively tight binding. And it should be possible
to elute the substance without destroying the sample or the ligand.
One of the most common forms of affinity chromatography is immunoaffinity
chromatography. The generation of antibodies that would be suitable
for use in accord with the present invention is discussed below.
 Synthetic Peptides
 The present invention also describes smaller TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR peptides for use
in various embodiments of the present invention. Because of their
relatively small size, the peptides of the invention can also be
synthesized in solution or on a solid support in accordance with
conventional techniques. Various automatic synthesizers are commercially
available and can be used in accordance with known protocols. See,
for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield,
(1986); and Barany and Merrifield (1979), each incorporated herein
by reference. Short peptide sequences, or libraries of overlapping
peptides, usually from about 6 up to about 35 to 50 amino acids,
which correspond to selected regions of the TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR proteins, can be readily synthesized
and then screened in screening assays designed to identify reactive
peptides. Alternatively, recombinant DNA technology may be employed
wherein a nucleotide sequence which encodes a peptide of the invention
is inserted into an expression vector, transformed or transfected
into an appropriate host cell, and cultivated under conditions suitable
 Antigen Compositions
 The present invention also provides for the use of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR 1, VEGF or bFGFR proteins
or peptides as antigens for the immunization of animals relating
to the production of antibodies. It is envisioned that either TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR proteins,
or portions thereof, will be coupled, bonded, bound, conjugated,
or chemically-linked to one or more agents via linkers, polylinkers,
or derivatized amino acids. This may be performed such that a bispecific
or multivalent composition or vaccine is produced. It is further
envisioned that the methods used in the preparation of these compositions
will be familiar to those of skill in the art and should be suitable
for administration to animals, i.e., pharmaceutically acceptable.
Preferred agents are the carriers are keyhole limpet hemocyanin
(KLH) or bovine serum albumin (BSA).
 Nucleic Acids
 The present invention also provides, in another embodiment,
genes encoding TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR. As discussed below, a "TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR gene" may contain a variety
of different bases and yet still produce a corresponding polypeptide
that is indistinguishable functionally, and in some cases structurally,
from the genes disclosed herein.
 Similarly, any reference to a nucleic acid should be read
as encompassing a host cell containing that nucleic acid and, in
some cases, capable of expressing the product of that nucleic acid.
In addition to therapeutic considerations, cells expressing nucleic
acids of the present invention may prove useful in the context of
screening for agents that induce, repress, inhibit, augment, interfere
with, block, abrogate, stimulate, or enhance the function of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR.
 Nucleic Acids Encoding TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR
 Nucleic acids according to the present invention may encode
an entire gene, a domain of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR that expresses a tumor suppressing function,
or any other fragment of the sequences set forth herein. The nucleic
acid may be derived from genomic DNA, i.e., cloned directly from
the genome of a particular organism. In preferred embodiments, however,
the nucleic acid would comprise complementary DNA (cDNA). Also contemplated
is a cDNA plus a natural intron or an intron derived from another
gene; such engineered molecules are sometime referred to as "mini-genes."
At a minimum, these and other nucleic acids of the present invention
may be used as molecular weight standards in, for example, gel electrophoresis.
 The term "cDNA" is intended to refer to DNA prepared
using messenger RNA (mRNA) as template. The advantage of using a
cDNA, as opposed to genomic DNA or DNA polymerized from a genomic,
non- or partially-processed RNA template, is that the cDNA primarily
contains coding sequences of the corresponding protein. There may
be times when the full or partial genomic sequence is preferred,
such as where the non-coding regions are required for optimal expression
or where non-coding regions such as introns are to be targeted in
an antisense strategy.
 It also is contemplated that TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR may be represented by natural variants that
have slightly different nucleic acid sequences but, nonetheless,
encode the same proteins (see Table 1 below).
 As used in this application, the term "a nucleic acid
encoding a TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR" refers to a nucleic acid molecule that has been isolated
free of total cellular nucleic acid. The term "functionally
equivalent codon" is used herein to refer to codons that encode
the same amino acid, such as the six codons for arginine or serine
(Table 1, below), and also refers to codons that encode biologically
equivalent amino acids, as discussed in the following pages.
1 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine
His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine
Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA
CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU
UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA
GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
 The DNA segments of the present invention include those
encoding biologically functional equivalent TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR 1, VEGF or bFGFR proteins and peptides, as
described above. Such sequences may arise as a consequence of codon
redundancy and amino acid functional equivalency that are known
to occur naturally within nucleic acid sequences and the proteins
thus encoded. Alternatively, functionally equivalent proteins or
peptides may be created via the application of recombinant DNA technology,
in which changes in the protein structure may be engineered, based
on considerations of the properties of the amino acids being exchanged.
Changes designed by man may be introduced through the application
of site-directed mutagenesis techniques or may be introduced randomly
and screened later for the desired function, as described below.
 Oligonucleotide Probes and Primers
 Naturally, the present invention also encompasses DNA segments
that are complementary, or essentially complementary, to the cDNA
sequences of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR. Nucleic acid sequences that are "complementary"
are those that are capable of base-pairing according to the standard
Watson-Crick complementary rules. As used herein, the term "complementary
sequences" means nucleic acid sequences that are complementary
to the extent that they are capable of hybridizing under relatively
stringent conditions such as those described herein. Such sequences
may encode the entire TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR protein or functional or non-functional fragments
 Alternatively, the hybridizing segments may be shorter oligonucleotides.
Sequences of 17 bases long should occur only once in the human genome
and, therefore, suffice to specify a unique target sequence. Although
shorter oligomers are easier to make and increase in vivo accessibility,
numerous other factors are involved in determining the specificity
of hybridization. Both binding affinity and sequence specificity
of an oligonucleotide to its complementary target increases with
increasing length. It is contemplated that exemplary oligonucleotides
of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more base
pairs will be used, although others are contemplated. Longer polynucleotides
encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or 3040 bases
and longer are contemplated as well. Such oligonucleotides will
find use, for example, as probes in Southern and Northern blots
and as primers in amplification reactions.
 Hybridization Conditions
 Suitable hybridization conditions will be well known to
those of skill in the art. In certain applications, for example,
substitution of amino acids by site-directed mutagenesis, it is
appreciated that lower stringency conditions are required. Under
these conditions, hybridization may occur even though the sequences
of probe and target strand are not perfectly complementary, but
are mismatched at one or more positions. Conditions may be rendered
less stringent by increasing salt concentration and decreasing temperature.
For example, a medium stringency condition could be provided by
about 0.1 to 0.25 M NaCl, at temperatures of about 37.degree. C.
to about 55.degree. C., while a low stringency condition could be
provided by about 0.15 M to about 0.9 M salt, at temperatures ranging
from about 20.degree. C. to about 55.degree. C. Thus, hybridization
conditions can be readily manipulated, and thus will generally be
a method of choice depending on the desired results.
 In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl,
3 mM MgCl.sub.2, 10 mM dithiothreitol, at temperatures between approximately
20.degree. C. to about 37.degree. C. Other hybridization conditions
utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50
mM KCl, 1.5 .mu.M MgCl.sub.2, at temperatures ranging from approximately
40.degree. C. to about 72.degree. C. Formamide and SDS (sodium dodecylsulphate)
also may be used to alter the hybridization conditions.
 One method of using probes and primers of the present invention
is in the search for genes related to TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR. Normally, the target DNA will
be a genomic or cDNA library, although screening may involve analysis
of RNA molecules. By varying the stringency of hybridization, and
the region of the probe, different degrees of homology may be discovered.
 Antisense Constructs
 Antisense technology may be used to "knock-out"
function of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR in the treatment of tamoxifen-resistant breast cancers
or in the development of cell lines or transgenic mice for research,
diagnostic and screening purposes.
 Antisense methodology takes advantage of the fact that nucleic
acids tend to pair with "complementary" sequences. By
complementary, it is meant that polynucleotides are those which
are capable of base-pairing according to the standard Watson-Crick
complementarity rules. That is, the larger purines will base pair
with the smaller pyrimidines to form combinations of guanine paired
with cytosine (G:C) and adenine paired with either thymine (A:T)
in the case of DNA, or adenine paired with uracil (A:U) in the case
of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine,
6-methyladenine, hypoxanthine and others in hybridizing sequences
does not interfere with pairing.
 Targeting double-stranded (ds) DNA with polynucleotides
leads to triple-helix formation; targeting RNA will lead to double-helix
formation. Antisense polynucleotides, when introduced into a target
cell, specifically bind to their target polynucleotide and interfere
with transcription, RNA processing, transport, translation and/or
stability. Antisense RNA constructs, or DNA encoding such antisense
RNAs, may be employed to inhibit gene transcription, or translation,
or both within a host cell, either in vitro or in vivo, such as
within a host animal, including a human subject.
 Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns, or even exon-intron boundaries
of a gene. It is contemplated that the most effective antisense
constructs will include regions complementary to intron/exon splice
junctions. Thus, it is proposed that a preferred embodiment includes
an antisense construct with complementarity to regions within about
50-200 bases of an intron-exon splice junction. It has been observed
that some exon sequences can be included in the construct without
seriously affecting the target selectivity thereof. The amount of
exonic material included will vary depending on the particular exon
and intron sequences used. One can readily test whether too much
exon DNA is included simply by testing the constructs in vitro to
determine whether normal cellular function is affected or whether
the expression of related genes having complementary sequences is
 As stated above, "complementary" or "antisense"
means polynucleotide sequences that are substantially complementary
over their entire length and have very few base mismatches. For
example, sequences of fifteen bases in length may be termed complementary
when they have complementary nucleotides at thirteen or fourteen
positions. Naturally, sequences which are completely complementary
will be sequences which are entirely complementary throughout their
entire length and have no base mismatches. Other sequences with
lower degrees of homology also are contemplated. For example, an
antisense construct which has limited regions of high homology,
but also contains a non-homologous region (e.g., ribozyme; see below)
could be designed. These molecules, though having less than 50%
homology, would bind to target sequences under appropriate conditions.
 It may be advantageous to combine portions of genomic DNA
with cDNA or synthetic sequences to generate specific constructs.
For example, where an intron is desired in the ultimate construct,
a genomic clone will need to be used. The cDNA or a synthesized
polynucleotide may provide more convenient restriction sites for
the remaining portion of the construct and, therefore, would be
used for the rest of the sequence.
 Another approach for addressing overexpression of genes
in breast cancer is through the use of ribozymes. Although proteins
traditionally have been used for catalysis of nucleic acids, another
class of macromolecules has emerged as useful in this endeavor.
Ribozymes are RNA-protein complexes that cleave nucleic acids in
a site-specific fashion. Ribozymes have specific catalytic domains
that possess endonuclease activity (Kim and Cech, 1987; Gerlach
et al., 1987; Forster and Symons, 1987). For example, a large number
of ribozymes accelerate phosphoester transfer reactions with a high
degree of specificity, often cleaving only one of several phosphoesters
in an oligonucleotide substrate (Cech et al., 1981; Michel and Westhof,
1990; Reinhold-Hurek and Shub, 1992). This specificity has been
attributed to the requirement that the substrate bind via specific
base-pairing interactions to the internal guide sequence ("IGS")
of the ribozyme prior to chemical reaction.
 Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic
acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No.
5,354,855 reports that certain ribozymes can act as endonucleases
with a sequence specificity greater than that of known ribonucleases
and approaching that of the DNA restriction enzymes. Thus, sequence-specific
ribozyme-mediated inhibition of gene expression may be particularly
suited to therapeutic applications (Scanlon et al., 1991; Sarver
et al., 1990). Recently, it was reported that ribozymes elicited
genetic changes in some cell lines to which they were applied; the
altered genes included the oncogenes H-ras, c-fos and genes of HIV.
Most of this work involved the modification of a target mRNA, based
on a specific mutant codon that is cleaved by a specific ribozyme.
 It is anticipated that particularly appropriate targets
for ribozyme or antisense directed therapies for tamoxifen-resistant
breast cancer would be the genes or gene products for TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR 1, VEGF or bFGFR.
 Vectors for Cloning, Gene Transfer and Expression
 Within certain embodiments expression vectors are employed
to express the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR polypeptide product, which can then be purified and,
for example, be used to vaccinate animals to generate antisera or
monoclonal antibody with which further studies may be conducted.
In other embodiments, the expression vectors are used in gene therapy.
 Expression requires that appropriate signals be provided
in the vectors, and which include various regulatory elements, such
as enhancers/promoters from both viral and mammalian sources that
drive expression of the genes of interest in host cells. Elements
designed to optimize messenger RNA stability and translatability
in host cells also are defined. The conditions for the use of a
number of dominant drug selection markers for establishing permanent,
stable cell clones expressing the products are also provided, as
is an element that links expression of the drug selection markers
to expression of the polypeptide.
 Regulatory Elements
 Throughout this application, the term "expression construct"
is meant to include any type of genetic construct containing a nucleic
acid coding for a gene product in which part or all of the nucleic
acid coding sequence is capable of being transcribed. The transcript
may be translated into a protein, but it need not be. In certain
embodiments, expression includes both transcription of a gene and
translation of mRNA into a gene product. In other embodiments, expression
only includes transcription of the nucleic acid encoding a gene
 In preferred embodiments, the nucleic acid encoding a gene
product is under transcriptional control of a promoter. A "promoter"
refers to a DNA sequence recognized by the synthetic machinery of
the cell, or introduced synthetic machinery, required to initiate
the specific transcription of a gene. The phrase "under transcriptional
control" means that the promoter is in the correct location
and orientation in relation to the nucleic acid to control RNA polymerase
initiation and expression of the gene.
 The term promoter will be used here to refer to a group
of transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for transcriptional
activator or repressor proteins.
 At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this
is the TATA box. However, in some promoters lacking a TATA box,
such as the promoter for the mammalian terminal deoxynucleotidyl
transferase gene and the promoter for the SV40 late genes, a discrete
element overlying the start site itself helps to fix the place of
 Additional promoter elements regulate the frequency of transcriptional
initiation. Typically, these are located in the region 30-110 bp
upstream of the start site, although a number of promoters have
recently been shown to contain functional elements downstream of
the start site as well. The spacing between promoter elements frequently
is flexible, so that promoter function is preserved when elements
are inverted or moved relative to one another. In the tk promoter,
the spacing between promoter elements can be increased to 50 bp
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either co-operatively
or independently to activate transcription.
 The particular promoter employed to control the expression
of a nucleic acid sequence of interest is not believed to be important,
so long as it is capable of directing the expression of the nucleic
acid in the targeted cell. Thus, where a human cell is targeted,
it is preferable to position the nucleic acid coding region adjacent
and under the control of a promoter that is capable of being expressed
in a human cell. Generally speaking, such a promoter might include
either a human or viral promoter.
 In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus long terminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphate
dehydrogenase promoter can be used to obtain high-level expression
of the coding sequence of interest. The use of other viral or mammalian
cellular or bacterial phage promoters which are well-known in the
art to achieve expression of a coding sequence of interest is contemplated
as well, provided that the levels of expression are sufficient for
a given purpose.
 By employing a promoter with well-known properties, the
level and pattern of expression of the protein of interest following
transfection or transformation can be optimized. Further, selection
of a promoter that is regulated in response to specific physiologic
signals can permit inducible expression of the gene product. Tables
2 and 3 list several elements/promoters which may be employed, in
the context of the present invention, to regulate the expression
of the gene of interest. This list is not intended to be exhaustive
of all the possible elements involved in the promotion of gene expression
but, merely, to be exemplary thereof.
 Enhancers are genetic elements that increase transcription
from a promoter located at a distant position on the same molecule
of DNA. Enhancers are organized much like promoters. That is, they
are composed of many individual elements, each of which binds to
one or more transcriptional proteins.
 The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to stimulate
transcription at a distance; this need not be true of a promoter
region or its component elements. On the other hand, a promoter
must have one or more elements that direct initiation of RNA synthesis
at a particular site and in a particular orientation, whereas enhancers
lack these specificities. Promoters and enhancers are often overlapping
and contiguous, often seeming to have a very similar modular organization.
 Below is a list of viral promoters, cellular promoters/enhancers,
and inducible promoters/enhancers that could be used in combination
with the nucleic acid encoding a gene of interest in an expression
construct (Table 2 and Table 3). Additionally, any promoter/enhancer
combination (as per the Eukaryotic Promoter Data Base EPDB) also
could be used to drive expression of the gene. Eukaryotic cells
can support cytoplasmic transcription from certain bacterial promoters
if the appropriate bacterial polymerase is provided, either as part
of the delivery complex or as an additional genetic expression construct.
2 TABLE 2 ENHANCER/PROMOTER Immunoglobulin Heavy Chain Immunoglobulin
Light Chain T-Cell Receptor HLA DQ .alpha. and DQ .beta. .beta.-Interferon
Interleukin-2 Interleukin-2 Receptor MHC Class II 5 MHC Class II
HLA-DR.alpha. .beta.-Actin Prealbumin (Transthyretin) Muscle Creatine
Kinase Elastase I Metallothionein Collagenase Albumin Gene .alpha.-Fetoprotein
.tau.-Globin .beta.-Globin e-fos c-HA-ras Insulin Neural Cell Adhesion
Molecule (NCAM) .alpha.1-Antitrypsin H2B (TH2B) Histone Mouse or
Type I Collagen Glucose-Regulated Proteins (GRP94 and GRP78) Rat
Growth Hormone Human Serum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived
Growth Factor Duchenne Muscular Dystrophy SV40 Polyoma Retroviruses
Papilloma Virus Hepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus
3TABLE 3 Element Inducer MT II Phorbol Ester (TPA) Heavy metals
MMTV (mouse mammary tumor Glucocorticoids virus) .beta.-Interferon
poly(rl)X, poly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA),
H.sub.2O.sub.2 Collagenase Phorbol Ester (TPA) Stromelysin Phorbol
Ester (TPA), IL-1 SV40 Phorbol Ester (TPA) Murine MX Gene Interferon,
Newcastle Disease Virus GRP78 Gene A23187 .alpha.-2-Macroglobulin
IL-6 Vimentin Serum MHC Class I Gene H-2kB Interferon HSP70 Ela,
SV40 Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis
Factor FMA Thyroid Stimulating Hormone .alpha. Thyroid Hormone Gene
Insulin E Box Glucose
 Where a cDNA insert is employed, typically one will typically
include a polyadenylation signal to effect proper polyadenylation
of the gene transcript. The nature of the polyadenylation signal
is not believed to be crucial to the successful practice of the
invention, and any such sequence may be employed, such as human
growth hormone and SV40 polyadenylation signals. Also contemplated
as an element of the expression construct is a terminator. These
elements can serve to enhance message levels and to minimize read
through from the construct into other sequences.
 Selectable Markers
 In certain embodiments of the invention, the cells containing
nucleic acid constructs of the present invention may be identified
in vitro or in vivo by including a marker in the expression construct.
Such markers would confer an identifiable change to the cell permitting
easy identification of cells containing the expression construct.
Usually the inclusion of a drug selection marker aids in cloning
and in the selection of transformants. For example, genes that confer
resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin,
and histidinol are useful selectable markers. Alternatively, enzymes
such as herpes simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be employed. Immunologic markers also
can be employed. The selectable marker employed is not believed
to be important, so long as it is capable of being expressed simultaneously
with the nucleic acid encoding a gene product. Further examples
of selectable markers are well known to one of skill in the art.
 Delivery of Expression Vectors
 There are a number of ways in which expression vectors may
introduced into cells. In certain embodiments of the invention,
the expression construct comprises a virus or engineered construct
derived from a viral genome. The ability of certain viruses to enter
cells via receptor-mediated endocytosis, to integrate into a host
cell genome, and express viral genes stably and efficiently have
made them attractive candidates for the transfer of foreign genes
into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988;
Baichwal and Sugden, 1986; Temin, 1986). Preferred gene therapy
vectors are generally viral vectors.
 Although some viruses that can accept foreign genetic material
are limited in the number of nucleotides they can accommodate and
in the range of cells they infect, these viruses have been demonstrated
to successfully effect gene expression. However, adenoviruses do
not integrate their genetic material into the host genome and therefore
do not require host replication for gene expression making them
ideally suited for rapid, efficient, heterologous gene expression.
Techniques for preparing replication infective viruses are well
known in the art.
 Of course in using viral delivery systems, one will desire
to purify the virion sufficiently to render it essentially free
of undesirable contaminants, such as defective interfering viral
particles or endotoxins and other pyrogens such that it will not
cause any untoward reactions in the cell, animal or individual receiving
the vector construct. A preferred means of purifying the vector
involves the use of buoyant density gradients, such as cesium chloride
 Viruses used as gene vectors such as DNA viruses may include
the papovaviruses (e.g., simian virus 40, bovine papilloma virus,
and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses
(Ridgeway, 1988; Baichwal and Sugden, 1986).
 One of the preferred methods for in vivo delivery involves
the use of an adenovirus expression vector. Although adenovirus
vectors are known to have a low capacity for integration into genomic
DNA, this feature is counterbalanced by the high efficiency of gene
transfer afforded by these vectors. "Adenovirus expression
vector" is meant to include those constructs containing adenovirus
sequences sufficient to (a) support packaging of the construct and
(b) to express an antisense polynucleotide that has been cloned
 The expression vector comprises a genetically engineered
form of adenovirus. Knowledge of the genetic organization of adenovirus,
a 36 kb, linear, double-stranded DNA virus, allows substitution
of large pieces of adenoviral DNA with foreign sequences up to 7
kb (Grunhaus and Horwitz, 1992). In contrast to retroviral infection,
the adenoviral infection of host cells does not result in chromosomal
integration because adenoviral DNA can replicate in an episomal
manner without potential genotoxicity. Also, adenoviruses are structurally
stable, and no genome rearrangement has been detected after extensive
amplification. Adenovirus can infect virtually all epithelial cells
regardless of their cell cycle stage. So far, adenoviral infection
appears to be linked only to mild disease such as acute respiratory
disease in humans.
 Adenovirus is particularly suitable for use as a gene transfer
vector because of its mid-sized genome, ease of manipulation, high
titer, wide target cell range and high infectivity. Both ends of
the viral genome contain 100-200 base pair inverted repeats (ITRs),
which are cis elements necessary for viral DNA replication and packaging.
The early (E) and late (L) regions of the genome contain different
transcription units that are divided by the onset of viral DNA replication.
The E1 region (E1A and E1B) encodes proteins responsible for the
regulation of transcription of the viral genome and a few cellular
genes. The expression of the E2 region (E2A and E2B) results in
the synthesis of the proteins for viral DNA replication. These proteins
are involved in DNA replication, late gene expression and host cell
shut-off (Renan, 1990). The products of the late genes, including
the majority of the viral capsid proteins, are expressed only after
significant processing of a single primary transcript issued by
the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is
particularly efficient during the late phase of infection, and all
the mRNAs issued from this promoter possess a 5'-tripartite leader
(TPL) sequence which makes them preferred mRNAs for translation.
 In currently used systems, recombinant adenovirus is generated
from homologous recombination between shuttle vector and provirus
vector. Due to the possible recombination between two proviral vectors,
wild-type adenovirus may be generated from this process. Therefore,
it is critical to isolate a single clone of virus from an individual
plaque and examine its genomic structure.
 Generation and propagation of adenovirus vectors which are
replication deficient depend on a unique helper cell line, designated
293, which is transformed from human embryonic kidney cells by Ad5
DNA fragments and constitutively expresses E1 proteins (Graham et
al., 1977). Since the E3 region is dispensable from the adenovirus
genome (Jones and Shenk, 1978), the current adenovirus vectors,
with the help of 293 cells, carry foreign DNA in either the E1,
the E3, or both regions (Graham and Prevec, 1991). In nature, adenovirus
can package approximately 105% of the wild-type genome (Ghosh-Choudhury
et al., 1987), providing capacity for about 2 extra kb of DNA. Combined
with the approximately 5.5 kb of DNA that is replaceable in the
E1 and E3 regions, the maximum capacity of the current adenovirus
vector is under 7.5 kb, or about 15% of the total length of the
vector. More than 80% of the adenovirus viral genome remains in
the vector backbone and is the source of vector-borne cytotoxicity.
Also, the replication deficiency of the E1-deleted virus is incomplete.
For example, leakage of viral gene expression has been observed
with the currently available vectors at high multiplicities of infection
(MOI) (Mulligan, 1993).
 Helper cell lines may be derived from human cells such as
human embryonic kidney cells, muscle cells, hematopoietic cells
or other human embryonic mesenchymal or epithelial cells. Alternatively,
the helper cells, may be derived from the cells of other mammalian
species that are permissive for human adenovirus. Such cells include,
e.g., Vero cells or other monkey embryonic mesenchymal or epithelial
cells. As discussed, the preferred helper cell line is 293.
 Recently, Racher et al. (1995) disclosed improved methods
for culturing 293 cells and propagating adenovirus. In one format,
natural cell aggregates are grown by inoculating individual cells
into 1 liter siliconized spinner flasks (Techne, Cambridge, UK)
containing 100-200 ml of medium. Following stirring at 40 rpm, the
cell viability is estimated with trypan blue. In another format,
Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) are employed
as follows. A cell innoculum, resuspended in 5 ml of medium, is
added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left
stationary, with occasional agitation, for 1 to 4 h. The medium
is then replaced with 50 ml of fresh medium and shaking is initiated.
For virus production, cells are allowed to grow to about 80% confluence,
after which time the medium is replaced (to 25% of the final volume)
and adenovirus added at an MOI of 0.05. Cultures are left stationary
overnight, following which the volume is increased to 100% and shaking
is commenced for another 72 h.
 Other than the requirement that the adenovirus vector be
replication defective, or at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovirus may be
of any of the 42 different known serotypes or subgroups A-F. Adenovirus
type 5 of subgroup C is the preferred starting material in order
to obtain the conditional replication-defective adenovirus vector
for use in the present invention. This is because Adenovirus type
5 is a human adenovirus about which a great deal of biochemical
and genetic information is known, and it has historically been used
for most constructions employing adenovirus as a vector.
 A typical vector applicable to practicing the present invention
is replication defective and will not have an adenovirus E1 region.
Thus, it will be most convenient to introduce the polynucleotide
encoding the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR gene at the position from which the E1-coding sequences
have been removed. However, the position of insertion of the construct
within the adenovirus sequences is not critical. The polynucleotide
encoding the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR gene may also be inserted in lieu of the deleted E3 region
in E3 replacement vectors as described by Karlsson et al., (1986)
or in the E4 region where a helper cell line or helper virus complements
the E4 defect.
 Adenovirus is easy to grow and manipulate and exhibits broad
host range in vitro and in vivo. This group of viruses can be obtained
in high titers, e.g., 10.sup.9-10.sup.11 plaque-forming units per
ml, and they are highly infective. The life cycle of adenovirus
does not require integration into the host cell genome. The foreign
genes delivered by adenovirus vectors are episomal and, therefore,
have low genotoxicity to host cells. No side effects have been reported
in studies of vaccination with wild-type adenovirus (Couch et al.,
1963; Top et al., 1971), demonstrating their safety and therapeutic
potential as in vivo gene transfer vectors.
 Adenovirus vectors have been used in eukaryotic gene expression
(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development
(Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently,
animal studies suggested that recombinant adenovirus could be used
for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet
et al., 1990; Rich et al., 1993). Studies in administering recombinant
adenovirus to different tissues include trachea instillation (Rosenfeld
et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et
al., 1993), peripheral intravenous injections (Herz and Gerard,
1993) and stereotactic inoculation into the brain (Le Gal La Salle
et al., 1993).
 Other gene transfer vectors may be constructed from retroviruses.
The retroviruses are a group of single-stranded RNA viruses characterized
by an ability to convert their RNA to double-stranded DNA in infected
cells by a process of reverse-transcription (Coffin, 1990). The
resulting DNA then stably integrates into cellular chromosomes as
a provirus and directs synthesis of viral proteins. The integration
results in the retention of the viral gene sequences in the recipient
cell and its descendants. The retroviral genome contains three genes,
gag, pol, and env. that code for capsid proteins, polymerase enzyme,
and envelope components, respectively. A sequence found upstream
from the gag gene contains a signal for packaging of the genome
into virions. Two long terminal repeat (LTR) sequences are present
at the 5' and 3' ends of the viral genome. These contain strong
promoter and enhancer sequences, and also are required for integration
in the host cell genome (Coffin, 1990).
 In order to construct a retroviral vector, a nucleic acid
encoding a TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR gene is inserted into the viral genome in the place of
certain viral sequences to produce a virus that is replication-defective.
In order to produce virions, a packaging cell line containing the
gag, pol, and env genes, but without the LTR and packaging components,
is constructed (Mann et al., 1983). When a recombinant plasmid containing
a cDNA, together with the retroviral LTR and packaging sequences
is introduced into this cell line (by calcium phosphate precipitation
for example), the packaging sequence allows the RNA transcript of
the recombinant plasmid to be packaged into viral particles, which
are then secreted into the culture media (Nicolas and Rubenstein,
1988; Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally concentrated,
and used for gene transfer. Retroviral vectors are capable of infecting
a broad variety of cell types. However, integration and stable expression
require the division of host cells (Paskind et al., 1975).
 A novel approach designed to allow specific targeting of
retrovirus vectors was recently developed based on the chemical
modification of a retrovirus by the chemical addition of lactose
residues to the viral envelope. This modification could permit the
specific infection of hepatocytes via sialoglycoprotein receptors.
 A different approach to targeting of recombinant retroviruses
has been designed in which biotinylated antibodies against a retroviral
envelope protein and against a specific cell receptor were used.
The antibodies were coupled via the biotin components by using streptavidin
(Roux et al., 1989). Using antibodies against major histocompatibility
complex class I and class II antigens, the infection of a variety
of human cells that bear those surface antigens with an ecotropic
virus in vitro was demonstrated (Roux et al., 1989).
 There are certain limitations to the use of retrovirus vectors.
For example, retrovirus vectors usually integrate into random sites
in the cell genome. This can lead to insertional mutagenesis through
the interruption of host genes or through the insertion of viral
regulatory sequences that can interfere with the function of flanking
genes (Varmus et al., 1981). Another concern with the use of defective
retrovirus vectors is the potential appearance of wild-type replication-competent
virus in the packaging cells. This may result from recombination
events in which the intact sequence from the recombinant virus inserts
upstream from the gag, pot, env sequence integrated in the host
cell genome. However, new packaging cell lines are now available
that should greatly decrease the likelihood of recombination (Markowitz
et al., 1988; Hersdorffer et al., 1990).
 Other viral vectors may be employed as expression constructs.
Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988;
Baichwal and Sugden, 1986; Coupar et al., 1988), adeno-associated
virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat
and Muzycska, 1984), and herpes viruses may be employed. They offer
several attractive features for various mammalian cells (Friedmann,
1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al.,
1988; Horwich et al., 1990).
 With the recent recognition of defective hepatitis B viruses,
new insight has been gained into the structure-function relationship
of different viral sequences. In vitro studies showed that the virus
could retain the ability for helper-dependent packaging and reverse
transcription despite the deletion of up to 80% of its genome (Horwich
et al., 1990). This suggests that large portions of the genome can
be replaced with foreign genetic material. The hepatotropism and
persistence (integration) are particularly attractive properties
for liver-directed gene transfer. Chang et al. (1991) recently introduced
the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis
B virus genome in the place of the polymerase, surface, and pre-surface
coding sequences. It was co-transfected with wild-type virus into
an avian hepatoma cell line. Culture media containing high titers
of the recombinant virus were used to infect primary duckling hepatocytes.
Stable CAT gene expression was detected for at least 24 days after
transfection (Chang et al., 1991).
 To effect expression of sense or antisense gene constructs,
the expression construct must be delivered into a cell. This delivery
may be accomplished in vitro, as in laboratory procedures for transforming
cells lines, or in vivo or ex vivo, as in the treatment of certain
disease states. One mechanism for delivery is via viral infection
where the expression construct is encapsidated in an infectious
 Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells also are contemplated by
the present invention. These include calcium phosphate precipitation
(Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al.,
1990), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et
al., 1986; Potter et al., 1984), direct microinjection (Harland
and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979) and lipofectamine-DNA complexes, cell sonication
(Fechheimer et al., 1987), gene bombardment using high velocity
microprojectiles (Yang et al., 1990), and receptor-mediated transfection
(Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may
be successfully adapted for in vivo or ex vivo use.
 Once the expression construct has been delivered into the
cell the nucleic acid encoding the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR gene may be positioned and expressed at different
sites. In certain embodiments, the nucleic acid encoding the gene
may be stably integrated into the genome of the cell. This integration
may be in the cognate location and orientation via homologous recombination
(gene replacement) or it may be integrated in a random, non-specific
location (gene augmentation). In yet further embodiments, the nucleic
acid may be stably maintained in the cell as a separate, episomal
segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication
independent of or in synchronization with the host cell cycle. How
the expression construct is delivered to a cell and where in the
cell the nucleic acid remains is dependent on the type of expression
 In yet another embodiment of the invention, the expression
construct may simply consist of naked recombinant DNA or plasmids.
Transfer of the construct may be performed by any of the methods
mentioned above which physically or chemically permeabilize the
cell membrane. This is particularly applicable for transfer in vitro
but it may be applied to in vivo use as well. Dubensky et al. (1984)
successfully injected polyomavirus DNA in the form of calcium phosphate
precipitates into liver and spleen of adult and newborn mice demonstrating
active viral replication and acute infection. Benvenisty and Neshif
(1986) also demonstrated that direct intraperitoneal injection of
calcium phosphate-precipitated plasmids results in expression of
the transfected genes. It is envisioned that DNA encoding a TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR gene may
also be transferred in a similar manner in vivo and express the
 In still another embodiment of the invention for transferring
a naked DNA expression construct into cells may involve particle
bombardment. This method depends on the ability to accelerate DNA-coated
microprojectiles to a high velocity allowing them to pierce cell
membranes and enter cells without killing them (Klein et al., 1987).
Several devices for accelerating small particles have been developed.
One such device relies on a high voltage discharge to generate an
electrical current, which in turn provides the motive force (Yang
et al., 1990). The microprojectiles used have consisted of biologically
inert substances such as tungsten or gold beads.
 In a further embodiment of the invention, the expression
construct may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and
an inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation
of closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated
are lipofectamine-DNA complexes.
 Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful. Wong et al., (1980)
demonstrated the feasibility of liposome-mediated delivery and expression
of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
Nicolau et al., (1987) accomplished successful liposome-mediated
gene transfer in rats after intravenous injection.
 In certain embodiments of the invention, the liposome may
be complexed with a hemagglutinating virus (HVJ). This has been
shown to facilitate fusion with the cell membrane and promote cell
entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in conjunction
with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al.,
1991). In yet further embodiments, the liposome may be complexed
or employed in conjunction with both HVJ and HMG-1. In that such
expression constructs have been successfully employed in transfer
and expression of nucleic acid in vitro and in vivo, then they are
applicable for the present invention. Where a bacterial promoter
is employed in the DNA construct, it also will be desirable to include
within the liposome an appropriate bacterial polymerase.
 Other expression constructs which can be employed to deliver
a nucleic acid encoding a TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR gene into cells are receptor-mediated delivery
vehicles. These take advantage of the selective uptake of macromolecules
by receptor-mediated endocytosis in almost all eukaryotic cells.
Because of the cell type-specific distribution of various receptors,
the delivery can be highly specific (Wu and Wu, 1993).
 Receptor-mediated gene targeting vehicles generally consist
of two components: a cell receptor-specific ligand and a DNA-binding
agent. Several ligands have been used for receptor-mediated gene
transfer. The most extensively characterized ligands are asialoorosomucoid
(ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1990).
Recently, a synthetic neoglycoprotein, which recognizes the same
receptor as ASOR, has been used as a gene delivery vehicle (Ferkol
et al., 1993; Perales et al., 1994) and epidermal growth factor
(EGF) has also been used to deliver genes to squamous carcinoma
cells (Myers, EPO 0273085).
 In other embodiments, the delivery vehicle may comprise
a ligand and a liposome. For example, Nicolau et al., (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated
into liposomes and observed an increase in the uptake of the insulin
gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding
a TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR
gene also may be specifically delivered into a cell type such as
lung, epithelial, or tumor cells, by any number of receptor-ligand
systems with or without liposomes. For example, epidermal growth
factor (EGF) may be used as the receptor for mediated delivery of
a nucleic acid encoding a gene in many tumor cells that exhibit
upregulation of EGF receptor. Mannose can be used to target the
mannose receptor on liver cells. Also, antibodies to CD5 (CLL),
CD22 (lymphoma), CD25 (T-cell leukemia), and MAA (melanoma) can
be used similarly as targeting moieties.
 In certain embodiments, gene transfer may more easily be
performed under ex vivo conditions. Ex vivo gene therapy refers
to the isolation of cells from an animal, the delivery of a nucleic
acid into the cells in vitro, and then the return of the modified
cells back into an animal. This may involve the surgical removal
of tissue/organs from an animal or the primary culture of cells
 Primary mammalian cell cultures may be prepared in various
ways. In order for the cells to be kept viable while in vitro and
in contact with the expression construct, it is necessary to ensure
that the cells maintain contact with the correct ratio of oxygen
and carbon dioxide and nutrients but are protected from microbial
contamination. Cell culture techniques are well documented and are
disclosed herein by reference (Freshner, 1992).
 Examples of useful mammalian host cell lines are Vero and
HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7,
293, HepG2, NIH3T3, RIN, and MDCK cells. In addition, a host cell
strain may be chosen that modulates the expression of the inserted
sequences, or modifies and processes the gene product in the manner
desired. Such modifications (e.g., glycosylation) and processing
(e.g., cleavage) of protein products may be important for the function
of the protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cell lines or host systems can be chosen
to insure the correct modification and processing of the foreign
 A number of selection systems may be used including, but
not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase
and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-
cells, respectively. Also, anti-metabolite resistance can be used
as the basis of selection for dhfr: that confers resistance to methotrexate;
gpt, that confers resistance to mycophenolic acid; neo, that confers
resistance to the aminoglycoside G418; and hygro, that confers resistance
 Animal cells can be propagated in vitro in two modes: as
non-anchorage dependent cells growing in suspension throughout the
bulk of the culture or as anchorage-dependent cells requiring attachment
to a solid substrate for their propagation (i.e., a monolayer type
of cell growth).
 Non-anchorage dependent or suspension cultures from continuous
established cell lines are the most widely used means of large scale
production of cells and cell products. However, suspension cultured
cells have limitations, such as tumorigenic potential and lower
protein production than adherent T-cells.
 Large scale suspension culture of mammalian cells in stirred
tanks is a common method for production of recombinant proteins.
Two suspension culture reactor designs are in wide use--the stirred
reactor and the airlift reactor. The stirred design has been used
successfully on an 8000 liter capacity for the production of interferon.
Cells are grown in a stainless steel tank with a height-to-diameter
ratio of 1:1 to 3:1. The culture usually is mixed with one or more
agitators, based on bladed disks or marine propeller patterns. Agitator
systems offering less shear forces than blades have been described.
Agitation may be driven either directly or indirectly by magnetically
coupled drives. Indirect drives reduce the risk of microbial contamination
through seals on stirrer shafts.
 The airlift reactor, also initially described for microbial
fermentation and later adapted for mammalian culture, relies on
a gas stream to both mix and oxygenate the culture. The gas stream
enters a riser section of the reactor and drives circulation. Gas
disengages at the culture surface, causing denser liquid which is
free of gas bubbles to travel downward in the downcomer section
of the reactor. The main advantage of this design is the simplicity
and lack of need for mechanical mixing. Typically, the height-to-diameter
ratio is 10:1. The airlift reactor scales up relatively easily,
has good mass transfer of gases and generates relatively low shear
 Generating Antibodies Reactive With TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR
 In another aspect, the present invention contemplates an
antibody that is immunoreactive with a TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR molecule of the present invention,
or any portion thereof. An antibody can be a polyclonal or a monoclonal
antibody. In a preferred embodiment, an antibody is a monoclonal
antibody. Means for preparing and characterizing antibodies are
well known in the art (see, e.g., Harlow and Lane, 1988).
 Briefly, a polyclonal antibody is prepared by immunizing
an animal with an immunogen comprising a polypeptide of the present
invention and collecting antisera from that immunized animal. A
wide range of animal species can be used for the production of antisera.
Typically an animal used for production of anti-antisera is a non-human
animal, for example, rabbits, mice, rats, hamsters, pigs or horses.
Because of the relatively large blood volume of rabbits, a rabbit
is a preferred choice for production of polyclonal antibodies.
 Antibodies, both polyclonal and monoclonal, specific for
isoforms of antigen may be prepared using conventional immunization
techniques, as will be generally known to those of skill in the
art. A composition containing antigenic epitopes of the compounds
of the present invention can be used to immunize one or more experimental
animals, such as a rabbit or mouse, which will then proceed to produce
specific antibodies against the compounds of the present invention.
Polyclonal antisera may be obtained, after allowing time for antibody
generation, simply by bleeding the animal and preparing serum samples
from the whole blood.
 It is proposed that the antibodies of the present invention
will find useful application in standard immunochemical procedures,
such as ELISA and Western blot methods and in immunohistochemical
procedures such as tissue staining, as well as in other procedures
which may utilize antibodies specific to TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR-related antigen epitopes.
 The antibodies of the present invention are also useful
for the isolation of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR polypeptides by immunoprecipitation. Immunoprecipitation
involves the separation of the target antigen component from a complex
mixture, and is used to discriminate or isolate minute amounts of
protein. For the isolation of membrane proteins cells must be solubilized
into detergent nicelles. Nonionic salts are preferred, since other
agents such as bile salts, precipitate at acid pH or in the presence
of bivalent cations. Antibodies are and their uses are discussed
 In general, both polyclonal and monoclonal antibodies against
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR may
be used in a variety of embodiments. For example, they may be employed
in antibody cloning protocols to obtain cDNAs or genes encoding
other isoforms of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR or related proteins. They also may be used in inhibition
studies to analyze the effects of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR-related peptides in cells or animals. A particularly
useful application of such antibodies is in purifying native or
recombinant TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR, for example, using an antibody affinity column. The operation
of all such immunological techniques will be known to those of skill
in the art in light of the present disclosure.
 Means for preparing and characterizing antibodies are well
known in the art (see, e.g., Harlow and Lane, 1988; incorporated
herein by reference). More specific examples of monoclonal antibody
preparation are give in the examples below.
 As is well known in the art, a given composition may vary
in its immunogenicity. It is often necessary, therefore, to boost
the host immune system, as may be achieved by coupling a peptide
or polypeptide immunogen to a carrier. Exemplary and preferred carriers
are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
Other albumins such as ovalbumin, mouse serum albumin or rabbit
serum albumin also can be used as carriers. Means for conjugating
a polypeptide to a carrier protein are well known in the art and
include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide
ester, carbodiimide and bis-biazotized benzidine.
 As also is well known in the art, the immunogenicity of
a particular immunogen composition can be enhanced by the use of
non-specific stimulators of the immune response, known as adjuvants.
Exemplary and preferred adjuvants include complete Freund's adjuvant
(a non-specific stimulator of the immune response containing killed
Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum
 The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen
as well as the animal used for immunization. A variety of routes
can be used to administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal). The production of
polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points following immunization. A second,
booster, injection also may be given. The process of boosting and
titering is repeated until a suitable titer is achieved. When a
desired level of immunogenicity is obtained, the immunized animal
can be bled and the serum isolated and stored, and/or the animal
can be used to generate monoclonal antibodies.
 Monoclonal antibodies may be readily prepared through use
of well-known techniques, such as those exemplified in U.S. Pat.
No. 4,196,265, incorporated herein by reference. Typically, this
technique involves immunizing a suitable animal with a selected
immunogen composition, e.g., a purified or partially purified TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR protein,
polypeptide, or peptide or a cell expressing high levels of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR. The immunizing
composition is administered in a manner effective to stimulate antibody
producing cells. Cells from rodents such as mice and rats are preferred,
however, the use of rabbit, sheep or frog cells is also possible.
The use of rats may provide certain advantages (Goding, 1986), but
mice are preferred, with the BALB/c mouse being most preferred as
this is most routinely used and generally gives a higher percentage
of stable fusions.
 Following immunization, somatic cells with the potential
for producing antibodies, specifically B-lymphocytes (B-cells),
are selected for use in the mAb generating protocol. These cells
may be obtained from biopsied spleens, tonsils or lymph nodes, or
from a peripheral blood sample. Spleen cells and peripheral blood
cells are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast stage,
and the latter because peripheral blood is easily accessible. Often,
a panel of animals will have been immunized and the spleen of the
animal with the highest antibody titer will be removed and the spleen
lymphocytes obtained by homogenizing the spleen with a syringe.
Typically, a spleen from an immunized mouse contains approximately
5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes.
 The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell, generally
one of the same species as the animal that was immunized. Myeloma
cell lines suited for use in hybridoma-producing fusion procedures
preferably are non-antibody-producing, have high fusion efficiency,
and enzyme deficiencies that render then incapable of growing in
certain selective media which support the growth of only the desired
fused cells (hybridomas).
 Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, 1986; Campbell, 1984).
For example, where the immunized animal is a mouse, one may use
P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U,
MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; for rats, one may use
R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,
LICR-LON-HMy2 and UC729-6 are all useful in connection with cell
 Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing somatic
cells with myeloma cells in a 2:1 ratio, though the ratio may vary
from about 20:1 to about 1:1, respectively, in the presence of an
agent or agents (chemical or electrical) that promote the fusion
of cell membranes. Fusion methods using Sendai virus (Kohler and
Milstein, 1975; 1976), and those using polyethylene glycol (PEG),
such as 37% (v/v) PEG, have been described by Gefter et al., (1977).
The use of electrically induced fusion methods is also appropriate
 Fusion procedures usually produce viable hybrids at low
frequencies, around 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are differentiated
from the parental, unfused cells (particularly the unfused myeloma
cells that would normally continue to divide indefinitely) by culturing
in a selective medium. The selective medium is generally one that
contains an agent that blocks the de novo synthesis of nucleotides
in the tissue culture media. Exemplary and preferred agents are
aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate
block de novo synthesis of both purines and pyrimidines, whereas
azaserine blocks only purine synthesis. Where aminopterin or methotrexate
is used, the media is supplemented with hypoxanthine and thymidine
as a source of nucleotides (HAT medium). Where azaserine is used,
the media is supplemented with hypoxanthine.
 The preferred selection medium is HAT. Only cells capable
of operating nucleotide salvage pathways are able to survive in
HAT medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT),
and they cannot survive. The B-cells can operate this pathway, but
they have a limited life span in culture and generally die within
about two wk. Therefore, the only cells that can survive in the
selective media are those hybrids formed from myeloma and B-cells.
 This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone dilution
in microtiter plates, followed by testing the individual clonal
supernatants (after about two to three wk) for the desired reactivity.
The assay should be sensitive, simple and rapid, such as radioimmunoassays,
enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding
assays, and the like.
 The selected hybridomas would then be serially diluted and
cloned into individual antibody-producing cell lines, which clones
can then be propagated indefinitely to provide mAbs. The cell lines
may be exploited for mAb production in two basic ways. A sample
of the hybridoma can be injected (often into the peritoneal cavity)
into a histocompatible animal of the type that was used to provide
the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific monoclonal antibody
produced by the fused cell hybrid. The body fluids of the animal,
such as serum or ascites fluid, can then be tapped to provide mnAbs
in high concentration. The individual cell lines also could be cultured
in vitro, where the mAbs are naturally secreted into the culture
medium from which they can be readily obtained in high concentrations.
mAbs produced by either means may be further purified, if desired,
using filtration, centrifugation, and various chromatographic methods
such as HPLC or affinity chromatography.
 Diagnosing Cancers Involving TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR
 The present inventors have determined that alterations in
expression of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR are associated with tamoxifen-resistant breast cancer and
may be associated with other malignancies. Therefore, TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR mRNAs and the corresponding
genes may be employed as a diagnostic or predictive indicator of
cancer, particularly tamoxifen-resistant breast cancer.
 Genetic Diagnosis
 One embodiment of the instant invention comprises a method
for detecting variation in the expression of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR. This may comprise determining
the level of expression of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR or determining specific alterations in the
expressed product in a biological sample. In particular, the present
invention relates to the diagnosis or prediction of tamoxifen-resistant
 The nucleic acid used in the disclosed methods is isolated
from cells contained in a biological sample, according to standard
methodologies (Sambrook et al., 1989). The nucleic acid may be genomic
DNA or fractionated or whole cell RNA. Where RNA is used, it may
be desirable to convert the RNA to a complementary DNA. In one embodiment,
the RNA is whole cell RNA; in another embodiment, it is poly-A RNA.
Normally, the nucleic acid is amplified.
 Depending on the format, the specific nucleic acid of interest
is identified directly in the sample using amplification or by hybridization
with a second, known nucleic acid following amplification. Next,
the identified product is detected. In certain applications, the
detection may be performed by visual means (e.g., ethidium bromide
staining of a gel). Alternatively, the detection may involve indirect
identification of the product via chemiluminescence, radioactive
scintigraphy of radiolabel or fluorescent label, or even via a system
using electrical or thermal impulse signals (Affymax Technology;
 Following detection, one compares the results obtained from
a patient with a sufficiently large reference group of normal patients,
patients with tamoxifen-sensitive breast cancer and patients with
tamoxifen-resistant breast cancer. In this way, it is possible to
correlate the amount of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR detected with various clinical states, such
as tamoxifen-resistance. In particular applications, such as breast
cancers, it is contemplated that different levels of progression
of breast cancer may be identified.
 Various types of defects are to be identified. Thus, "alterations"
should be read as including deletions, insertions, point mutations
and duplications. Point mutations result in stop codons, frameshift
mutations or amino acid substitutions. Somatic mutations are those
occurring in non-germline tissues. Germ-line mutations can occur
in reproductive tissue and are inherited. Mutations in and outside
the coding region also may affect the amount of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR 1, VEGF or bFGFR produced, both by altering
the transcription of the gene or in destabilizing or otherwise altering
the processing of either the transcript (mRNA) or protein.
 A variety of different assays are contemplated in this regard,
including but not limited to, fluorescent in situ hybridization
(FISH), direct DNA sequencing, PFGE analysis, Southern or Northern
blotting, single-stranded conformation polymorphism (SSCP), RNAse
protection assay, allele-specific oligonucleotide (ASO), dot blot
analysis, denaturing gradient gel electrophoresis, RFLP and PCR.TM.-SSCP.
 An alternative method for detection of mutations in the
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR sequences
involves the recently developed protein truncation assay (PTT) to
detect mutations affecting the length of the protein. This method
is based on RT-PCR.TM. using an upstream PCR.TM. primer containing
a RNA polymerase promoter and a eukaryotic translation initiation
signal. Approximately 200 ng of the PCR.TM. product is used directly
for the coupled in vitro transcription/translation reaction (coupled
TNT T7 reticulocyte system, Promega) which is substituted with .sup.35S
methionine. The amplified oligonucleotide products may be sequenced
by standard techniques known to those skilled in the art.
 Primers and Probes
 The term primer, as defined herein, is meant to encompass
any nucleic acid that is capable of priming the synthesis of a nascent
nucleic acid in a template-dependent process. Typically, primers
are oligonucleotides from ten to twenty base pairs in length, but
longer sequences can be employed. Primers may be provided in double-stranded
or single-stranded form, although the single-stranded form is preferred.
Probes are defined differently, although they may act as primers.
Probes, while perhaps capable of priming, are designed to bind to
the target DNA or RNA and need not be used in an amplification process.
 In preferred embodiments, the probes or primers are labeled
with radioactive species (.sup.32P, .sup.14C, .sup.35S, .sup.3H,
or other label), with a fluorophore (rhodamine, fluorescein), or
a chemilluminescent moiety (luciferase).
 Template Dependent Amplification Methods
 A number of template dependent processes are available to
amplify the marker sequences present in a given template sample.
One of the best known amplification methods is the polymerase chain
reaction (referred to as PCR.TM.) which is described in detail in
U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis
et al., 1990, each of which is incorporated herein by reference
in its entirety.
 Briefly, in PCR.TM., two primer sequences are prepared that
are complementary to regions on opposite complementary strands of
the marker sequence. An excess of deoxynucleoside triphosphates
are added to a reaction mixture along with a DNA polymerase, e.g.,
Taq polymerase. If the marker sequence is present in a sample, the
primers will bind to the marker and the polymerase will cause the
primers to be extended along the marker sequence by adding on nucleotides.
By raising and lowering the temperature of the reaction mixture,
the extended primers will dissociate from the marker to form reaction
products, excess primers will bind to the marker and to the reaction
products and the process is repeated.
 A reverse transcriptase PCR.TM. amplification procedure
may be performed in order to quantify the amount of mRNA amplified.
Methods of reverse transcribing RNA into cDNA are well known and
described in Sambrook et al., 1989. Alternative methods for reverse
transcription utilize thermostable, RNA-dependent DNA polymerases.
These methods are described in WO 90/07641 filed Dec. 21, 1990.
Polymerase chain reaction methodologies are well known in the art.
 Another method for amplification is the ligase chain reaction
("LCR"), disclosed in EPO No. 320 308, incorporated herein
by reference in its entirety. In LCR, two complementary probe pairs
are prepared, and in the presence of the target sequence, each pair
will bind to opposite complementary strands of the target such that
they abut. In the presence of a ligase, the two probe pairs will
link to form a single unit. By temperature cycling, as in PCR.TM.,
bound ligated units dissociate from the target and then serve as
"target sequences" for ligation of excess probe pairs.
U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding
probe pairs to a target sequence.
 Qbeta Replicase, described in PCT Application No. PCT/US87/00880,
may also be used as still another amplification method in the present
invention. In this method, a replicative sequence of RNA that has
a region complementary to that of a target is added to a sample
in the presence of an RNA polymerase. The polymerase will copy the
replicative sequence that can then be detected.
 An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification
of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates
in one strand of a restriction site also may be useful in the amplification
of nucleic acids in the present invention, Walker et al., (1992).
 Strand Displacement Amplification (SDA) is another method
of carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis, i.e.,
nick translation. A similar method, called Repair Chain Reaction
(RCR), involves annealing several probes throughout a region targeted
for amplification, followed by a repair reaction in which only two
of the four bases are present. The other two bases can be added
as biotinylated derivatives for easy detection. A similar approach
is used in SDA. Target specific sequences also can be detected using
a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5'
sequences of non-specific DNA and a middle sequence of specific
RNA is hybridized to DNA that is present in a sample. Upon hybridization,
the reaction is treated with RNase H, and the products of the probe
identified as distinctive products that are released after digestion.
The original template is annealed to another cycling probe and the
reaction is repeated.
 Still other amplification methods described in GB Application
No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of
which is incorporated herein by reference in its entirety, may be
used in accordance with the present invention. In the former application,
"modified" primers are used in a PCR.TM.-like, template-
and enzyme-dependent synthesis. The primers may be modified by labeling
with a capture moiety (e.g., biotin) and/or a detector moiety (e.g.,
enzyme). In the latter application, an excess of labeled probes
is added to a sample. In the presence of the target sequence, the
probe binds and is cleaved catalytically. After cleavage, the target
sequence is released intact to be bound by excess probe. Cleavage
of the labeled probe signals the presence of the target sequence.
 Other nucleic acid amplification procedures include transcription-based
amplification systems (TAS), including nucleic acid sequence based
amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al.,
PCT Application WO 88/10315, incorporated herein by reference in
their entirety). In NASBA, the nucleic acids can be prepared for
amplification by standard phenol/chloroform extraction, heat denaturation
of a clinical sample, treatment with lysis buffer, and minispin
columns for isolation of DNA and RNA or guanidinium chloride extraction
of RNA. These amplification techniques involve annealing a primer
which has target specific sequences. Following polymerization, DNA/RNA
hybrids are digested with RNase H while double stranded DNA molecules
are heat denatured again. In either case the single stranded DNA
is made fully double stranded by addition of second target specific
primer, followed by polymerization. The double-stranded DNA molecules
are then multiply transcribed by an RNA polymerase such as T7 or
SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed
into single stranded DNA, which is then converted to double stranded
DNA, and then transcribed once again with an RNA polymerase such
as T7 or SP6. The resulting products, whether truncated or complete,
indicate target specific sequences.
 Davey et al., EPO No. 329 822 (incorporated herein by reference
in its entirety) disclose a nucleic acid amplification process involving
cyclically synthesizing single-stranded RNA ("ssRNA"),
ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance
with the present invention. The ssRNA is a template for a first
primer oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from the
resulting DNA:RNA duplex by the action of ribonuclease H (RNase
H, an RNase specific for RNA in duplex with either DNA or RNA).
The resultant ssDNA is a template for a second primer, which also
includes the sequences of an RNA polymerase promoter (exemplified
by T7 RNA polymerase) 5' to its homology to the template. This primer
is then extended by DNA polymerase (exemplified by the large "Klenow"
fragment of E. coli DNA polymerase I), resulting in a double-stranded
DNA ("dsDNA") molecule having a sequence identical to
that of the original RNA between the primers, and having additionally,
at one end, a promoter sequence. This promoter sequence can be used
by the appropriate RNA polymerase to make many RNA copies of the
DNA. These copies can then re-enter the cycle leading to very swift
amplification. With proper choice of enzymes, this amplification
can be done isothermally without addition of enzymes at each cycle.
Because of the cyclical nature of this process, the starting sequence
can be chosen to be in the form of either DNA or RNA.
 Miller et al., PCT Application WO 89/06700 (incorporated
herein by reference in its entirety) disclose a nucleic acid sequence
amplification scheme based on the hybridization of a promoter/primer
sequence to a target single-stranded DNA ("ssDNA") followed
by transcription of many RNA copies of the sequence. This scheme
is not cyclic, i.e., new templates are not produced from the resultant
RNA transcripts. Other amplification methods include "RACE"
and "one-sided PCR.TM." (Frohman, 1990; Ohara et al.,
1989; each herein incorporated by reference in their entirety).
 Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the resulting
"di-oligonucleotide", thereby amplifying the di-oligonucleotide,
may also be used in the amplification step of the present invention
as described in Wu et al., (1989), incorporated herein by reference
in its entirety.
 Separation Methods
 It normally is desirable, at one stage or another, to separate
the amplification product from the template and the excess primer
for the purpose of determining whether specific amplification has
occurred. In one embodiment, amplification products are separated
by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis
using standard methods. (See Sambrook et al., 1989)
 Alternatively, chromatographic techniques may be employed
to effect separation. There are many kinds of chromatography which
may be used in the present invention: adsorption, partition, ion-exchange
and molecular sieve, and many specialized techniques for using them
including column, paper, thin-layer and gas chromatography (Freifelder,
 Detection Methods
 Products may be visualized in order to confirm amplification
of the marker sequences and to measure the relative amounts of amplification
products as a measure of gene expression levels. One typical visualization
method involves staining of a gel with ethidium bromide and visualization
under UV light. Alternatively, if the amplification products are
integrally labeled with radio- or fluorometrically-labeled nucleotides,
the amplification products can then be exposed to X-ray film or
visualized under the appropriate stimulating spectra, following
 In one embodiment, visualization is achieved indirectly.
Following separation of amplification products, a labeled nucleic
acid probe is brought into contact with the amplified marker sequence.
The probe preferably is conjugated to a chromophore but may be radiolabeled.
In another embodiment, the probe is conjugated to a binding partner,
such as an antibody or biotin, and the other member of the binding
pair carries a detectable moiety.
 In one embodiment, detection is by a labeled probe. The
techniques involved are well known to those of skill in the art
and can be found in many standard books on molecular protocols.
(See Sambrook et al., 1989) For example, chromophore or radiolabel
probes or primers identify the target during or following amplification.
 One example of the foregoing is described in U.S. Pat. No.
5,279,721, incorporated by reference herein, which discloses an
apparatus and method for the automated electrophoresis and transfer
of nucleic acids. The apparatus permits electrophoresis and blotting
without external manipulation of the gel and is ideally suited to
carrying out methods according to the present invention.
 In addition, the amplification products described above
may be subjected to sequence analysis to identify specific kinds
of variations using standard sequence analysis techniques. General
techniques for determination of the DNA sequence of amplification
products are well known in the art and include standard dideoxy
sequencing by the Sanger technique (See Sambrook et al., 1989).
Within certain methods, exhaustive analysis of genes is carried
out by sequence analysis using primer sets designed for optimal
sequencing (Pignon et al., 1994).
 The present invention may utilize any or all of these types
of analyses. Using the sequences disclosed herein, oligonucleotide
primers, may be designed to permit the amplification of sequences
throughout the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR genes that may then be analyzed by direct sequencing.
The amplified sequences may also be identified and quantitated,
using techniques well known in the art and further described herein.
The expression levels of the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR genes or mutants thereof may be used in the
methods disclosed herein to determine degree of malignancy, cell
tumorigenicity, and potential diagnosis and prediction of cancers
such as tamoxifen-resistant breast cancers.
 Southern/Northern Blotting
 Blotting techniques are well known to those of skill in
the art. Southern blotting involves the use of DNA as a target,
whereas Northern blotting involves the use of RNA as a target. Each
provide different types of information, although cDNA blotting is
analogous, in many aspects, to blotting RNA species.
 Briefly, a probe is used to target a DNA or RNA species
that has been immobilized on a suitable matrix, often a filter of
nitrocellulose. The different species should be spatially separated
to facilitate analysis. This often is accomplished by gel electrophoresis
of nucleic acid species followed by transfer of the separated nucleic
acids ("blotting") on to the filter.
 Subsequently, the blotted target is incubated with a probe
(usually labeled) under conditions that promote denaturation and
rehybridization. Because the probe is designed to base pair with
the target, the probe will bind a portion of the target sequence
under renaturing conditions. Unbound probe is then removed, and
the labeled probe detected and quantified using standard techniques
known to those skilled in the art.
 Kit Components
 All the essential materials and reagents required for detecting,
measuring, or sequencing TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR and variants thereof may be assembled together
in a kit. This generally will comprise preselected primers and probes.
Also included may be enzymes suitable for amplifying nucleic acids
including various polymerases (RT, Taq, Sequenase.TM. etc.), deoxynucleotides
and buffers to provide the necessary reaction mixture for amplification.
Such kits also generally will comprise, in suitable means, distinct
containers for each individual reagent and enzyme as well as for
each primer or probe.
 Chip Technologies
 Specifically contemplated by the present inventors are chip-based
DNA technologies such as those described by Hacia et al. (1996)
and Shoemaker et al. (1996). Briefly, these techniques involve quantitative
methods for analyzing large numbers of genes rapidly and accurately.
By tagging genes with oligonucleotides or using fixed probe arrays,
one can employ chip technology to segregate target molecules as
high density arrays and screen these molecules on the basis of hybridization.
See also Chen et al., 1998); Pease et al. (1994); Fodor et al. (1991).
 A preferred embodiment utilizes cDNA array technology, exemplified
by the CLONTECH Atlas.TM. human cDNA expression array (CLONTECH
Laboratories, Inc.). cDNA arrays offer the potential to simultaneously
quantify expression of many genes. Advances in cDNA array technology
to address array size, probe density, probe content and readout
make this technology suitable for application in the laboratory
(Marshall and Hodgson, 1998). However, the novelty of this technology
means that there are no well-established and widely accepted standards
to guide analysis and interpretation of the data. cDNA arrays have
most often been utilized in paired comparisons (e.g. control vs.
tumor) to identify differentially expressed genes in only a few
types of cancer, such as melanoma (DeRisi et al., 1996), Ewing's
sarcoma (Welford et al., 1998), alveolar rhabdomyosarcoma (Khan
et al., 1998) and gastrointestinal tumors (Zhang et al., 1997).
After standardization, rules for gene selection have typically been
based on ratios of expression, for example, greater than two-fold
difference (Schena et al., 1996), greater than three standard deviations
of control genes ratio (DeRisi et al., 1996), or an arbitrary percent.
 Due to expense, limited amounts of RNA and other considerations,
array experiments have previously involved few replications and
have orders of magnitude more variables (genes and ESTs) than observations.
The study illustrated in the EXAMPLES section of the present disclosure
shows the application of principal components analysis, coupled
with robust estimates of 99% prediction regions or higher order
components, as a practical approach to screening array data. The
method presumes that the vast majority of genes will be altered
very little and uses information from all genes to obtain more stable
estimates of variability. The method is not limited to pairwise
comparisons, but can be used to study several tumor types or experimental
conditions simultaneously. This approach is capable of reliably
identifying 60-85% of genes exhibiting moderate degrees of differential
expression (2-2.5 fold) without increasing the number of spuriously
 Antibodies of the present invention can be used in characterizing
the TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR
content of healthy and diseased tissues, through techniques such
as ELISA and Western blotting. This may provide a screen for the
presence or absence of malignancy or as a predictor of cancer progression
and patient survival.
 The use of antibodies of the present invention, in an ELISA
assay is contemplated. For example, anti-TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR antibodies are immobilized
onto a selected surface, preferably a surface exhibiting a protein
affinity such as the wells of a polystyrene microtiter plate. After
washing to remove incompletely adsorbed material, it is desirable
to bind or coat the assay plate wells with a non-specific protein
that is known to be antigenically neutral with regard to the test
antisera, such as bovine serum albumin (BSA), casein or solutions
of powdered milk. This allows for blocking of non-specific adsorption
sites on the immobilizing surface and thus reduces the background
caused by non-specific binding of antigen onto the surface.
 After binding of antibody to the well, coating with a non-reactive
material to reduce background, and washing to remove unbound material,
the immobilizing surface is contacted with the sample to be tested
in a manner conducive to immune complex (antigen/antibody) formation.
 Following formation of specific immunocomplexes between
the test sample and the bound antibody, and subsequent washing,
the occurrence and even amount of immunocomplex formation may be
determined by subjecting the same to a second antibody having specificity
for TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR
that differs from that of the first antibody. Appropriate conditions
preferably include diluting the sample with diluents such as BSA,
bovine gamma globulin (BGG), and phosphate buffered saline (PBS)/Tween.RTM..
These added agents also tend to assist in the reduction of nonspecific
background. The layered antisera is then allowed to incubate for
from about 2 to about 4 h, at temperatures preferably on the order
of about 25.degree. to about 27.degree. C. Following incubation,
the antisera-contacted surface is washed so as to remove non-immunocomplexed
material. A preferred washing procedure includes washing with a
solution such as PBS/Tween.RTM. or borate buffer.
 To provide a detecting means, the second antibody will preferably
have an associated enzyme that will generate a color development
upon incubating with an appropriate chromogenic substrate. Thus,
for example, one will desire to contact and incubate the second
antibody-bound surface with a urease or peroxidase-conjugated anti-IgG
for a period of time and under conditions which favor the development
of immunocomplex formation (e.g., incubation for 2 h at room temperature
in a PBS-containing solution such as PBS/Tween.RTM.).
 After incubation with the second enzyme-tagged antibody,
and subsequent to washing to remove unbound material, the amount
of label is quantified by incubation with a chromogenic substrate
such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic
acid (ABTS) and H.sub.2O.sub.2, in the case of peroxidase as the
enzyme label. Quantitation is then achieved by measuring the degree
of color generation, e.g., using a visible spectrum spectrophotometer.
 The preceding format may be altered by first binding the
sample to the assay plate. Then, primary antibody is incubated with
the assay plate, followed by detecting of bound primary antibody
using a labeled second antibody with specificity for the primary
 The antibody compositions of the present invention will
find great use in immunoblot or Western blot analysis. The antibodies
may be used as high-affinity primary reagents for the identification
of proteins immobilized onto a solid support matrix, such as nitrocellulose,
nylon or combinations thereof. In conjunction with immunoprecipitation,
followed by gel electrophoresis, these may be used as a single step
reagent for use in detecting antigens against which secondary reagents
used in the detection of the antigen cause an adverse background.
Immunologically-based detection methods for use in conjunction with
Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged
secondary antibodies against TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR proteins or the primary antibodies.
 Methods for Screening Active Compounds
 The present invention also contemplates the use of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR and active
fragments, and nucleic acids coding therefor, in the screening of
compounds for activity in blocking the effect of overexpression
of these genes. These assays may make use of a variety of different
formats and may depend on the kind of "activity" for which
the screen is being conducted. Contemplated functional "read-outs"
include binding to a compound, inhibition of binding to a substrate,
ligand, receptor or other binding partner by a compound, phosphatase
activity, anti-phosphatase activity, phosphorylation or dephosphorylation
of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR,
or inhibition or stimulation of angiogenesis, growth, metastasis,
cell division, apoptosis, tumor progression or other malignant phenotype.
Preferred embodiments include assay of cell replication by incorporation
of radiolabeled thymidine or colony formation.
 In Vitro Assays
 In one embodiment, the invention is to be applied for the
screening of compounds that bind to the TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR molecules or a fragment thereof.
The polypeptide or fragment may be either free in solution, fixed
to a support, or expressed in or on the surface of a cell. Either
the polypeptide or the compound may be labeled, thereby permitting
the determination of binding.
 In another embodiment, the assay may measure the inhibition
of binding of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR to a natural or artificial substrate or binding partner.
Competitive binding assays can be performed in which one of the
agents is labeled. Usually, the polypeptide will be the labeled
species. One may measure the amount of free label versus bound label
to determine binding or inhibition of binding.
 Another technique for high throughput screening of compounds
is described in WO 84/03564, the contents of which are incorporated
herein by reference. Large numbers of small peptide test compounds
are synthesized on a solid substrate, such as plastic pins or some
other surface. The peptide test compounds are reacted with TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR and washed.
Bound polypeptide is detected by various methods.
 Purified TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR can be coated directly onto plates for use in the
aforementioned drug screening techniques. However, non-neutralizing
antibodies to the polypeptide can be used to immobilize the polypeptide
to a solid phase. Also, fusion proteins containing a reactive region
(preferably a terminal region) may be used to link the TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR active region to
a solid phase.
 Various cell lines containing wild-type or natural or engineered
mutations in TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR can be used to study various functional attributes of these
proteins and how a candidate compound affects these attributes.
Methods for engineering mutations are described elsewhere in this
document. In such assays, the compound would be formulated appropriately,
given its biochemical nature, and contacted with a target cell.
Depending on the assay, culture may be required. The cell may then
be examined by virtue of a number of different physiologic assays.
Alternatively, molecular analysis may be performed in which the
function of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR, or related pathways, may be explored. This may involve
assays such as those for protein expression, enzyme function, substrate
utilization, phosphorylation states of various molecules, cAMP levels,
mRNA expression (including differential display of whole cell or
polyA RNA) and others.
 In Vivo Assays
 The present invention also encompasses the use of various
animal models. By developing or isolating mutant cells lines that
show differential expression of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR, one can generate cancer models in mice that
will be predictive of cancers in humans and other mammals. These
models may employ the orthotopic or systemic administration of tumor
cells to mimic primary and/or metastatic cancers. Alternatively,
one may induce cancers in animals by providing agents known to be
responsible for certain events associated with malignant transformation
and/or tumor progression. Finally, transgenic animals (discussed
below) that differentially express a wild-type TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR may be utilized as models for
cancer development and treatment.
 Treatment of animals with test compounds will involve the
administration of the compound, in an appropriate form, to the animal.
Administration will be by any route that could be utilized for clinical
or non-clinical purposes, including but not limited to oral, nasal,
buccal, rectal, vaginal or topical. Alternatively, administration
may be by intratracheal instillation, bronchial instillation, intradermal,
subcutaneous, intramuscular, intraperitoneal or intravenous injection.
Specifically contemplated are systemic intravenous injection, regional
administration via blood or lymph supply and intratumoral injection.
 Determining the effectiveness of a compound in vivo may
involve a variety of different criteria. Such criteria include,
but are not limited to, survival, reduction of tumor burden or mass,
arrest or slowing of tumor progression, elimination of tumors, inhibition
or prevention of metastasis, increased activity level, improvement
in immune effector function and improved food intake.
 Rational Drug Design
 The goal of rational drug design is to produce structural
analogs of biologically active polypeptides or compounds with which
they interact (agonists, antagonists, inhibitors, binding partners,
etc.). By creating such analogs, it is possible to fashion drugs
which are more active or stable than the natural molecules, which
have different susceptibility to alteration or which may affect
the function of various other molecules. In one approach, one would
generate a three-dimensional structure for TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR or a fragment thereof. This
could be accomplished by x-ray crystallography, computer modeling
or by a combination of both approaches. In addition, knowledge of
the polypeptide sequences permits computer employed predictions
of structure-function relationships. An alternative approach, an
"alanine scan," involves the random replacement of residues
throughout a protein or peptide molecule with alanine, followed
by determining the resulting effect(s) on protein function.
 It also is possible to isolate a TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR specific antibody, selected
by a functional assay, and then solve its crystal structure. In
principle, this approach yields a pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein crystallography
altogether by generating anti-idiotypic antibodies to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of an anti-idiotype antibody would be expected
to be an analog of the original antigen. The anti-idiotype could
then be used to identify and isolate peptides from banks of chemically-
or biologically-produced peptides. Selected peptides would then
serve as the pharmacore. Anti-idiotypes may be generated using the
methods described herein for producing antibodies, using an antibody
as the antigen.
 Thus, one may design drugs which have improved TIE-2, EDNRA,
TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR activity or which
act as stimulators, inhibitors, agonists, or antagonists of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR or molecules
affected by TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR function.
 Methods for Treating TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR Related Malignancies
 The present invention also contemplates, in another embodiment,
the treatment of cancer. The types of cancer that may be treated,
according to the present invention, are limited only by the involvement
of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR.
By involvement is meant that, it is not even a requirement that
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR be
mutated or abnormal--the overexpression or underexpression of these
proteins may be a primary factor in the development of tamoxifen-resistance.
Thus, it is contemplated that tumors may be treated using antisense
or expression therapy targeted to TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR.
 In many contexts, it is not necessary that the tumor cell
be killed or induced to undergo normal cell death or "apoptosis."
Rather, to accomplish a meaningful treatment, all that is required
is that the tumor growth be slowed to some degree. It may be that
the tumor growth is completely blocked, however, or that some tumor
regression is achieved. Clinical terminology such as "remission"
and "reduction of tumor" burden also are contemplated
given their normal usage.
 Genetic Based Therapies
 One of the therapeutic embodiments contemplated by the present
inventors is the intervention, at the molecular level, in the events
involved in the tumorigenesis of some cancers. Specifically, the
present inventors intend to provide, to a cancer cell, an antisense
construct capable of inhibiting expression of TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR, or an expression construct
capable of increasing expression of VEGF or bFGFR in that cell.
The lengthy discussion of expression vectors and the genetic elements
employed therein is incorporated into this section by reference.
Particularly preferred expression vectors are viral vectors such
as adenovirus, adeno-associated virus, herpes virus, vaccinia virus
and retrovirus. Also preferred is liposomally-encapsulated expression
 Those of skill in the art are well aware of how to apply
gene delivery to in vivo and ex vivo situations. For viral vectors,
one generally will prepare a viral vector stock. Depending on the
kind of virus and the titer attainable, one will deliver between
about 1.times.10.sup.4 and 1.times.10.sup.12 infectious particles
to the patient. Similar figures may be extrapolated for liposomal
or other non-viral formulations by comparing relative uptake efficiencies.
Formulation as a pharmaceutically acceptable composition is discussed
 Various routes are contemplated for various tumor types.
The section below on routes contains an extensive list of possible
routes. For practically any tumor, systemic delivery is contemplated.
This will prove especially important for attacking microscopic or
metastatic cancer. Where discrete tumor mass may be identified,
a variety of direct, local and regional approaches may be taken.
For example, the tumor may be injected directly with the expression
vector. A tumor bed may be treated prior to, during or after resection.
Following resection, one generally will deliver the vector by a
catheter left in place following surgery. One may utilize the tumor
vasculature to introduce the vector into the tumor by injecting
a supporting vein or artery. A more distal blood supply route also
may be utilized.
 In a different embodiment, ex vivo gene therapy is contemplated.
This approach is particularly suited, although not limited, to treatment
of bone marrow associated cancers. In an ex vivo embodiment, cells
from the patient are removed and maintained outside the body for
at least some period of time. During this period, a therapy is delivered,
after which the cells are reintroduced into the patient. Preferably,
any tumor cells in the sample have been killed.
 Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a targeting
agent. Alternatively, the effector may be a lymphocyte carrying
a surface molecule that interacts, either directly or indirectly,
with a tumor cell target. Various effector cells include cytotoxic
T cells and NK cells.
 According to the present invention, native or wild type
TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR may
be likely targets for an immune effector. It is possible TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR may be targeted
by immunotherapy, either using antibodies, antibody conjugates,
or immune effector cells.
 Alternatively, immunotherapy could be used as part of a
combined therapy, in conjunction with TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR-targeted gene therapy. The
general approach for combined therapy is discussed below. Generally,
the tumor cell must bear some marker that is amenable to targeting,
i.e., is not present on the majority of other cells. Many tumor
markers exist and any of these may be suitable for targeting in
the context of the present invention. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,
HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
 Combined Therapy With Immunotherapy, Traditional Chemo-
 Tumor cell resistance to DNA damaging agents represents
a major problem in clinical oncology. One goal of current cancer
research is to find ways to improve the efficacy of chemo- and radiotherapy.
One way is by combining such traditional therapies with gene therapy.
For example, the herpes simplex-thymidine kinase (HS-tk) gene, when
delivered to brain tumors by a retroviral vector system, successfully
induced susceptibility to the antiviral agent ganciclovir (Culver
et al., 1992). In the context of the present invention, it is contemplated
that TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR
gene therapy could be used similarly in conjunction with chemo-
or radiotherapeutic intervention.
 To kill cells, inhibit cell growth, inhibit metastasis,
inhibit angiogenesis or otherwise reverse or reduce the malignant
phenotype of tumor cells, using the methods and compositions of
the present invention, one would generally contact a "target"
cell with an antisense construct of TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR, or an expression construct of VEGF or bFGFR
and at least one other agent. These compositions would be provided
in a combined amount effective to kill or inhibit proliferation
of the cell. This process may involve contacting the cells with
the antisense or expression construct and the agent(s) or factor(s)
at the same time. This may be achieved by contacting the cell with
a single composition or pharmacological formulation that includes
both agents, or by contacting the cell with two distinct compositions
or formulations simultaneously, wherein one composition includes
the antisense or expression construct and the other includes the
 Alternatively, the gene therapy treatment may precede or
follow the other agent treatment by intervals ranging from min to
wk. In embodiments where the other agent and expression construct
are applied separately to the cell, one would generally ensure that
a significant period of time did not expire between the time of
each delivery, such that the agent and expression construct would
still be able to exert an advantageously combined (e.g., synergistic)
effect on the cell. In such instances, it is contemplated that one
would contact the cell with both modalities within about 12-24 h
of each other and, more preferably, within about 6-12 h of each
other, with a delay time of only about 12 h being most preferred.
In some situations, it may be desirable to extend the duration of
treatment with only the therapeutic agent significantly, for example,
where several days (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3,
4, 5, 6, 7 or 8) lapse between the respective administrations.
 It also is conceivable that more than one administration
of either TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR or the other agent will be desired. Various combinations
may be employed, where TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR is "A" and the other agent is "B",
as exemplified below:
4 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B
A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A
A/A/B/A A/B/B/B B/A/B/B B/B/A/B
 In addition, other combinations are contemplated. For instance,
constructs targeted to two or more of the TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGFR1, VEGF or bFGFR genes may be employed simultaneously
to achieve an improved antiangiogenic effect. In a preferred embodiment,
the agent "B" would comprise tamoxifen. To achieve cell
killing, both agents are delivered to a cell in a combined amount
effective to kill the cell.
 Agents or factors suitable for use in a combined therapy
are any chemical compound or treatment method that induces DNA damage
when applied to a cell. Such agents and factors include radiation
and waves that induce DNA damage such as .beta.-irradiation, X-rays,
UV-irradiation, microwaves, electronic emissions, and the like.
A variety of chemical compounds, also described as "chemotherapeutic
agents," function to induce DNA damage, all of which are intended
to be of use in the combined treatment methods disclosed herein.
Chemotherapeutic agents contemplated to be of use include, e.g.,
adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin,
actinomycin-D, mitomycin C cisplatin (CDDP) and even hydrogen peroxide.
The invention also encompasses the use of a combination of one or
more DNA damaging agents, whether radiation-based or actual compounds,
such as the use of X-rays with cisplatin or the use of cisplatin
 Particularly prefered for this embodiment is adjunct therapy
with compounds that have reported antiangiogenic activity, such
as angiotensin, laminin peptides, fibronectin peptides, plasminogen
activator inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin 12, platelet factor 4, IP-10, Gro-.beta., thrombospondin,
2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,
CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,
paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,
AGM-1470, platelet factor 4 or minocycline. It is anticipated that
such agents may be used in combination with either tamoxifen therapy
and/or gene therapy targeted to TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR.
 In treating cancer according to the invention, one would
contact the tumor cells with an agent in addition to the antisense
construct. This may be achieved by irradiating the localized tumor
site with radiation such as X-rays, UV-light, .beta.-rays or even
microwaves. Alternatively, the tumor cells may be contacted with
the agent by administering to the subject a therapeutically effective
amount of a pharmaceutical composition comprising a compound such
as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D,
mitomycin C, or more preferably, tamoxifen. The agent may be prepared
and used as a combined therapeutic composition, or kit, by combining
it with an TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF
or bFGFR construct, as described above.
 Agents that directly cross-link nucleic acids, specifically
DNA, are envisaged to facilitate DNA damage leading to a synergistic,
antineoplastic combination with TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR. Agents such as cisplatin, and other DNA alkylating
agents may be used. Cisplatin has been widely used to treat cancer,
with efficacious doses used in clinical applications of 20 mg/m.sup.2
for 5 days every three wk for a total of three courses. Cisplatin
is not absorbed orally and must therefore be delivered via injection
intravenously, subcutaneously, intratumorally or intraperitoneally.
 Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as doxorubicin,
etoposide, verapamil, podophyllotoxin, and the like. Widely used
in a clinical setting for the treatment of neoplasms, these compounds
are administered intravenously through bolus injections at doses
ranging from 25-75 mg/m.sup.2 at 21 day intervals for adriamycin,
to 35-50 mg/m.sup.2 for etoposide intravenously or double the intravenous
 Agents that disrupt the synthesis and fidelity of nucleic
acid precursors and subunits also lead to DNA damage. A number of
nucleic acid precursors have been developed for this purpose. Particularly
useful are agents that have undergone extensive testing and are
readily available, such as 5-fluorouracil (5-FU). Although quite
toxic, 5-FU is applicable in a wide range of carriers, including
topical. However intravenous administration with doses ranging from
3 to 15 mg/kg/day is commonly used.
 Other factors that cause DNA damage and have been used extensively
include .gamma.-rays, X-rays, and/or the directed delivery of radioisotopes
to tumor cells. Other forms of DNA damaging factors also are contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage to DNA, on the precursors
of DNA, the replication and repair of DNA, and the assembly and
maintenance of chromosomes. Dosage ranges for X-rays range from
daily doses of 50 to 200 roentgens for prolonged periods of time
(3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges
for radioisotopes vary widely, and depend on the half-life of the
isotope, the strength and type of radiation emitted, and the uptake
by the neoplastic cells.
 The skilled artisan is directed to "Remington's Pharmaceutical
Sciences" 15th Edition, chapter 33, and in particular to pages
624-652. Some variation in dosage will necessarily occur depending
on the condition of the subject being treated. The person responsible
for administration will, in any event, determine the appropriate
dose for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, and general safety
and purity standards as required by the FDA Office of Biologics
 The inventors propose that the regional delivery of TIE-2,
EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1, VEGF or bFGFR constructs
to patients with breast cancer will be a very efficient method for
delivering a therapeutically effective gene to counteract the clinical
disease. Similarly, chemo- or radiotherapy may be directed to a
particular, affected region of the subject's body. Alternatively,
systemic delivery of expression construct and/or the agent may be
appropriate in certain circumstances, for example, where extensive
metastasis has occurred.
 In addition to combining TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III,
VEGFR1, VEGF or bFGFR-targeted therapies with chemo- and radiotherapies,
it also is contemplated that combination with other gene therapies
will be advantageous. For example, simultaneous targeting of therapies
directed toward TIE-2, EDNRA, TGF.beta.3, TGFR.beta.III, VEGFR1,
VEGF or bFGFR and p53, BRCA1 or BRCA2 mutations may produce an improved
anti-cancer treatment. Any other tumor-related gene conceivably
can be targeted in this manner, for example, p21, Rb, APC, DCC,
NF-1, NF-2, p16, FHIT, WT-1, MEN-I, MEN-II, VHL, FCC, MCC, ras,
myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.
 Formulations and Routes for Administration to Patients
 Where clinical applications are contemplated, it will be
necessary to prepare pharmaceutical compositions--antisense vectors,
virus stocks, proteins, antibodies and drugs--in a form appropriate
for the intended application. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as other
impurities that could be harmful to humans or animals.
 One generally will desire to employ appropriate salts and
buffers to render delivery vectors stable and allow for uptake by
target cells. Buffers also will be employed when recombinant cells
are introduced into a patient. Aqueous compositions of the present
invention comprise an effective amount of the vector to cells, dissolved
or dispersed in a pharmaceutically acceptable carrier or aqueous
medium. Such compositions also are referred to as innocula. The
phrase "pharmaceutically or pharmacologically acceptable"
refers to molecular entities and compositions that do not produce
adverse, allergic, or other untoward reactions when administered
to an animal or a human. As used herein, "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutically active substances is well know in the
art. Except insofar as any conventional media or agent is incompatible
with the vectors or cells of the present invention, its use in therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions.
 The active compositions of the present invention may include
classic pharmaceutical preparations. Administration of these compositions
according to the present invention will be via any common route
so long as the target tissue is available via that route. This includes
oral, nasal, buccal, rectal, vaginal or topical. Alternatively,
administration may be by orthotopic, intradermal, subcutaneous,
intramuscular, intraperitoneal or intravenous injection. Such compositions
normally would be administered as pharmaceutically acceptable compositions,
 The active compounds also may be administered parenterally
or intraperitoneally. Solutions of the active compounds as free
base or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary conditions
of storage and use, these preparations contain a preservative to
prevent the growth of microorganisms.
 The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be preserved
against the contaminating action of microorganisms, such as bacteria
and fungi. The carrier can be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
 Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate solvent
with various other ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a sterile
vehicle which contains the basic dispersion medium and the required
other ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum-drying and freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
 For oral administration the polypeptides of the present
invention may be incorporated with excipients and used in the form
of non-ingestible mouthwashes and dentifrices. A mouthwash may be
prepared incorporating the active ingredient in the required amount
in an appropriate solvent, such as a sodium borate solution (Dobell's
Solution). Alternatively, the active ingredient may be incorporated
into an antiseptic wash containing sodium borate, glycerin and potassium
bicarbonate. The active ingredient may also be dispersed in dentifrices,
including: gels, pastes, powders and slurries. The active ingredient
may be added in a therapeutically effective amount to a paste dentifrice
that may include water, binders, abrasives, flavoring agents, foaming
agents, and humectants.
 The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids
as acetic, oxalic, tartaric, mandelic, and the like. Salts formed
with the free carboxyl groups can also be derived from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium,
or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
 Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in
an aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered isotonic
with sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular, subcutaneous
and intraperitoneal administration. In this connection, sterile
aqueous media which can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage could be dissolved in 1 ml of isotonic NaCl solution and
either added to 1000 ml of hypodermoclysis fluid or injected at
the proposed site of infusion, (see for example, "Remnington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur depending
on the condition of the subject being treated. The person responsible
for administration will, in any event, determine the appropriate
dose for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics standards.
 The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those
of skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific embodiments
which are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
 Portions of this work are recited in Hilsenbeck et al. (1999),
the entire text of which is incorporated herein by reference.
 Materials and Methods Utilized in Examples 1 Through 4
 Tumors and Microarray Hybridization
 MCF-7 tumors were inoculated into the mammary fat pads of
athymic nude mice supplemented with an estrogen pellet as described
previously (Osborne et al., 1985) until tumors arose. The estrogen
pellets were removed and the animals were treated with tamoxifen.
Tumor volumes then declined and remained stable for several months.
Invariably, however, after initial growth suppression, the tumors
became resistant and growth resumed. Animals were sacrificed at
various times to obtain cells from estrogen-stimulated (ES) tumors
prior to tamoxifen treatment, from tamoxifen-sensitive (TS) tumors
during tamoxifen treatment but prior to acquired resistance, and
from tamoxifen-resistant (TR) tumors after tumor growth had resumed.
 RNA was prepared from these tumors (n=5 tumors per group)
using RNeasy kits (Qiagen Inc., Valencia, Calif.), and mRNA was
isolated on Dynabeads (Dyne, Oslo, Norway) according to manufacturer's
instructions. The RNAs were pooled in each group and used to synthesize
32P-radiolabeled cDNAs for hybridization to the Atlas.TM. Human
cDNA Expression Array 1 according to the manufacturer's instructions
(CLONTECH Laboratories, Inc., 1997) with SuperScriptII RT (Gibco
BRL, Gaithersburg, Md.). The CLONTECH Atlas.TM. Human cDNA Expression
Array comprises a positively charged 8.times.12 cm nylon membrane,
duplicately spotted with 200-600 BP cDNA fragments representing
588 genes and 21 housekeeping genes or control sequences (CLONTECH
Laboratories, Inc., 1997). Genes are arrayed in six quadrants with
genes of like function (i.e. oncogenes, assorted receptors, etc.)
grouped together geographically. The hybridization data were collected
with a Molecular Dynamics Phosphoimager.TM. (Sunnyvale, Calif.).
 Western Blot Analysis
 Pulverized, frozen tumors were manually homogenized in a
5% SDS solution. After boiling and microcentrifugation, clear supernatants
were collected and the protein concentration was determined by the
bicinchoninic acid method (Pierce, Rockford, Ill.) as previously
described (Tandon et al., 1989). Twenty-five .mu.g of protein were
separated on an acrylamide denaturing gel and transferred by electroblotting
onto nitrocellulose membranes (Schleicher & Schuell, Keene,
N.H.). The blots were first stained with StainAll Dye (Alpha Diagnostic
Intl., Inc., San Antonio, Tex.) to confirm uniform transfer of all
samples, and then incubated in blocking solution [5% non-fat dry
milk in Tris-buffered saline-Tween (TBST:50 mM Tris-HCL pH 7.5,
150 mM NaCl, 0.05% Tween-20)]. After brief washes with TBST, the
filters then were reacted with primary antibodies to erk-2 (UBI,
Lake Placid, N.Y.) or HSF-1 (Stressgen, Victoria, Canada) for 1
h at room temperature followed by extensive washes with TBST. Blots
were then incubated with horseradish peroxidase-conjugated secondary
antibody (Amersham, Arlington Heights, Ill.) for 1 h washed with
TBST, and developed using the ECL procedure (Amersham).
 Statistical Considerations
 Each hybridization (m=3) resulted in expression values for
588 genes and 21 controls (putative housekeeping genes and negative
controls). The controls, which were more difficult to quantitate
reliably, were not included in the statistical analyses. Expression
of the highest and lowest expressed genes on the array varied by
2-3 orders of magnitude. Logarithmic transformation of the raw data
reduced this range and helped equalize variability. This also means
that additive effects on the log scale can be interpreted as fold
changes in actual expression.
 Due to expense, limited amounts of RNA and other considerations,
array studies usually have few replications and invariably have
orders of magnitude more variables (genes and expressed sequence
tags) than observations (hybridizations). Here, the roles of variables
and observations were switched by treating each tumor type as a
variable (m=3) and each expressed gene sequence as an observation
 Principal Components Analysis (PCA) of mean-centered log-transformed
data, based on the variance-covariance matrix (Tatsuoka, 1971),
was then used to standardize across the three hybridizations and
to extract three new axes (components P1, P2, and P3), expressed
as linear combinations of the original axes (ES, TS, and TR).
 In PCA, the coefficients (A's, B's, C's) are chosen so that
the first component (P1) explains the maximal amount of variance
in the data. The second component (P2) is perpendicular to the first
and explains the maximal residual squared variation, and the third
component (P3) is perpendicular to the first two. Meaning was ascribed
to the new axes by visual examination of the coefficients. In these
array studies, P1 represents the average level of expression across
the tumor types. P2 and P3 represent differences between tumor types.
A bivariate analysis, which results in two new axes (P1 and P2),
was also performed to compare TS and TR. The coefficients do not
always have a biologically sensible interpretation, although the
higher order components can still be used to identify outlier genes,
regardless of interpretation.
 P2 (and P3 in the higher-order analysis) were used to identify
"outlier" genes that might represent true alterations
in gene expression. In the bivariate PCA of TS vs. TR, a normal
approximation was used to construct a 99% prediction region for
P2 (i.e. 0.+-.2.57*SD.sub.r). A robust estimate of the standard
deviation (SD.sub.r=interquartile range/1.35) was used to reduce
the variance inflating effects of outliers (Venables et al., 1994
). Genes outside the region were identified for further study. Analogously,
in a trivariate PCA (ES, TS, TR) a 99% bivariate normal prediction
ellipse was computed (Tatsuoka, 1971; Anderson, 1958) for P2 vs.
P3 and genes outside the ellipse were selected for investigation.
 This "robust prediction interval" approach seems
justified on the following basis. While the distribution of P1 is
highly skewed, higher order components are roughly symmetric. When
there is no differential expression, as in a bivariate analysis
of two array hybridizations using the same pool of RNA, the higher
order components are approximately normally distributed. In studies
comparing different pools of RNA, where some genes may be differentially
expressed, the observed distribution of each higher order component
(P2, P3, etc.) should comprise a mixture of central (.mu.=0) and
noncentral (.mu..noteq.0) distributions. A robust estimator that
focuses on the middle of the observed distribution, which should
represent primarily unaltered genes, was used to increase sensitivity
to identify truly altered genes. The prediction level (99%), which
is analogous to the specificity of a diagnostic test, was chosen
arbitrarily as representing a reasonable balance between identifying
too many spuriously "significant" genes, versus missing
true alterations. For display purposes, the data was back-transformed
by exponentiating P2 and P3 so that the data are shown as approximate
fold-increases or decreases in expression.
 The ability of this methodology to detect true alterations
was examined in a small simulation study. Log transformed values
from a hypothetical bivariate array study with 588 genes were generated
to have a common log-normally distributed component for level of
expression (i.e. exp(X)+8, where X.about.N(.mu.=0, .sigma.=6)),
and independent normally distributed errors (i.e. log.sub.e(Control)=exp(X)+8+Y,
log.sub.e(Experimental)=exp(X)+8+Z, where Y,Z.about.N(.mu.=0, .sigma.=0.17)).
The distributional parameters were chosen to mimic data seen in
real studies. A small percentage of truly altered genes (2% or 4%)
were created by shifting the error distribution for the experimental
member of the pair up or down (with 50% probability) to represent
an average 2 or 2.5-fold change from baseline (i.e. log.sub.e(Experimental)=-
exp(X)+8+W, where W.about.N(.mu.=.+-.0.7, .sigma.=0.17)). The generated
data were then analyzed as described above, and the number of truly
altered and spuriously-altered genes falling outside the 99% prediction
region was tabulated. Each scenario was replicated 100 times and
the results were summarized over all replications. All analyses
were performed using SAS (Version 6.11, Cary, N.C.).
 FIG. 1 shows the three bivariate log-log scatterplots that
arise from pairwise comparisons of the data from the three hybridizations
(ES, TS, TR). Each of the 588 genes on the array (excluding housekeeping
and control genes) is represented by a point on the scatterplots.
The individual values ranged over 2-3 orders of magnitude, indicating
that the most highly expressed genes were expressed at 100 or 1,000-fold
higher levels than the lowest expressed genes. For example, heat
shock protein 27 (hsp27) was the most highly expressed gene on the
array in all three tumor types. This is consistent with the previously
published result that hsp27 is amplified and overexpressed in the
late-passage MCF-7 cells used in this model (Fuqua et al., 1994).
Similarly, the array results are consistent with previous findings
(Tang et al., 1996) that heregulin .alpha. is expressed at relatively
low levels in all three types of tumor cells.
 In each scatterplot, most genes lie fairly close to a diagonal
line of "identity". This line may not be centered on the
graph if there are differences in the average level of radioactivity
of probes used in each hybridization. Distance along this line denotes
differences in level of expression between genes, such as seen between
hsp27 and heregulin a, while perpendicular distance away from the
line denotes differences in expression within the same gene between
 Principal Components Analysis (PCA) of the log-transformed
expression data was used to produce a new set of axes (FIG. 2).
For TS vs. TR tumors (FIG. 2A), the new x-axis or first principal
component (P1) roughly corresponds to the line of "identity"
and represents level of expression. The second principal component
(P2) is perpendicular to the first, and represents difference in
expression between tumor types. In the bivariate analysis, more
than 97% of the total variation in the log-transformed data was
associated with P1, leaving about 3% for P2. The two components
are, by definition, not correlated (p=0). The distribution of P1
is skewed, as many genes on the array are expressed at low to moderate
levels, while only a few are expressed at extremely high levels.
The distribution of P2 is roughly symmetric, and a 99% robust prediction
interval identified 35 outlier genes that may be over- or under-expressed
in TR relative to TS tumors (FIG. 2B).
 Bivariate PCA could be performed for each pair of tumor
types, however, a more comprehensive three-way analysis is preferred
and is more biologically relevant. PCA of the mean-centered log-transformed
data (ES, TS, TR) yields three new axes (P1, P2, P3), which account
for 90.5%, 8%, and 1.5% of the variation in the data, respectively.
By inspection of the coefficients, the first principal component
(P1) is again interpreted as the "average level of expression"
since the coefficients were all positive and similar in value (0.63,
0.55, 0.55, respectively). The second principal component (P2) clearly
contrasts ES to the average of TS and TR because the P2 coefficient
for ES is negative (-0.78) and roughly equal to the sum of the TS
and TR coefficients (0.46, 0.43, respectively). The third principal
component (P3) represents primarily differences between TS and TR
because the P3 coefficient for ES is small (0.02) and the TS and
TR coefficients are nearly equal, but opposite in sign (0.69 and
-0.72, respectively). FIG. 3 shows a scatterplot of P2 versus P3.
Points near the center represent genes that were similarly expressed
in all three tumor types while points on the periphery exhibit alterations
in expression. Data have been back-transformed to show approximate
fold changes in expression. A bivariate normal approximation with
robust estimates of standard deviations was used to compute a 99%
prediction ellipse. Genes lying outside the region may exhibit real
alterations in level of expression that are associated with the
biologic effects during the transition from ES to TS and TS to TR.
 In addition, different regions of the P2.times.P3 plane
correspond to different temporal patterns of expression alteration.
For example, genes in the far right of FIG. 3 (i.e. near erk-2)
are unregulated by tamoxifen relative to ES, but unchanged in TR
relative to TS, while genes in the lower right (i.e. near HSF-1)
are unregulated in TS relative to ES, but downregulated in TR tumors.
Confirmation of Gene Expression by Western Blot Analysis
 Two genes just outside of the 99% prediction ellipse (erk-2
and heat shock transcription factor 1 or HSF-1) were selected for
quantitation by Western blot. These two were chosen based on their
relatively low expression (FIG. 1) and modest alteration so that
sensitivity questions could be addressed, and on the ready availability
of specific antibodies. The erk-2 kinase is a known mediator of
growth factor pathway signaling, and it has been shown that ER can
activate its activity in MCF-7 cells (Migliaccio et al., 1996).
HSF-1 is involved in cellular stress responses (Rabindran et al.,
1991), and is thus a potential marker of tamoxifen-induced stress.
The relative levels of erk-2 and HSF-1 predicted in the array study
were indeed confirmed in an independent set of individual tumors
(numbered 1-15 in FIG. 4) from the athymic nude mouse model. As
predicted by FIG. 3A and FIG. 1A, Western blot results for HSF-1
indicate a significant upregulation in TS cells relative to ES,
which is followed by down-regulation in TR to near ES levels (FIG.
4). Similarly for erk-2, there is a significant upregulation in
TS relative to ES (FIG. 4) but relatively less change between TS
and TR as reflected by the approximate fold increase in TR over
TS around 1:1 (FIG. 4).
Identification of Angiogenic Factors and Receptors as Markers for
Tamoxifen-Resistant Breast Cancer
 The techniques described in Examples 1-3 above were used
to identify seven genes encoding angiogenic factors or angiogenic
receptors as differentially expressed in tamoxifen-resistant breast
cancer versus estrogen-stimulated or tamoxifen sensitive breast
cancers, using the athymic mouse model and array screening to identify
differentially expressed genes. Although angiogenic factors and
receptors were known as a bad prognostic marker for breast cancer
(Folkman, 1995a), this unexpected result is the first report of
a correlation between expression levels for angiogenic factors and
receptors and tamoxifen-resistant breast cancer.
 The marker genes for tamoxifen-resistant breast cancer identified
in the present application are tyrosine protein kinase receptor
(TIE-2), endothelin-1 receptor (EDNRA), transforming growth factor
.beta.3 (TGF.beta.3), transforming growth factor receptor Om (TGFR.beta.III),
vascular permeability factor receptor (VEGFR1), vascular endothelin
growth factor (VEGF) and basic fibroblast growth factor receptor
 As shown in FIG. 6, both VEGF and bFGFR exhibited a decreased
expression in breast cancers treated with tamoxifen. Expression
was significantly inhibited in comparison with estrogen-stimulated
breast cancer. While VEGF expression was significantly higher in
tamoxifen-resistant compared to tamoxifen-sensitive breast cancers,
no significant difference in bFGFR expression levels was observed
between tamoxifen-sensitive and tamoxifen-resistant breast cancers.
 The remaining markers all showed a significant increase
in expression in tamoxifen-resistant breast cancer, when compared
to either estrogen-stimulated or tamoxifen-sensitive breast cancers.
In FIG. 5, expression levels for TGF.beta.3, TIE-2, EDNRA, TGF.beta.III
and VEGFR1 are elevated in tamoxifen-resistant (TR) tumors, compared
to estrogen-stimulated (E2) or tamoxifen sensitive (TS) breast cancers.
 The results of array analysis were confirmed in part by
Western blotting. As shown in FIGS. 7-9, both the TIE-2 and VEGF
proteins showed increased expression in tamoxifen-resistant tumors,
compared to tamoxifen-sensitive and estrogen-stimulated breast cancers.
In addition, a higher molecular weight form of a putative TIE-2
related protein was observed only in TR tumors.
 These results demonstrate that TIE-2, EDNRA, TGF.beta.3,
TGFR.beta.III, VEGF and VEGFR 1 are all positive markers for tamoxifen-resistant
breast cancer. Thus, assays for increased expression of these markers
may be used to differentiate between tamoxifen-resistant and tamoxifen-sensitive
forms of breast cancer, allowing more efficient clinical application
of antiestrogen therapy. Significantly, these results suggest that
antiangiogenic agents or treatment with antisense or "knock-out"
constructs directed against these six genes may be used as adjuvants
to tamoxifen treatment and can potentially be applied to convert
tamoxifen-resistant breast cancers to tamoxifen-sensitive tumors.
Further, application of antiangiogenic agents could potentially
be used to prolong the sensitivity of tamoxifen-sensitive breast
cancer to antiestrogen therapy. bFGFR may be important for angiogenesis
to proceed but not necessarily a marker for tamoxifen resistance.
 All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred embodiments,
it will be apparent to those of skill in the art that variations
may be applied to the composition, methods and in the steps or in
the sequence of steps of the method described herein without departing
from the concept, spirit and scope of the invention. More specifically,
it will be apparent that certain agents which are both chemically
and physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those skilled
in the art are deemed to be within the spirit, scope and concept
of the invention as defined by the appended claims.