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Cancer Patent Abstract
Gene sequences as shown in SEQ ID NO:1-18 have been discovered and
isolated, and found to be significantly associated with metastatic
spread of breast and colon cancer cells to other organs. Methods
are provided for determining the risk of metastasis of a breast
or colon tumor, which involve determining whether a tissue sample
from a tumor expresses a polypeptide encoded by a gene as shown
in SEQ ID NOS:1-18, or a substantial portion thereof. One of the
gene sequences encodes a novel aspartyl protease termed CSP56, which
can be used to provide reagents and methods for determining which
tumors are likely to metastasize and for suppressing metastases
of these tumors. Clinicians can use this information to predict
which tumors will metastasize to other organs and to provide relevant
therapies to appropriate patients.
Cancer Patent Claims
The invention claimed is:
1. A cDNA molecule which is at least 85% identical to a polynucleotide
comprising of SEQ ID NO:1, wherein said polynucleotide is expressed
at a higher level in metastatic breast cancer tissue relative to
non-metastatic breast cancer tissue.
2. The cDNA molecule of claim 1 which is at least 95% identical
to a polynucleotide comprising of SEQ ID NO:1.
3. The cDNA molecule of claim 1 which is at least 85% identical
to the nucleotide sequence shown in SEQ ID NO: 1.
4. The cDNA molecule of claim 1 which is at least 90% identical
to a polynucleotide comprising SEQ ID NO:1.
5. A method of making a recombinant vector comprising inserting
a cDNA molecule of claim 1 into a vector in operable linkage to
a promoter.
6. A recombinant vector produced according to the method of claim
5.
7. A method of making a recombinant host cell comprising introducing
the recombinant vector of claim 6 into a host cell.
8. A recombinant host cell produced according to the method of
claim 7.
9. A method of producing a polypeptide comprising culturing the
recombinant host cell of claim 8 under conditions such that the
polypeptide is expressed, and recovering said polypeptide.
10. A eDNA molecule which encodes at least 500 contiguous amino
acids of a protein encoded by a polynucleotide comprising SEQ ID
NO:1.
11. The cDNA molecule of claim 10 which encodes at least 550 contiguous
amino acids of a protein encoded by a polynucleotide comprising
SEQ ID NO:1.
12. The cDNA molecule of claim 10 which encodes at least 600 contiguous
amino acids of a protein encoded by a polynucleotide comprising
SEQ ID NO:1.
13. A cDNA molecule comprising a polynucleotide selected from the
group consisting of: (a) at least 1450 contiguous nucleotides of
SEQ ID NO:1; (b) at least 1500 contiguous nucleotides of SEQ ID
NO:1; (c) at least 1550 contiguous nucleotides of SEQ ID NO:1; and
(d) at least 1600 contiguous nucleotides of SEQ ID NO:1.
14. The cDNA molecule of claim 13 wherein the polynucleotide is
expressed at a higher level in metastatic breast cancer tissue relative
to non-metaslatic breast cancer tissue.
15. An isolated and purified polynucleotide comprising a nucleotide
segment selected from the group consisting of: (a) a segment of
at least 1450 contiguous nucleotides which hybridizes under stringent
conditions to a nucleotide sequence from SEQ ID NO:1; and (b) a
segment of at least 1500 contiguous nucleotides which hybridizes
under stringent conditions to a nucleotide sequence from of SEQ
ID NO:1, wherein said polynueleotide is expressed at a higher level
in metastatic breast cancer tissue relative to non-metastatic breast
cancer tissue, wherein said stringent conditions are selected from
the group consisting of 4.times.SSC at 65.degree. C.; 50% formamide,
4.times.SSC at 42.degree. C.; or 0.5.times.SSC, 0.1% SDS at 65.degree.
C.
16. A construct comprising: a promoter; and a polynucleotide segment
comprising a nucleotide sequence of SEQ ID NO:1, wherein the polynucleotide
segment is located downstream from the promoter, wherein transcription
of the polynucleotide segment initiates at the promoter.
17. A recombinant host cell comprising a construct which comprises:
a promoter and: a polynucleotide segment comprising a nucleotide
sequence of SEQ ID NO:1.
18. A recombinant host cell comprising a transcription initiation
unit, wherein the transcription initiation unit comprises in 5'
to 3' order: (a) an exogenous regulatory sequence; (b) an exogenous
exon; and (c) a splice donor site, wherein the transcription initiation
unit is located upstream of a coding sequence of SEQ ID NO:1, wherein
the exogenous regulatory sequence controls transcription of the
coding sequence.
19. A polynucleotide probe comprising polynucleotide selected from
the group consisting of: (a) at least 1450 contiguous nucleotides
of SEQ ID NO:1; (b) at least 1500 contiguous nucleotides of SEQ
ID NO:1; (c) at least 1550 contiguous nucleotides of SEQ ID NO:1;
and (d) at least 1600 contiguous nucleotides of SEQ ID NO:1; said
polynucleotide probe further comprising a detectable label.
20. A polynucleotide array comprising at least one single-stranded
polynucleotide selected from the group consisting of: (a) at least
1450 contiguous nucleotides SEQ ID NO:1; (b) at least 1500 contiguous
nucleotides of SEQ ID NO:1; (c) at least 1550 contiguous nucleotides
of SEQ ID NO:1; and (d) at least 1600 contiguous nucleotides of
SEQ ID NO:1.
21. The polynucleotide array of claim 20 wherein the polynucleotide
comprises the sequence of SEQ ID NO:1.
22. An isolated nucleic acid molecule which is at least 85% identical
to a polynucleotide comprising SEQ ID NO:1, wherein said polynucleotide
is expressed at a higher level in metastatic breast cancer tissue
relative to non-metastatic breast cancer tissue.
23. The isolated nucleic acid molecule of claim 22 which is at
least 90% identical to a polynucleotide comprising SEQ ID NO:1.
24. The isolated nucleic acid molecule of claim 22 which is at
least 95% identical to a polynucleotide comprising SEQ ID NO:1.
25. The isolated nucleic acid molecule of claim 22 which is at
least 99% identical to a polynucleotide comprising SEQ ID NO:1.
26. The isolated nucleic acid molecule of claim 22 which is SEQ
ID NO:1.
Cancer Patent Description
TECHNICAL FIELD OF THE INVENTION
This invention relates to methods for predicting the behavior of
tumors and in particular, but not exclusively, to methods in which
a tumor sample is examined for expression of a specified gene sequence
which indicates propensity for metastatic spread.
BACKGROUND OF THE INVENTION
Despite use of a number of histochemical, genetic, and immunological
markers, clinicians still have a difficult time predicting which
tumors will metastasize to other organs. Some patients are in need
of adjuvant therapy to prevent recurrence and metastasis and others
are not. Distinguishing between these subpopulations of patients
is not straightforward. Thus the course of treatment is not easily
charted. There is therefore a need in the art for new markers for
distinguishing between tumors of differing metastatic potential.
SUMMARY OF THE INVENTION
It is an object of the invention to provide reagents and methods
for determining which tumors are likely to metastasize and for suppressing
metastases of these tumors. These and other objects of the invention
are provided by one or more of the embodiments described below.
One embodiment of the invention is an isolated and purified protein
having an amino acid sequence which is at least 85% identical to
an amino acid sequence encoded by a polynucleotide comprising a
nucleotide sequence selected from the group consisting of SEQ ID
NOS:1-18. Percent identity is determined using a Smith-Waterman
homology search algorithm using an affine gap search with a gap
open penalty of 12 and a gap extension penalty of 1.
Another embodiment of the invention is an isolated and purified
polypeptide which consists of at least 8 contiguous amino acids
of a protein having an amino acid sequence encoded by a polynucleotide
comprising a nucleotide sequence selected from the group consisting
of SEQ ID NOS:1-18.
Yet another embodiment of the invention is a fusion protein which
comprises a first protein segment and a second protein segment fused
to each other by means of a peptide bond. The first protein segment
consists of at least 8 contiguous amino acids selected from an amino
acid sequence encoded by a polynucleotide comprising a nucleotide
sequence selected from the group consisting of SEQ ID NOS:1-18.
Still another embodiment of the invention is a preparation of antibodies
which specifically bind to a protein with an amino acid sequence
encoded by a polynucleotide comprising a nucleotide sequence selected
from the group consisting of SEQ ID NOS:1-18.
Even another embodiment of the invention is a cDNA molecule which
encodes an isolated and purified protein having an amino acid sequence
which is at least 85% identical to an amino acid sequence encoded
by a polynucleotide comprising a nucleotide sequence selected from
the group consisting of SEQ ID NO:1-18. Percent identity is determined
using a Smith-Waterman homology search algorithm using an affine
gap search with a gap open penalty of 12 and a gap extension penalty
of 1.
Another embodiment of the invention is a cDNA molecule which encodes
at least 8 contiguous amino acids of a protein encoded by a polynucleotide
comprising a nucleotide sequence selected from the group consisting
of SEQ ID NOS:1-18.
Even another embodiment of the invention is a cDNA molecule comprising
at least 12 contiguous nucleotides of a nucleotide sequence selected
from the group consisting of SEQ ID NOS:1-18.
Still another embodiment of the invention is a cDNA molecule which
is at least 85% identical to a nucleotide sequence selected from
the group consisting of SEQ ID NOS:1-18. Percent identity is determined
using a Smith-Waterman homology search algorithm using an affine
gap search with a gap open penalty of 12 and a gap extension penalty
of 1.
A further embodiment of the invention is an isolated and purified
subgenomic polynucleotide comprising a nucleotide segment which
hybridizes to a nucleotide sequence selected from the group consisting
of SEQ ID NOS:1-18 after washing with 0.2.times.SSC at 65.degree.
C.
Another embodiment of the invention is a construct comprising a
promoter and a polynucleotide segment encoding at least 8 contiguous
amino acids of a protein encoded by a polynucleotide comprising
a nucleotide sequence selected from the group consisting of SEQ
ID NOS:1-18. The polynucleotide segment is located downstream from
the promoter, wherein transcription of the polynucleotide segment
initiates at the promoter.
Yet another embodiment of the invention is a host cell comprising
a construct which comprises a promoter and a polynucleotide segment
encoding at least 8 contiguous amino acids of a protein encoded
by a polynucleotide comprising a nucleotide sequence selected from
the group consisting of SEQ ID NOS:1-18.
Even another embodiment of the invention is a recombinant host
cell comprising a new transcription initiation unit. The new transcription
initiation unit comprises in 5' to 3' order (a) an exogenous regulatory
sequence, (b) an exogenous exon, and (c) a splice donor site. The
new transcription initiation unit is located upstream of a coding
sequence of a gene. The coding sequence comprises a nucleotide sequence
selected from the group consisting of SEQ ID NOS:1-18. The exogenous
regulatory sequence controls transcription of the coding sequence
of the gene.
Still another embodiment of the invention is a polynucleotide probe
comprising (a) at least 12 contiguous nucleotides selected from
the group consisting of SEQ ID NOS:1-18 and (b) a detectable label.
Even another embodiment of the invention is a method for identifying
a metastatic tissue or metastatic potential of a tissue. An expression
product of a gene comprising a nucleotide sequence selected from
the group consisting of SEQ ID NOS:1-4, 6-13, and 15-18 is measured
in a tissue sample. A tissue sample which expresses a product of
a gene comprising a nucleotide sequence selected from the group
consisting of SEQ ID NOS:1, 4, 11, 16, 17, and 18 or which does
not express a product of a gene comprising a nucleotide sequence
selected from the group consisting of SEQ ID NOS:2, 3, 6, 7, 8,
9, 10, 12, 13, and 15 is identified as metastatic or as having metastatic
potential.
Still another embodiment of the invention is a method of screening
test compounds for the ability to suppress the metastatic potential
of a tumor. A biological sample is contacted with a test compound.
Synthesis of a protein having an amino acid sequence encoded by
a polynucleotide comprising a nucleotide sequence selected from
the group consisting of SEQ ID NOS:1-4, 6-13, and 15-18 is measured
in the biological sample. A test compound which decreases synthesis
of a protein encoded by a polynucleotide comprising SEQ ID NOS:1,
4, 11, 16, 17, or 18 or which increases synthesis of a protein encoded
by a polynucleotide comprising SEQ ID NOS:2, 3, 6, 7, 8, 9, 10,
12, 13, or 15 is identified as a potential agent for suppressing
the metastatic potential of a tumor.
Another embodiment of the invention is a method of predicting propensity
for high-grade or low-grade metastatic spread of a colon tumor.
An expression product of a gene having a sequence selected from
the group consisting of SEQ ID NO:16 and 17 is measured in a colon
tumor sample. A colon tumor sample which expresses the product of
SEQ ID NO:16 is categorized as having a high propensity to metastasize
and a colon tumor sample which expresses the product of SEQ ID NO:17
is categorized as having a low propensity to metastasize.
Still another embodiment of the invention is a set of primers for
amplifying at least a portion of a gene having a coding sequence
selected from the group consisting of the nucleotide sequences shown
in SEQ ID NOS:1-18.
Even another embodiment of the invention is a polynucleotide array
comprising at least one single-stranded polynucleotide which comprises
at least 12 contiguous nucleotides of a nucleotide sequence selected
from the group consisting of SEQ ID NOS:1-18.
A further embodiment of the invention is a method of identifying
a metastatic tissue or metastatic potential of a tissue. A tissue
sample comprising single-stranded polynucleotide molecules is contacted
with a polynucleotide array comprising at least one single-stranded
polynucleotide probe. The at least one single-stranded polynucleotide
probe comprises at least 12 contiguous nucleotides of a nucleotide
sequence selected from the group consisting of SEQ ID NOS:1-4, 6-13,
and 15-18. The tissue sample is suspected of being metastatic or
of having metastatic potential. Double-stranded polynucleotides
bound to the polynucleotide array are detected. Detection of a double-stranded
polynucleotide comprising contiguous nucleotides selected from the
group consisting of SEQ ID NOS:1-4, 11, 16, 17, and 18 or lack of
detection of a double-stranded polynucleotide comprising contiguous
nucleotides selected from the group consisting of SEQ ID NOS:2,
3, 6, 7, 8, 9, 10, 12, 13, and 15 identifies the tissue sample as
metastatic or of having metastatic potential.
The invention thus provides the art with a number of genes and
proteins, which can be used as markers of metastasis. These are
useful for more rationally prescribing the course of therapy for
cancer patients, especially those with breast or colon cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Arbitrary primer-based differential display and confirmation
by RNA blot analysis of different human breast cancer cell line.
FIG. 1A. Autoradiograph of a differential display gel depicting
two bands of approximately 1.2 kb in size in the human breast cancer
cell line MDA-MB-435. Differential display reactions were prepared
and run in duplicates. FIG. 1B. Northern blot analysis verifying
the expression pattern in MDA-MB-435. cDNA isolated from the differential
display gel hybridized to two transcripts of approximately 2.0 kb
and 2.5 kb in size. Equal amounts of RNA in each lane were loaded
as judged by staining of the membrane with methylene blue and hybridization
of the membrane with a human .beta.-actin probe.
FIG. 2. Nucleotide sequence and deduced amino acid sequence of
CSP56 (SEQ ID NOS:18 and 19, respectively). FIG. 2A. The 518 amino
acid long sequence is shown in single-letter code below the nucleotide
sequence of 1855 base pairs. The active site residue (D) and flanking
amino acid residues characteristic of aspartyl proteases are underlined.
The putative propeptide is boxed. The putative signal peptide at
the N-terminus and the transmembrane domain at the C-terminus are
underlined. FIG. 2B. Expressed sequence tags (SEQ ID NO:27) extending
the nucleotide sequence of CSP56 to 2606 base pairs in length. FIG.
2C. Schematic representation of CSP56. SS, signal sequence; Pro,
propeptide; TM transmembrane domain. The asterisks indicate the
active sites.
FIG. 3. Multiple amino acid sequence alignment of CSP56 (SEQ ID
NO:19) with other members of the pepsin family of aspartyl proteases.
Identical amino acid residues are indicated by black boxes. The
aspartyl protease active residues (D-S/T-G) are indicated by a bar
on top. The cysteine residues characteristic for aspartyl protease
in members of the pepsin family are indicted by asterisks. The putative
membrane attachment domain is underlined. Gaps are indicated by
dots. Cat-E, cathepsin E (SEQ ID NO:22); Pep-A, pepsinogen A (SEQ
ID NO:23); Pep-C, pepsinogen C (SEQ ID NO:24); Cat-D, cathepsin
D (SEQ ID NO:25); and Renin (SEQ ID NO:26).
FIG. 4. CSP56 expression in primary tumor and metastases isolated
from scid mice. Northern blot analysis using RNA isolated from primary
tumors (PT) and metastatic tissues (Met) of mice injected with different
human breast cancer cell lines. Equal amounts of RNA in each lane
were loaded as judged by staining of the membrane with methylene
blue and hybridization of the membrane with a human .beta.-action
probe.
FIG. 5. CSP56 is up-regulated in patient breast tumor samples.
FIG. 5A. Northern blot analysis using RNA isolated from tumor and
normal breast tissue from the same patient. FIG. 5B. Northern blot
analysis using RNA isolated from three different human breast tumor
patients and normal breast tissue.
FIG. 6. In situ hybridization analysis of CSP56 expression in breast
and colon tumors. Adjacent or near-adjacent sections through normal
breast tissue (A-C) and the primary breast tissue (D-F) of one patient
and through normal colon tissue (G, H), the primary colon tumor
(J, K), and the liver metastatis (L, M) of another patient. Sections
A, D, G, J, and L were stained with haematoxylin and eosin (H &
E). Sections B, E, H, K, and M were hybridized with the antisense
CSP56 probe, and sections C and F were hybridized with the CSP56
sense control probe. d, lactiferous duct; f, fatty connective tissue;
ly, lymphocytes; m, colon mucosa; met, metastatic tissue; PT, primary
tumor; st, stroma; tc, tumor cells.
FIG. 7. Expression of CSP56 in human tissues. RNA blot analysis
depicting two CSP56 transcripts of 2.0 kb and 2.5 kb in various
human tissues. sk. muscle, skeletal muscle; sm. intestine, small
intestine; p.b. lymphocytes, peripheral blood lymphocytes.
DETAILED DESCRIPTION OF THE INVENTION
It is a discovery of the present invention that a number of genes
are differentially expressed between cancer cells and non-metastatic
cancer cells (Table 1). This information can be utilized to make
diagnostic reagents specific for the expression products of the
differentially displayed genes. It can also be used in diagnostic
and prognostic methods which will help clinicians in planning appropriate
treatment regimes for cancers, especially of the breast or colon.
Some of the metastatic markers disclosed herein, such as clone
122, are up-regulated in metastatic cells relative to non-metastatic
cells. Some of the metastatic markers, such as clones 337 and 280,
are down-regulated in metastatic cells relative to non-metastatic
cells. Identification of these relationships and markers permits
the formulation of reagents and methods as further described below.
In addition, homologies to known proteins have been identified which
suggest functions for the disclosed proteins. For example, transcript
280 is homologous to human N-acetylglucosamine-6-sulfatase precursor,
transcript 245 is homologous to bifunctional ATP sulfurylase-adenosine
5'-phosphosulfate kinase, and transcript 122 is homologous to human
pepsinogen c, an aspartyl protease.
It is another discovery of the present invention that a novel aspartyl-type
protease, CSP56, is over-expressed in highly metastatic cancer,
particularly in breast and colon cancer, and is associated with
the progression of primary tumors to a metastatic state. This information
can be utilized to make diagnostic reagents specific for expression
products of the CSP56 gene. It can also be used in diagnostic and
prognostic methods which will help clinicians to plan appropriate
treatment regimes for cancers, especially of the breast and colon.
The amino acid sequence of CSP56 protein is shown in SEQ ID NO:19.
Amino acid sequences encoded by novel polynucleotides of the invention
can be predicted by running a translation program for each of the
three reading frames for a particular polynucleotide sequence. A
metastatic marker protein encoded by a polynucleotide comprising
a nucleotide sequence as shown in SEQ ID NOS:1-17, the CSP56 protein
shown in SEQ ID NO:19, or naturally or non-naturally occurring biologically
active protein variants of metastatic marker proteins, including
CSP56, can be used in diagnostic and therapeutic methods of the
invention. Biologically active metastatic marker protein variants,
including CSP56 variants, retain the same biological activities
as the proteins encoded by polynucleotides comprising SEQ ID NOS:1-18.
Biological activities of metastatic marker proteins include differential
expression between tumors and normal tissue, particularly between
tumors with high metastatic potential and normal tissue. Biological
activity of CSP56 also includes the ability to permit metastases
and aspartyl-type protease activity.
Biological activity of a metastatic marker protein variant, including
a CSP56 variant, can be readily determined by one of skill in the
art. Differential expression of the variant, for example, can be
measured in cell lines which vary in metastatic potential, such
as the breast cancer cell lines MDA-MB-231 (Brinkley et al., Cancer
Res. 40, 3118-29, 1980), MDA-MB-435 (Brinkley et al., 1980), MCF-7,
BT-20, ZR-75-1, MDA-MB-157, MDA-MB-361, MDA-MB-453, Alab and MDA-MB-468,
or colon cancer cell lines Km12C and Km12L4A. The MDA-MB-231 cell
line was deposited at the ATCC on May 15, 1998 (ATCC CRL-12532).
The Km12C cell line was deposited at the ATCC on May 15, 1998 (ATCC-CRL-12533).
The Km12L4A cell line was deposited at the ATCC on Mar. 19, 1998
(ATCC CRL-12496). The MDA-MB-435 cell line was deposited at the
ATCC on Oct. 9, 1998 (ATCC CRL 12583). The MCF-7 cell line was deposited
at the ATCC on Oct. 9, 1998 (ATCC CRL-12584).
Expression in a non-cancerous cell line, such as the breast cell
line Hs58Bst, can be compared with expression in cancerous cell
lines. Alternatively, a breast cancer cell line with high metastatic
potential, such as MDA-MB-231 or MDA-MB-435, can be contacted with
a polynucleotide encoding a variant and assayed for lowered metastatic
potential, for example by monitoring cell division or protein or
DNA synthesis, as is known in the art. Aspartyl protease activity
of a potential CSP56 variant can also be measured, for example,
as taught in Wright et al., J. Prot. Chem. 16, 171-81 (1997).
Naturally occurring biologically active metastatic marker protein
variants, including variants of CSP56, are found in humans or other
species and comprise amino acid sequences which are substantially
identical to the amino acid sequences encoded by polynucleotides
comprising nucleotide sequences of SEQ ID NOS:1-18. Non-naturally
occurring biologically active metastatic marker protein variants
can be constructed in the laboratory, using standard recombinant
DNA techniques.
Preferably, naturally or non-naturally occurring biologically active
metastatic marker protein variants have amino acid sequences which
are at least 65%, 75%, 85%, 90%, or 95% identical to the amino acid
sequences encoded by polynucleotides comprising nucleotide sequences
of SEQ ID NOS:1-18 and have similar differential expression patterns,
though these properties may differ in degree. Naturally or non-naturally
occurring biologically active CSP56 variants also have aspartyl-type
protease activity. More preferably, the variants are at least 98%
or 99% identical. Percent sequence identity is determined using
computer programs which employ the Smith-Waterman algorithm using
an affine gap search with the following parameters: a gap open penalty
of 12 and a gap extension penalty of 1. The Smith-Waterman homology
search algorithm is taught in Smith and Waterman, Adv. Appl. Math.
(1981) 2:482-489.
Guidance in determining which amino acid residues may be substituted,
inserted, or deleted without abolishing biological or immunological
activity may be found using computer programs well known in the
art, such as DNASTAR software. Preferably, amino acid changes in
biologically active metastatic marker protein variants are conservative
amino acid changes, i.e., substitutions of similarly charged or
uncharged amino acids. A conservative amino acid change involves
substitution of one of a family of amino acids which are related
in their side chains. Naturally occurring amino acids are generally
divided into four families: acidic (aspartate, glutamate), basic
(lysine, arginine, histidine), non-polar (alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), and
uncharged polar (glycine, asparagine, glutamine, cystine, serine,
threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and
tyrosine are sometimes classified jointly as aromatic amino acids.
It is reasonable to expect that an isolated replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on
the biological properties of the resulting metastatic marker protein
variant. For example, isolated conservative amino acid substitutions
are not expected to have a major effect on the aspartyl protease
activity of CSP56, especially if the replacement is not at the catalytic
domains of the protease.
Metastatic marker protein variants also include allelic variants,
species variants, muteins, glycosylated forms, aggregative conjugates
with other molecules, and covalent conjugates with unrelated chemical
moieties which retain biological activity. Covalent metastatic marker
variants can be prepared by linkage of functionalities to groups
which are found in the amino acid chain or at the N- or C-terminal
residue, as is known in the art. Truncations or deletions of regions
which do not affect the expression patterns of metastatic marker
proteins or, for example, the aspartyl protease activity of CSP56,
are also biologically active variants.
A subset of mutants, called muteins, is a group of proteins in
which neutral amino acids, such as serine, are substituted for cysteine
residues which do not participate in disulfide bonds. These mutants
may be stable over a broader temperature range than naturally occurring
proteins. See Mark et al., U.S. Pat. No. 4,959,314.
Metastatic marker polypeptides contain fewer amino acids than full-length
metastatic marker proteins. Metastatic marker protein polypeptides
can contain at least 8, 10, 12, 15, 25, 50, 75, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, or 700 contiguous amino
acids encoded by a polynucleotide comprising SEQ ID NO:1; at least
8, 10, 12, 15, 25, 50, 75, 100, or 125 contiguous amino acids encoded
by a polynucleotide comprising SEQ ID NOS:2 or 9; at least 8, 10,
12, 15, 25, 50, 75, or 100 contiguous amino acids encoded by a polynucleotide
comprising SEQ ID NOS:3, 4, 5, 8, or 10; at least 8, 10, 12, 15,
25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, or 800 contiguous amino acids encoded by a polynucleotide
comprising SEQ ID NO:6; at least 8, 10, 12, 14, 25, 50, 55, or 60
contiguous amino acids encoded by a polynucleotide comprising SEQ
ID NO:7; 8, 10, 12, 15, 25, 50, 75, 100, 150, or 160 contiguous
amino acids encoded by a polynucleotide comprising SEQ ID NO:11;
at least 8, 10, 12, 15, 25, 50, 75, 100, 125, or 130 contiguous
amino acids encoded by a polynucleotide comprising SEQ ID NO:12;
at least 8, 10, 12, 15, 25, 50, 75, or 100 contiguous amino acids
encoded by a polynucleotide comprising SEQ ID NO:13; at least 8,
10, 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,
or 300 contiguous amino acids encoded by a polynucleotide comprising
SEQ ID NO:14; at least 8, 10, 12, 15, 25, 50, 75, 100, or 150 contiguous
amino acids encoded by a polynucleotide comprising SEQ ID NO:15;
at least 8, 10, 12, 15, 25, 50, 75, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,
1050, or 1100 contiguous amino acids encoded by a polynucleotide
comprising SEQ ID NO:16; or at least 8, 10, 12, 15, 25, 50, 75,
100, 150, 200, 250, 300, 350, 400, 450, or 500 contiguous amino
acids encoded by a polynucleotide comprising SEQ ID NO:17 in the
same order as found in the full-length protein or biologically active
variant. CSP56 polypeptides can contain at least 8, 10, 11, 12,
13, 14, 15, 16, 17, 20, 21, 23, 25, 28, 29, 30, 31, 32, 33, 35,
40, 50, 60, 75, 100, 111, 112, 120, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, or 500 or more amino acids
of a CSP56 protein or biologically active variant. Preferred CSP56
polypeptides comprise at least amino acids 106-115, 105-116, 104-117,
100-120, 297-306, 296-307, 295-308, 290-320, 8-20, 7-21, 6-22, 1-30,
461-489, 460-490, 459-491, and 407-518 of SEQ ID NO:19. Polypeptide
molecules having substantially the same amino acid sequence as the
amino acid sequences encoded by polynucleotides comprising nucleotide
sequences of SEQ ID NOS:1-18 thereof but possessing minor amino
acid substitutions which do not substantially affect the biological
properties of a particular metastatic marker polypeptide variant
are within the definition of metastatic marker polypeptides.
Metastatic marker proteins or polypeptides can be isolated from,
for example, human cells, using biochemical techniques well known
to the skilled artisan. A preparation of isolated and purified metastatic
marker protein is at least 80% pure; preferably, the preparations
are at least 90%, 95%, 98%, or 99% pure. Metastatic marker proteins
and polypeptides can also be produced by recombinant DNA methods
or by synthetic chemical methods. For production of recombinant
metastatic marker proteins or polypeptides, coding sequences selected
from SEQ ID NOS:1-18 can be expressed in known prokaryotic or eukaryotic
expression systems. Bacterial, yeast, insect, or mammalian expression
systems can be used, as is known in the art. Alternatively, synthetic
chemical methods, such as solid phase peptide synthesis, can be
used to synthesize metastatic marker protein or polypeptides. Biologically
active protein or polypeptide variants can be similarly produced.
Fusion proteins comprising contiguous amino acids of metastatic
marker proteins of the invention can also be constructed. Fusion
proteins are useful for generating antibodies against metastatic
marker protein amino acid sequences and for use in various assay
systems. For example, CSP56 fusion proteins can be used to identify
proteins which interact with CSP56 protein and influence, for example,
its aspartyl protease activity, its differential expression, or
its ability to permit metastases. Physical methods, such as protein
affinity chromatography, or library-based assays for protein-protein
interactions, such as the yeast two-hybrid or phage display systems,
can also be used for this purpose. Such methods are well known in
the art and can also be used as drug screens.
A fusion protein comprises two protein segments fused together
by means of a peptide bond. The first protein segment consists of
at least 8, 10, 12, 15, 25, 50, 75, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, or 700 contiguous amino acids encoded
by a polynucleotide comprising SEQ ID NO:1; at least 8, 10, 12,
15, 25, 50, 75, 100, or 125 contiguous amino acids encoded by a
polynucleotide comprising SEQ ID NOS:2 or 9; at least 8, 10, 12,
15, 25, 50, 75, or 100 contiguous amino acids encoded by a polynucleotide
comprising SEQ ID NOS:3, 4, 5, 8, or 10; at least 8, 10, 12, 15,
25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, or 800 contiguous amino acids encoded by a polynucleotide
comprising SEQ ID NO:6; at least 8, 10, 12, 14, 25, 50, 55, or 60
contiguous amino acids encoded by a polynucleotide comprising SEQ
ID NO:7; 8, 10, 12, 15, 25, 50, 75, 100, 150, or 160 contiguous
amino acids encoded by a polynucleotide comprising SEQ ID NO:11;
at least 8, 10, 12, 15, 25, 50, 75, 100, 125, or 130 contiguous
amino acids encoded by a polynucleotide comprising SEQ ID NO:12;
at least 8, 10, 12, 15, 25, 50, 75, or 100 contiguous amino acids
encoded by a polynucleotide comprising SEQ ID NO:13; at least 8,
10, 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,
or 300 contiguous amino acids encoded by a polynucleotide comprising
SEQ ID NO:14; at least 8, 10, 12, 15, 25, 50, 75, 100, or 150 contiguous
amino acids encoded by a polynucleotide comprising SEQ ID NO:15;
at least 8, 10, 12, 15, 25, 50, 75, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,
1050, or 1100 contiguous amino acids encoded by a polynucleotide
comprising SEQ ID NO:16 ; or at least 8, 10, 12, 15, 25, 50, 75,
100, 150, 200, 250, 300, 350, 400, 450, or 500 contiguous amino
acids encoded by a polynucleotide comprising SEQ ID NO:17, or at
least 8, 10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 23, 25, 28, 29,
30, 31, 32, 33, 35, 40, 50, 60, 75, 100, 111, 112, 120, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500
contiguous amino acids of a CSP56 protein. The amino acids can be
selected from the amino acid sequences encoded by polynucleotides
comprising SEQ ID NOS:1-18 or from a biologically active variants
of those sequences. The first protein segment can also be a full-length
metastatic marker protein. The first protein segment can be N-terminal
or C-terminal, as is convenient.
The second protein segment can be a full-length protein or a protein
fragment or polypeptide. Proteins commonly used in fusion protein
construction include .beta.-galactosidase, .beta.-glucuronidase,
green fluorescent protein (GFP), autofluorescent proteins, including
blue fluorescent protein (BFP), glutathione-S-transferase (GST),
luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase
(CAT). Additionally, epitope tags are used in fusion protein constructions,
including histidine (His) tags, FLAG tags, influenza hemagglutinin
(HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other
fusion constructions can include maltose binding protein (MBP),
S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding
domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
These fusions can be made, for example, by covalently linking two
protein segments or by standard procedures in the art of molecular
biology. Recombinant DNA methods can be used to prepare fusion proteins,
for example, by making a DNA construct which comprises coding sequences
selected from SEQ ID NOS:1-18 in proper reading frame with nucleotides
encoding the second protein segment and expressing the DNA construct
in a host cell, as is known in the art. Many kits for constructing
fusion proteins are available from companies that supply research
labs with tools for experiments, including, for example, Promega
Corporation (Madison, Wiss.), Stratagene (La Jolla, Calif.), Clontech
(Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.),
MBL International Corporation (MIC; Watertown, Mass.), and Quantum
Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
Isolated metastatic marker proteins, polypeptides, biologically
active variants, or fusion proteins can be used as immunogens, to
obtain a preparation of antibodies which specifically bind to epitopes
of metastatic marker protein. The antibodies can be used, inter
alia, to detect metastatic marker proteins, such as CSP56, in human
tissue, particularly in human tumors, or in fractions thereof. The
antibodies can also be used to detect the presence of mutations
in metastatic marker protein genes, such as the CSP56 gene, which
result in under- or over-expression of a metastatic marker protein
or in expression of a metastatic marker protein with altered size
or electrophoretic mobility. By binding to CSP56, for example, antibodies
can also prevent CSP56 aspartyl-type protease activity or the ability
of CSP56 to permit metastases.
Antibodies which specifically bind to epitopes of metastatic marker
proteins, polypeptides, fusion proteins, or biologically active
variants can be used in immunochemical assays, including but not
limited to Western Blots, ELISAs, radioimmunoassays, immunohistochemical
assays, immunoprecipitations, or other immunochemical assays known
in the art. Typically, antibodies of the invention provide a detection
signal at least 5-, 10-, or 20-fold higher than a detection signal
provided with other proteins when used in such immunochemical assays.
Preferably, antibodies which specifically bind to epitopes of a
particular metastatic marker protein do not detect other proteins
in immunochemical assays and can immunoprecipitate that metastatic
marker protein or polypeptide fragments of the metastatic marker
protein from solution.
Metastatic marker protein-specific antibodies specifically bind
to epitopes present in a metastatic marker protein having an amino
acid sequence encoded by a polynucleotide comprising a nucleotide
sequence of SEQ ID NOS:1-18 or to biologically active variants of
those amino acid sequences. Typically, at least 6, 8, 10, or 12
contiguous amino acids are required to form an epitope. However,
epitopes which involve non-contiguous amino acids may require more,
e.g., at least 15, 25, or 50 amino acids. Preferably, metastatic
marker protein epitopes are not present in other human proteins.
Epitopes of a metastatic marker protein which are particularly
antigenic can be selected, for example, by routine screening of
polypeptide fragments of the metastatic marker protein for antigenicity
or by applying a theoretical method for selecting antigenic regions
of a protein to the amino acid sequence of the metastatic marker
protein. Such methods are taught, for example, in Hopp and Wood,
Proc. Natl. Acad. Sci. U.S.A. 78, 3824-28 (1981), Hopp and Wood,
Mol. Immunol. 20, 483-89 (1983), and Sutcliffe et al., Science 219,
660-66 (1983). By reference to FIG. 3, antigenic regions of CSP56
which could also bind to antibodies which crossreact with other
aspartyl proteases can be avoided.
Any type of antibody known in the art can be generated to bind
specifically to metastatic marker protein epitopes. For example,
preparations of polyclonal and monoclonal antibodies can be made
using standard methods which are well known in the art. Similarly,
single-chain antibodies can also be prepared. Single-chain antibodies
which specifically bind to metastatic marker protein epitopes can
be isolated, for example, from single-chain immunoglobulin display
libraries, as is known in the art. The library is "panned"
against a metastatic marker protein amino acid sequence, and a number
of single chain antibodies which bind with high-affinity to different
epitopes of the metastatic marker protein can be isolated. Hayashi
et al., 1995, Gene 160:129-30. Single-chain antibodies can also
be constructed using a DNA amplification method, such as the polymerase
chain reaction (PCR), using hybridoma cDNA as a template. Thirion
et al., 1996, Eur. J. Cancer Prev. 5:507-11.
Single-chain antibodies can be mono- or bispecific, and can be
bivalent or tetravalent. Construction of tetravalent, bispecific
single-chain antibodies is taught, for example, in Coloma and Morrison,
1997, Nat. Biotechnol. 15:159-63. Construction of bivalent, bispecific
single-chain antibodies is taught inter alia in Mallender and Voss,
1994, J. Biol. Chem. 269:199-206.
A nucleotide sequence encoding a single-chain antibody can be constructed
using manual or automated nucleotide synthesis, cloned into an expression
construct using standard recombinant DNA methods, and introduced
into a cell to express the coding sequence, as described below.
Alternatively, single-chain antibodies can be produced directly
using, for example, filamentous phage technology. Verhaar et al.,
1995, Int. J. Cancer 61:497-501; Nicholls et al., 1993, J. Immunol.
Meth. 165:81-91.
Monoclonal and other antibodies can also be "humanized"
in order to prevent a patient from mounting an immune response against
the antibody when it is used therapeutically. Such antibodies may
be sufficiently similar in sequence to human antibodies to be used
directly in therapy or may require alteration of a few key residues.
Sequence differences between, for example, rodent antibodies and
human sequences can be minimized by replacing residues which differ
from those in the human sequences, for example, by site directed
mutagenesis of individual residues, or by grating of entire complementarity
determining regions. Alternatively, one can produce humanized antibodies
using recombinant methods, as described in GB2188638B. Antibodies
which specifically bind to epitopes of a metastatic marker protein
can contain antigen binding sites which are either partially or
fully humanized, as disclosed in U.S. Pat. No. 5,565,332.
Other types of antibodies can be constructed and used therapeutically
in methods of the invention. For example, chimeric antibodies can
be constructed as disclosed, for example, in WO 93/03151. Binding
proteins which are derived from immunoglobulins and which are multivalent
and multispecific, such as the "diabodies" described in
WO 94/13804, can also be prepared.
Antibodies of the invention can be purified by methods well known
in the art. For example, antibodies can be affinity purified by
passing the antibodies over a column to which a metastatic marker
protein, polypeptide, variant, or fusion protein is bound. The bound
antibodies can then be eluted from the column, using a buffer with
a high salt concentration.
The invention also provides subgenomic polynucleotides which: encode
metastatic marker proteins, polypeptides, variants, or fusion proteins.
Subgenomic polynucleotides contain less than a whole chromosome.
Preferably, the subgenomic polynucleotides are intron-free. An isolated
metastatic marker protein subgenomic polynucleotide comprises at
least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,
40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,
1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, or 2200 contiguous
nucleotides of SEQ ID NO:1; at least 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200,
250, 300, 350, or 400 contiguous nucleotides of SEQ ID NOS:2 or
9; at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,
30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,
1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,
1700, 1750, 1800, 1850, 1900, 1950, 2000, 2250, or 2500 contiguous
nucleotides of SEQ ID NO:6; at least 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 contiguous
nucleotides of SEQ ID NO:7, at least 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200,
250, 300, or 350 contiguous nucleotides of SEQ ID NO:8; at least
8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50,
75, 100, 125, 150, 175, 200, 250, 300, or 350 contiguous nucleotides
of SEQ ID NO:12; at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, or
300 contiguous nucleotides of SEQ ID NOS:3, 4, 5, or 10; at least
8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50,
75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 contiguous
nucleotides of SEQ ID NO:11; at least 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175,
200, 250, or 300 contiguous nucleotides of SEQ ID NO:13; at least
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50,
75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, or 950 contiguous nucleotides
of SEQ ID NO:14; at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300,
350, 400, or 450 contiguous nucleotides of SEQ ID NO:15; at least
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50,
75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,
1750, 1800, 1850, 1900, 1950, 2000, 2250, 2500, 2750, 3000, 3250,
or 3500 contiguous nucleotides of SEQ ID NO:16; or at least 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75,
100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,
1250, 1300, 1350, 1400, 1450, or 1500 contiguous nucleotides of
SEQ ID NO:17 or can comprise one of SEQ ID NOS:1-17.
A CSP56 polynucleotide can comprise a contiguous sequence of at
least 10, 11, 12, 15, 20, 24, 25, 30, 32, 33, 35, 36, 40, 42, 45,
48, 50, 51, 54, 60, 63, 69, 70, 74, 75, 80, 84, 87, 90, 93, 96,
99, 100, 105, 114, 120, 125, 150, 225, 300, 333, 336, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,
1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,
1650, 1700, 1750, 1800, or 1850 nucleotides selected from SEQ ID
NO:18 or can comprise SEQ ID NO:18. An isolated CSP56 polynucleotide
encodes at least 8, 10, 12, 14, 15, 17, 18, 20, 25, 29, 30, 31,
32, 40, 50, 75, 100 or 111 contiguous amino acids of SEQ ID NO:19
and can encode the entire amino acid sequence shown in SEQ ID NO:19.
Preferred CSP56 polynucleotides encode at least amino acids 1-30,
8-20, 7-21, 6-22, 106-115, 105-116, 104-117, 100-120, 297-306, 296-307,
295-308, 290-320, 461-489, 460-490, 459-491, and 407-518 of SEQ
ID NO:19.
The complements of the nucleotide sequences shown in SEQ ID NOS:1-18
are contiguous nucleotide sequences which form Watson-Crick base
pairs with a contiguous nucleotide sequence as shown in SEQ ID NOS:1-18.
The complements of SEQ ID NOS:1-18 are also polynucleotides of the
invention. Complements of coding sequences can be used to provide
antisense oligonucleotides and probes. Antisense oligonucleotides
and probes of the invention can consist of at least 11, 12, 15,
20, 25, 30, 50, or 100 contiguous nucleotides. A complement of an
entire coding sequence can also be used. Double-stranded polynucleotides
which comprise all or a portion of the nucleotide sequences shown
in SEQ ID NOS:1-18, as well as polynucleotides which encode metastatic
marker protein-specific antibodies or ribozymes, are also polynucleotides
of the invention.
Degenerate nucleotide sequences encoding amino acid sequences of
metastatic marker proteins and or variants, as well as homologous
nucleotide sequences which are at least 65%, 75%, 85%, 90%, 95%,
98%, or 99% identical to the nucleotide sequences shown in SEQ ID
NOS:1-18, are also polynucleotides of the invention. Percent sequence
identity can be determined using computer programs which employ
the Smith-Waterman algorithm, for example as implemented in the
MPSRCH program (Oxford Molecular), using an affine gap search with
the following parameters: a gap open penalty of 12 and a gap extension
penalty of 1.
Typically, homologous polynucleotide sequences of the invention
can be confirmed by hybridization under stringent conditions, as
is known in the art. For example, using the following wash conditions--2.times.SSC,
0.1% SDS, room temperature twice, 30 minutes each; then 2.times.SSC,
0.1% SDS, 50.degree. C. once for 30 minutes; then 2.times.SSC, room
temperature twice, 10 minutes each--homologous sequences can be
identified that contain at most about 25-30% basepair mismatches.
More preferably, homologous nucleic acid strands contain 15-25%
basepair mismatches, even more preferably 5-15%, 2-10%, or 1-5%
basepair mismatches. Degrees of homology of polynucleotides of the
invention can be selected by varying the stringency of the wash
conditions for identification of clones from gene libraries (or
other sources of genetic material), as is well known in the art
and described, for example, in manuals such as. Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989).
Species homologs of subgenomic polynucleotides of the invention
can also be identified by making suitable probes or primers and
screening cDNA expression libraries or genomic libraries from other
species, such as mice, monkeys, yeast, or bacteria. Complete polynucleotide
sequences can be obtained by chromosome walking, screening of libraries
for overlapping clones, 5' RACE, or other techniques well known
in the art. It is well known that the T.sub.m of a double-stranded
DNA decreases by 1-1.5.degree. C. with every 1% decrease in homology
(Bonner et al., J. Mol. Biol. 81, 123 (1973). Homologous human polynucleotides
or polynucleotides of other species can therefore be identified,
for example, by hybridizing a putative homologous polynucleotide
with a polynucleotide having a nucleotide sequence of SEQ ID NOS:1-18,
comparing the melting temperature of the test hybrid with the melting
temperature of a hybrid comprising a polynucleotide having a nucleotide
sequence of SEQ ID NOS:1-18 and a polynucleotide which is perfectly
complementary to the nucleotide sequence, and calculating the number
of basepair mismatches within the test hybrid.
Nucleotide sequences which hybridize to the nucleotide sequences
shown in SEQ ID NOS:1-18 following stringent hybridization and/or
wash conditions are also subgenomic polynucleotides of the invention.
Stringent wash conditions are well known and understood in the art
and are disclosed, for example, in Sambrook et al., 1989, at pages
9.50-9.51.
Typically, for stringent hybridization conditions a combination
of temperature and salt concentration should be chosen that is approximately
12-20.degree. C. below the calculated T.sub.m of the hybrid under
study. The T.sub.m of a hybrid between a polynucleotide sequence
shown in SEQ ID NOS:1-18 and a polynucleotide sequence which is
65%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that
sequence can be calculated, for example, using the equation of Bolton
and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): T.sub.m=81.5.degree.
C.-16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-0.63(% formamide)-600/l),
where l=the length of the hybrid in basepairs. Stringent wash conditions
include, for example, 4.times.SSC at 65.degree. C., or 50% formamide,
4.times.SSC at 42.degree. C., or 0.5.times.SSC, 0.1% SDS at 65.degree.
C. Highly stringent wash conditions include, for example, 0.2.times.SSC
at 65.degree. C.
Subgenomic polynucleotides can be purified free from other nucleotide
sequences using standard nucleic acid purification techniques. For
example, restriction enzymes and probes can be used to isolate polynucleotides
which comprise nucleotide sequences encoding metastatic marker proteins.
Alternatively, PCR can be used to synthesize and amplify such polynucleotides.
At least 90% of a preparation of isolated and purified polynucleotides
comprises metastatic marker protein encoding polynucleotides.
Complementary DNA (cDNA) molecules which encode metastatic marker
proteins are also subgenomic polynucleotides of the invention. cDNA
molecules can be made with standard molecular biology techniques,
using mRNA as a template. cDNA molecules can thereafter be replicated
using molecular biology techniques known in the art and disclosed
in manuals such as Sambrook et al., 1989. An amplification technique,
such as the polymerase chain reaction (PCR), can be used to obtain
additional copies of subgenomic polynucleotides of the invention,
using either human genomic DNA or cDNA as a template.
Alternatively, synthetic chemistry techniques can be used to synthesize
subgenomic polynucleotide molecules of the invention. The degeneracy
of the genetic code allows alternate nucleotide sequences to be
synthesized which will encode a metastatic marker protein having
an amino acid sequence encoded by a polynucleotide comprising a
nucleotide sequence selected from SEQ ID NOS:1-17, a CSP56 amino
acid sequence as shown in SEQ ID NO:19, or a biologically active
variant of those sequences. All such nucleotide sequences are within
the scope of the present invention.
The invention also provides polynucleotide probes which can be
used to detect metastatic marker polypeptide sequences, for example,
in hybridization protocols such as Northern or Southern blotting
or in situ hybridizations. Polynucleotide probes of the invention
comprise at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40
or more contiguous nucleotides selected from SEQ ID NOS:1-18. Polynucleotide
probes of the invention can comprise a detectable label, such as
a radioisotopic, fluorescent, enzymatic, or chemiluminescent label.
Isolated polynucleotides can be used, for example, as primers to
obtain additional copies of the polynucleotides or as probes for
detecting mRNA. Polynucleotides can also be used to express metastatic
marker protein mRNA, protein, polypeptides, biologically active
variants, single-chain antibodies, ribozymes, or fusion proteins.
Any of the polynucleotides described above can be present in a
construct, such as a DNA or RNA construct. The construct can be
a vector and can be used to transfer the polynucleotide into a cell,
for example, for propagation of the polynucleotide. Constructs can
be linear or circular molecules. They can be on autonomously replicating
molecules or on molecules without replication sequences, and they
can be regulated by their own or by other regulatory sequences,
as is known in the art.
A construct can also be an expression construct. An expression
construct comprises a promoter which is functional in a selected
host cell. For example, the skilled artisan can readily select an
appropriate promoter from the large number of cell type-specific
promoters known and used in the art. The expression construct can
also contain a transcription terminator which is functional in the
host cell. The expression construct comprises a polynucleotide segment
which encodes, for example, all or a portion of a metastatic marker
protein, polypeptide, biologically active variant, antibody, ribozyme,
or fusion protein. The polynucleotide segment is located downstream
from the promoter. Transcription of the polynucleotide segment initiates
at the promoter. The expression construct can be linear or circular
and can contain sequences, if desired, for autonomous replication.
Subgenomic polynucleotides can be propagated in vectors and cell
lines using techniques well known in the art. Expression systems
in bacteria include those described in Chang et al., Nature (1978)
275: 615, Goeddel et al., Nature (1979) 281: 544, Goeddel et al.,
Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S. Pat. No. 4,551,433,
deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25, and
Siebenlist et al., Cell (1980) 20: 269.
Expression systems in yeast include those described in Hinnen et
al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al., J.
Bacteriol. (1983) 153: 163; Kurtz et al., Mol. Cell. Biol. (1986)
6: 142; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Gleeson
et al., J. Gen. Microbiol. (1986) 132: 3459, Roggenkamp et al.,
Mol. Gen. Genet. (1986) 202 :302) Das et al., J. Bacteriol. (1984)
158: 1165; De Louvencourt et al., J. Bacteriol. (1983) 154: 737,
Van den Berg et al., Bio/Technology (1990) 8: 135; Kunze et al.,
J. Basic Microbiol. (1985) 25: 141; Cregg et al., Mol. Cell. Biol.
(1985) 5: 3376, U.S. Pat. Nos. 4,837,148, 4,929,555; Beach and Nurse,
Nature (1981) 300: 706; Davidow et al., Curr. Genet. (1985)10: 380,
Gaillardin et al., Curr. Genet. (1985) 10: 49, Ballance et al.,
Biochem. Biophys. Res. Commun. (1983) 112: 284-289; Tilburn et al.,
Gene (1983) 26: 205-221, Yelton et al., Proc. Natl. Acad. Sci. USA
(1984) 81: 1470-1474, Kelly and Hynes, EMBO J. (1985) 4: 475479;
EP 244,234, and WO 91/00357.
Expression of subgenomic polynucleotides in insects can be accomplished
as described in U.S. Pat. No. 4,745,051, Friesen et al. (1986) "The
Regulation of Baculovirus Gene Expression" in: THE MOLECULAR
BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476,
and Vlak et al., J. Gen. Virol. (1988) 69: 765-776, Miller et al.,
Ann. Rev. Microbiol. (1988) 42: 177, Carbonell et al., Gene (1988)
73: 409, Maeda et al., Nature (1985) 315: 592-594, Lebacq-Verheyden
et al., Mol. Cell. Biol. (1988) 8: 3129; Smith et al., Proc. Natl.
Acad. Sci. USA (1985) 82: 8404, Miyajima et al., Gene (1987) 58:
273; and Martin et al., DNA (1988) 7:99. Numerous baculoviral strains
and variants and corresponding permissive insect host cells from
hosts are described in Luckow et al., Bio/Technology (1988) 6: 47-55,.
Miller et al., in GENETIC ENGINEERING (Setlow, J. K. et al. eds.),
Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al.,
Nature, (1985) 315: 592-594.
Mammalian expression of subgenomic polynucleotides can be accomplished
as described in Dijkema et al., EMBO J. (1985) 4: 761, Gorman et
al., Proc. Natl. Acad. Sci. USA (1982b) 79: 6777, Boshart et al.,
Cell (1985) 41: 521 and U.S. Pat. No. 4,399,216. Other features
of mammalian expression can be facilitated as described in Ham and
Wallace, Meth. Enz. (1979) 58: 44, Barnes and Sato, Anal. Biochem.
(1980) 102: 255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,
4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.
Subgenomic polynucleotides can be on linear or circular molecules.
They can be on autonomously replicating molecules or on molecules
without replication sequences. They can be regulated by their own
or by other regulatory sequences, as is known in the art. Subgenomic
polynucleotides can be introduced into suitable host cells using
a variety of techniques which are available in the art, such as
transferrin-polycation-mediated DNA transfer, transfection with
naked or encapsulated nucleic acids, liposome-mediated DNA transfer,
intracellular transportation of DNA-coated latex beads, protoplast
fusion, viral infection, electroporation, and calcium phosphate-mediated
transfection.
Polynucleotides of the invention can also be used in gene delivery
vehicles, for the purpose of delivering an mRNA or oligonucleotide
(either with the sequence of a native mRNA or its complement), full-length
protein, fusion protein, polypeptide, or ribozyme, or single-chain
antibody, into a cell, preferably a eukaryotic cell. According to
the present invention, a gene delivery vehicle can be, for example,
naked plasmid DNA, a viral expression vector comprising a polynucleotide
of the invention, or a polynucleotide of the invention in conjunction
with a liposome or a condensing agent.
In one embodiment of the invention, the gene delivery vehicle comprises
a promoter and one of the polynucleotides disclosed herein. Preferred
promoters are tissue-specific promoters and promoters which are
activated by cellular proliferation, such as the thymidine kinase
and thymidylate synthase promoters. Other preferred promoters include
promoters which are activatable by infection with a virus, such
as the .alpha.- and .beta.-interferon promoters, and promoters which
are activatable by a hormone, such as estrogen. Other promoters
which can be used include the Moloney virus LTR, the CMV promoter,
and the mouse albumin promoter.
A gene delivery vehicle can comprise viral sequences such as a
viral origin of replication or packaging signal. These viral sequences
can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus,
papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, retrovirus,
togavirus or adenovirus. In a preferred embodiment, the gene delivery
vehicle is a recombinant retroviral vector. Recombinant retroviruses
and various uses thereof have been described in numerous references
including, for example, Mann et al., Cell 33:153, 1983, Cane and
Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al.,
Human Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712, 4,861,719,
and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349,
and WO 90/02,806. Numerous retroviral gene delivery vehicles can
be utilized in the present invention, including for example those
described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698;
WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile
and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer
Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamiya
et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg.
79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242
and WO91/02805).
Particularly preferred retroviruses are derived from retroviruses
which include avian leukosis virus (ATCC Nos. VR-535 and VR-247),
bovine leukemia virus (VR-1315), murine leukemia virus (MLV), mink-cell
focus-inducing virus (Koch et al., J. Vir. 49:828, 1984; and Oliff
et al., J. Vir. 48:542, 1983), murine sarcoma virus (ATCC Nos. VR-844,
45010 and 45016), reticuloendotheliosis virus (ATCC Nos VR-994,
VR-770 and 45011), Rous sarcoma virus, Mason-Pfizer monkey virus,
baboon endogenous virus, endogenous feline retrovirus (e.g. RD114),
and mouse or rat gL30 sequences used as a retroviral vector.
Particularly preferred strains of MLV from which recombinant retroviruses
can be generated include 4070A and 1504A (Hartley and Rowe, J. Vir.
19:19, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245),
Graffi (Ru et al., J. Vir. 67:4722, 1993; and Yantchev Neoplasma
26:397, 1979), Gross (ATCC No. VR-590), Kirsten (Albino et al.,
J. Exp. Med. 164:1710, 1986), Harvey sarcoma virus (Manly et al.,
J. Vir. 62:3540, 1988; and Albino et al., J. Exp. Med. 164:1710,
1986) and Rauscher (ATCC No. VR-998), and Moloney MLV (ATCC No.
VR-190).
A particularly preferred non-mouse retrovirus is Rous sarcoma virus.
Preferred Rous sarcoma viruses include Bratislava (Manly et al.,
J. Vir. 62:3540, 1988; and Albino et al., J. Exp. Med. 164:1710,
1986), Bryan high titer (e.g., ATCC Nos. VR-334, VR-657, VR-726,
VR-659, and VR-728), Bryan standard (ATCC No. VR-140), Carr-Zilber
(Adgighitov et al., Neoplasma 27:159, 1980), Engelbreth-Holm (Laurent
et al., Biochem Biophys Acta 908:241, 1987), Harris, Prague (e.g.,
ATCC Nos. VR-772, and 45033), and Schmidt-Ruppin (e.g. ATCC Nos.
VR-724, VR-725, VR-354) viruses.
Any of the above retroviruses can be readily utilized in order
to assemble or construct retroviral gene delivery vehicles given
the disclosure provided herein and standard recombinant techniques
(e.g., Sambrook et al., 1989, and Kunkle, Proc. Natl. Acad. Sci.
U.S.A. 82:488, 1985) known in the art. Portions of retroviral expression
vectors can be derived from different retroviruses. For example,
retrovector LTRs can be derived from a murine sarcoma virus, a tRNA
binding site from a Rous sarcoma virus, a packaging signal from
a murine leukemia virus, and an origin of second strand synthesis
from an avian leukosis virus. These recombinant retroviral vectors
can be used to generate transduction competent retroviral vector
particles by introducing them into appropriate packaging cell lines
(see Ser. No. 07/800,921, filed Nov. 29, 1991). Recombinant retroviruses
can be produced which direct the site-specific integration of the
recombinant retroviral genome into specific regions of the host
cell DNA. Such site-specific integration can be mediated by a chimeric
integrase incorporated into the retroviral particle (see Ser. No.
08/445,466 filed May 22, 1995). It is preferable that the recombinant
viral gene delivery vehicle is a replication-defective recombinant
virus.
Packaging cell lines suitable for use with the above-described
retroviral gene delivery vehicles can be readily prepared (see Ser.
No. 08/240,030, filed May 9, 1994; see also WO 92/05266) and used
to create producer cell lines (also termed vector cell lines or
"VCLs") for production of recombinant viral particles.
In particularly preferred embodiments of the present invention,
packaging cell lines are made from human (e.g., mHT1080 cells) or
mink parent cell lines, thereby allowing production of recombinant
retroviral gene delivery vehicles which are capable of surviving
inactivation in human serum. The construction of recombinant retroviral
gene delivery vehicles is described in detail in WO 91/02805. These
recombinant retroviral gene delivery vehicles can be used to generate
transduction competent retroviral particles by introducing them
into appropriate packaging cell lines (see Ser. No. 07/800,921).
Similarly, adenovirus gene delivery vehicles can also be readily
prepared and utilized given the disclosure provided herein (see
also Berkner, Biotechniques 6:616-627, 1988, and Rosenfeld et al.,
Science 252:431-434, 1991, WO 93/07283, WO 93/06223, and WO 93/07282).
A gene delivery vehicle can also be a recombinant adenoviral gene
delivery vehicle. Such vehicles can be readily prepared and utilized
given the disclosure provided herein (see Berkner, Biotechniques
6:616, 1988, and Rosenfeld et al., Science 252:431, 1991, WO 93/07283,
WO 93/06223, and WO 93/07282). Adeno-associated viral gene delivery
vehicles can also be constructed and used to deliver proteins or
polynucleotides of the invention to cells in vitro or in vivo. The
use of adeno-associated viral gene delivery vehicles in vitro is
described in Chatterjee et al., Science 258: 1485-1488 (1992), Walsh
et al., Proc. Nat'l. Acad. Sci. 89: 7257-7261 (1992), Walsh et al.,
J. Clin. Invest. 94: 1440-1448 (1994), Flotte et al., J. Biol. Chem.
268: 3781-3790 (1993), Ponnazhagan et al., J. Exp. Med. 179: 733-738
(1994), Miller et al., Proc. Nat'l Acad. Sci. 91: 10183-10187 (1994),
Einerhand et al., Gene Ther. 2: 336-343 (1995), Luo et al., Exp.
Hematol. 23: 1261-1267 (1995), and Zhou et al., Gene Therapy 3:
223-229 (1996). In vivo use of these vehicles is described in Flotte
et al., Proc. Nat'l Acad. Sci. 90: 10613-10617 (1993), and Kaplitt
et al., Nature Genet. 8:148-153 (1994).
In another embodiment of the invention, a gene delivery vehicle
is derived from a togavirus. Preferred togaviruses include alphaviruses,
in particular those described in U.S. Ser. No. 08/405,627, filed
Mar. 15, 1995, WO 95/07994. Alpha viruses, including Sindbis and
ELVS viruses can be gene delivery vehicles for polynucleotides of
the invention. Alpha viruses are described in WO 94/21792, WO 92/10578
and WO 95/07994. Several different alphavirus gene delivery vehicle
systems can be constructed and used to deliver polynucleotides to
a cell according to the present invention. Representative examples
of such systems include those described in U.S. Pat. Nos. 5,091,309
and 5,217,879. Particularly preferred alphavirus gene delivery vehicles
for use in the present invention include those which are described
in WO 95/07994, and U.S. Ser. No. 08/405,627.
Preferably, the recombinant viral vehicle is a recombinant alphavirus
viral vehicle based on a Sindbis virus. Sindbis constructs, as well
as numerous similar constructs, can be readily prepared essentially
as described in U.S. Ser. No. 08/198,450. Sindbis viral gene delivery
vehicles typically comprise a 5' sequence capable of initiating
Sindbis virus transcription, a nucleotide sequence encoding Sindbis
non-structural proteins, a viral junction region inactivated so
as to prevent fragment transcription, and a Sindbis RNA polymerase
recognition sequence. Optionally, the viral junction region can
be modified so that polynucleotide transcription is reduced, increased,
or maintained. As will be appreciated by those in the art, corresponding
regions from other alphaviruses can be used in place of those described
above.
The viral junction region of an alphavirus-derived gene delivery
vehicle can comprise a first viral junction region which has been
inactivated in order to prevent transcription of the polynucleotide
and a second viral junction region which has been modified such
that polynucleotide transcription is reduced. An alphavirus-derived
vehicle can also include a 5' promoter capable of initiating synthesis
of viral RNA from cDNA and a 3' sequence which controls transcription
termination.
Other recombinant togaviral gene delivery vehicles which can be
utilized in the present invention include those derived from Semliki
Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC
VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan
equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249;
ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309 and
5,217,879 and in WO 92/10578. The Sindbis vehicles described above,
as well as numerous similar constructs, can be readily prepared
essentially as described in U.S. Ser. No. 08/198,450.
Other viral gene delivery vehicles suitable for use in the present
invention include, for example, those derived from poliovirus (Evans
et al., Nature 339:385, 1989, and Sabin et al., J. Biol. Standardization
1:115,1973) (ATCC VR-58); rhinovirus (Arnold et al., J. Cell. Biochem.
L401, 1990) (ATCC VR-1110); pox viruses, such as canary pox virus
or vaccinia virus (Fisher-Hoch et al., PROC NATL. ACAD. SCI. U.S.A.
86:317, 1989; Flexner et al, Ann. N.Y. Acad. Sci. 569:86, 1989;
Flexner et al., Vaccine 8:17, 1990; U.S. Pat. No. 4,603,112 and
U.S. Pat. No. 4,769,330; WO 89/01973) (ATCC VR-111; ATCC VR-2010);
SV40 (Mulligan et al., Nature 277:108, 1979) (ATCC VR-305), (Madzak
et al., J. Gen. Vir. 73:1533, 1992); influenza virus (Luytjes et
al., Cell 59:1107, 1989; McMicheal et al., The New England Journal
of Medicine 309:13, 1983; and Yap et al., Nature 273:238, 1978)
(ATCC VR-797); parvovirus such as adeno-associated virus (Samulski
et al., J. Vir. 63:3822, 1989, and Mendelson et al., Virology 166:154,
1988) (ATCC VR-645); herpes simplex virus (Kit et al., Adv. Exp.
Med. Biol. 215:219, 1989) (ATCC VR-977; ATCC VR-260); Nature 277:
108, 1979); human immunodeficiency virus (EPO 386,882, Buchschacher
et al., J. Vir. 66:2731, 1992); measles virus (EPO 440,219) (ATCC
VR-24); A (ATCC VR-67; ATCC VR-1247), Aura (ATCC VR-368), Bebaru
virus (ATCC VR-600; ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya
virus (ATCC VR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924), Getah
virus (ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro
(ATCC VR-66), Mucambo virus (ATCC VR-580; ATCC VR-1244), Ndumu (ATCC
VR-371), Pixuna virus (ATCC VR-372; ATCC VR-1245), Tonate (ATCC
VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCC
VR-926), Y-62-33 (ATCC VR-375), O'Nyong virus, Eastern encephalitis
virus (ATCC VR-65; ATCC VR-1242), Western encephalitis virus (ATCC
VR-70; ATCC VR-1251; ATCC VR-622; ATCC VR-1252), and coronavirus
(Hamre et al., Proc. Soc. Exp. Biol. Med. 121:190, 1966) (ATCC VR-740).
A polynucleotide of the invention can also be combined with a condensing
agent to form a gene delivery vehicle. In a preferred embodiment,
the condensing agent is a polycation, such as polylysine, polyarginine,
polyornithine, protamine, spermine, spermidine, and putrescine.
Many suitable methods for making such linkages are known in the
art (see, for example, Ser. No. 08/366,787, filed Dec. 30, 1994).
In an alternative embodiment, a polynucleotide is associated with
a liposome to form a gene delivery vehicle. Liposomes are small,
lipid vesicles comprised of an aqueous compartment enclosed by a
lipid bilayer, typically spherical or slightly elongated structures
several hundred Angstroms in diameter. Under appropriate conditions,
a liposome can fuse with the plasma membrane of a cell or with the
membrane of an endocytic vesicle within a cell which has internalized
the liposome, thereby releasing its contents into the cytoplasm.
Prior to interaction with the surface of a cell, however, the liposome
membrane acts as a relatively impermeable barrier which sequesters
and protects its contents, for example, from degradative enzymes.
Because a liposome is a synthetic structure, specially designed
liposomes can be produced which incorporate desirable features.
See Stryer, Biochemistry, pp. 236-240, 1975 (W. H. Freeman, San
Francisco, Calif.); Szoka et al., Biochim. Biophys. Acta 600:1,
1980; Bayer et al., Biochim. Biophys. Acta. 550:464, 1979; Rivnay
et al., Meth. Enzymol. 149:119, 1987; Wang et al., Proc. Natl. Acad.
Sci. U.S.A. 84: 7851, 1987, Plant et al., Anal. Biochem. 176:420,
1989, and U.S. Pat. No. 4,762,915. Liposomes can encapsulate a variety
of nucleic acid molecules including DNA, RNA, plasmids, and expression
constructs comprising polynucleotides such those disclosed in the
present invention.
Liposomal preparations for use in the present invention include
cationic (positively charged), anionic (negatively charged) and
neutral preparations. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl.
Acad Sci. USA 84:7413-7416, 1987), mRNA (Malone et al., Proc. Natl.
Acad Sci. USA 86:6077-6081, 1989), and purified transcription factors
(Debs et al., J. Biol. Chem. 265:10189-10192, 1990), in functional
form. Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium
(DOTMA) liposomes are available under the trademark Lipofectin,
from GIBCO BRL, Grand Island, N.Y. See also Felgner et al., Proc.
Natl. Acad Sci. USA 91. 5148-5152.87, 1994. Other commercially available
liposomes include Transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
Other cationic liposomes can be prepared from readily available
materials using techniques well known in the art. See, e.g., Szoka
et al., Proc. Natl. Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092
for descriptions of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)
liposomes.
Similarly, anionic and neutral liposomes are readily available,
such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily
prepared using readily available materials. Such materials include
phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl
choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be mixed
with the DOTMA and DOTAP starting materials in appropriate ratios.
Methods for making liposomes using these materials are well known
in the art.
The liposomes can comprise multilammelar vesicles (MLVs), small
unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
The various liposome-nucleic acid complexes are prepared using methods
known in the art. See, e.g., Straubinger et al., METHODS OF IMMUNOLOGY
(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci.
USA 87:3410-3414, 1990; Papahadjopoulos et al., Biochim. Biophys.
Acta 394:483, 1975; Wilson et al., Cell 17:77, 1979; Deamer and
Bangham, Biochim. Biophys. Acta 443:629, 1976; Ostro et al., Biochem.
Biophys. Res. Commun. 76:836, 1977; Fraley et al., Proc. Natl. Acad.
Sci. USA 76:3348, 1979; Enoch and Strittmatter, Proc. Natl. Acad
Sci. USA 76:145, 1979; Fraley et al., J. Biol. Chem. 255:10431,
1980; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA 75:145,
1979; and Schaefer-Ridder et al., Science 215:166, 1982.
In addition, lipoproteins can be included with a polynucleotide
of the invention for delivery to a cell. Examples of such lipoproteins
include chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments,
or fusions of these proteins can also be used. Modifications of
naturally occurring lipoproteins can also be used, such as acetylated
LDL. These lipoproteins can target the delivery of polynucleotides
to cells expressing lipoprotein receptors. Preferably, if lipoproteins
are included with a polynucleotide, no other targeting ligand is
included in the composition.
In another embodiment, naked polynucleotide molecules are used
as gene delivery vehicles, as described in WO 90/11092 and U.S.
Pat. No. 5,580,859. Such gene delivery vehicles can be either DNA
or RNA and, in certain embodiments, are linked to killed adenovirus.
Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other suitable
vehicles include DNA-ligand (Wu et al., J. Biol. Chem. 264:16985-16987,
1989) lipid-DNA combinations (Feigner et al., Proc. Natl. Acad.
Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc. Natl.
Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams et
al., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).
One can increase the efficiency of naked polynucleotide uptake
into cells by coating the polynucleotides onto biodegradable latex
beads. This approach takes advantage of the observation that latex
beads, when incubated with cells in culture, are efficiently transported
and concentrated in the perinuclear region of the cells. The beads
will then be transported into cells when injected into muscle. Polynucleotide-coated
latex beads will be efficiently transported into cells after endocytosis
is initiated by the latex beads and thus increase gene transfer
and expression efficiency. This method can be improved further by
treating the beads to increase their hydrophobicity, thereby facilitating
the disruption of the endosome and release of polynucleotides into
the cytoplasm.
The invention also provides a method of detecting metastatic marker
genes expression in a biological sample, such as a tissue sample
of the breast or colon. Detection of metastatic marker genes expression
is useful, for example, for identifying metastatic tissue and identifying
metastatic potential of a tissue, to identify patients who are at
risk for developing metastatic cancers in other organs of the body.
The tissue sample can be, for example, a solid tissue or a fluid
sample. Protein or nucleic acid expression products can be detected
in the tissue sample. In one embodiment, the tissue sample is assayed
for the presence of a metastatic marker proteins. The metastatic
marker protein has a sequence encoded by polynucleotides comprising
SEQ ID NOS:1-18 and can be detected using the metastatic marker
protein-specific antibodies of the present invention. The antibodies
can be labeled, for example, with a radioactive, fluorescent, biotinylated,
or enzymatic tag and detected directly, or can be detected using
indirect immunochemical methods, using a labeled secondary antibody.
The presence of the metastatic marker proteins can be assayed, for
example, in tissue sections by immunocytochemistry, or in lysates,
using Western blotting, as is known in the art.
In another embodiment, the tissue sample is assayed for the presence
of metastatic marker protein mRNA. Metastatic marker protein mRNA
can be detected by in situ hybridization in tissue sections or in
Northern blots containing poly A+ mRNA. Metastatic marker protein-specific
probes may be generated using the cDNA sequences disclosed in SEQ
ID NOS:1-18. The probes are preferably 15 to 50 nucleotides in length,
although they may be 8, 10, 11, 12, 20, 25, 30, 35, 40, 45, 60,
75, or 100 nucleotides in length. The probes can be synthesized
chemically or can be generated from longer polynucleotides using
restriction enzymes. The probes can be labeled, for example, with
a radioactive, biotinylated, or fluorescent tag. If desired, the
tissue sample can be subjected to a nucleic acid amplification process.
A tissue sample in which an expression product of a polynucleotide
comprising SEQ ID NOS:1, 4, 11, 16, 17, or 18 is detected is identified
as metastatic or as having metastatic potential. A tissue sample
in which an expression product of a polynucleotide comprising SEQ
ID NOS:2, 3, 6, 7, 8, 9, 10, 12, 13, or 15 is identified as not
metastatic or as having a low metastatic potential.
Propensity for high- or low-grade metastasis of a colon tumor can
also be predicted, by measuring in a colon tumor sample an expression
product of a gene comprising the nucleotide sequence of SEQ ID NOS:16
or 17. A colon tumor sample which expresses a product of a gene
comprising the nucleotide sequence of SEQ ID NO:16 is categorized
as having a high propensity to metastasize. A colon tumor sample
which expresses a product of a gene comprising the nucleotide sequence
of SEQ ID NO:17 is categorized as having a low propensity to metastasize.
Optionally, the level of a particular metastatic marker expression
product in a tissue sample can be quantitated. Quantitation can
be accomplished, for example, by comparing the level of expression
product detected in the tissue sample with the amounts of product
present in a standard curve. A comparison can be made visually or
using a technique such as densitometry, with or without computerized
assistance. For use as controls, tissue samples can be isolated
from other humans, other non-cancerous organs of the patient being
tested, or preferably non-metastatic breast or colon cancer from
the patient being tested.
Polynucleotides encoding metastatic marker-specific reagents of
the invention, such as antibodies and nucleotide probes, can be
supplied in a kit for detecting them in a biological sample. The
kit can also contain buffers or labeling components, as well as
instructions for using the reagents to detect the metastatic marker
expression products in the biological sample.
Metastatic marker gene expression in a cell can be increased or
decreased, as desired. Metastatic marker genes expression can be
altered for therapeutic purposes, as described below, or can be
used to identify therapeutic agents.
In one embodiment of the invention, expression of a metastatic
marker gene whose expression is upregulated in metastatic cancer
is decreased using a ribozyme, an RNA molecule with catalytic activity.
See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, 1990, Ann.
Rev. Biochem. 59:543-568; Cech, 1992, Curr. Opin. Struct. Biol.
2: 605-609; Couture and Stinchcomb, 1996, Trends Genet. 12: 510-515.
Ribozymes can be used to inhibit gene function by cleaving an RNA
sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat.
No. 5,641,673).
The coding sequence of the metastatic marker genes can be used
to generate a ribozyme which will specifically bind to mRNA transcribed
from a metastatic marker genes. Methods of designing and constructing
ribozymes which can cleave other RNA molecules in trans in a highly
sequence specific manner have been developed and described in the
art (see Haseloff et al. (1988), Nature 334:585-591). For example,
the cleavage activity of ribozymes can be targeted to specific RNAs
by engineering a discrete "hybridization" region into
the ribozyme. The hybridization region contains a sequence complementary
to the target RNA and thus specifically hybridizes with the target
(see, for example, Gerlach et al., EP 321,201). Longer complementary
sequences can be used to increase the affinity of the hybridization
sequence for the target. The hybridizing and cleavage regions of
the ribozyme can be integrally related; thus, upon hybridizing to
the target RNA through the complementary regions, the catalytic
region of the ribozyme can cleave the target.
Ribozymes can be introduced into cells as part of a DNA construct,
as is known in the art. The DNA construct can also include transcriptional
regulatory elements, such as a promoter element, an enhancer or
UAS element, and a transcriptional terminator signal, for controlling
the transcription of the ribozyme in the cells.
Mechanical methods, such as microinjection, liposome-mediated transfection,
electroporation, or calcium phosphate precipitation, can be used
to introduce the ribozyme-containing DNA construct into cells whose
division it is desired to decrease, as described above. Alternatively,
if it is desired that the DNA construct be stably retained by the
cells, the DNA construct can be supplied on a plasmid and maintained
as a separate element or integrated into the genome of the cells,
as is known in the art.
As taught in Haseloff et al., U.S. Pat. No. 5,641,673, the ribozyme
can be engineered so that its expression will occur in response
to factors which induce expression of the metastatic marker genes.
The ribozyme can also be engineered to provide an additional level
of regulation, so that destruction of mRNA occurs only when both
the ribozyme and the metastatic marker genes are induced in the
cells.
Expression of the metastatic marker genes can also be altered using
an antisense oligonucleotide sequence. The antisense sequence is
complementary to at least a portion of the coding sequence of a
metastatic marker genes having the nucleotide sequence shown in
SEQ ID NO:1-18. The complement of the nucleotide sequence shown
in SEQ ID NO:1-18 consists of a contiguous sequence of nucleotides
which form Watson-Crick basepairs with the contiguous nucleotide
sequence shown in SEQ ID NO:1-18.
Preferably, the antisense oligonucleotide sequence is at least
six nucleotides in length, but can be about 8, 12, 15, 20, 25, 30,
35, 40, 45, or 50 nucleotides long. Longer sequences can also be
used. Antisense oligonucleotide molecules can be provided in a DNA
construct and introduced into cells whose division is to be decreased,
as described above.
Antisense oligonucleotides can be composed of deoxyribonucleotides,
ribonucleotides, or a combination of both. Oligonucleotides can
be synthesized manually or by an automated synthesizer, by covalently
linking the 5' end of one nucleotide with the 3' end of another
nucleotide with non-phosphodiester internucleotide linkages such,
alkylphosphonates, phosphorothioate, phosphorodithioates, alkylphosphonothioates,
alkylphosphonates, phosphoramidates, phosphate esters, carbamates,
acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveauy, 1994, Meth.
Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev. 90:543-583.
Precise complementarity is not required for successful duplex formation.
between an antisense molecule and the complementary coding sequence
of a metastatic marker gene. Antisense molecules which comprise,
for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides
which are precisely complementary to a portion of a coding sequence
of a metastatic marker gene, each separated by a stretch of contiguous
nucleotides which are not complementary to adjacent coding sequences,
can provide targeting specificity for mRNA of a metastatic marker
gene. Preferably, each stretch of contiguous nucleotides is at least
4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary
intervening sequences are preferably 1, 2, 3, or 4 nucleotides in
length. One skilled in the art can easily use the calculated melting
point of an antisense-sense pair to determine the degree of mismatching
which will be tolerated between a particular antisense oligonucleotide
and a particular metastatic marker gene coding sequence.
Antisense oligonucleotides can be modified without affecting their
ability to hybridize to a metastatic marker protein coding sequence.
These modifications can be internal or at one or both ends of the
antisense molecule. For example, internucleoside phosphate linkages
can be modified by adding cholesteryl or diamine moieties with varying
numbers of carbon residues between the amino groups and terminal
ribose. Modified bases and/or sugars, such as arabinose instead
of ribose, or a 3', 5'-substituted oligonucleotide in which the
3' hydroxyl group or the 5' phosphate group are substituted, can
also be employed in a modified antisense oligonucleotide. These
modified oligonucleotides can be prepared by methods well known
in the art. Agrawal et al., 1992, Trends Biotechnol. 10:152-158;
Uhlmann et al., 1990, Chem. Rev. 90:543-584; Uhlmann et al., 1987,
Tetrahedron. Lett. 215:3539-3542.
Antibodies of the invention which specifically bind to a metastatic
marker protein can also be used to alter metastatic marker gene
expression. Specific antibodies bind to the metastatic marker proteins
and prevent the protein from functioning in the cell. Polynucleotides
encoding specific antibodies of the invention can be introduced
into cells, as described above.
To increase expression of metastatic marker genes which are down-regulated
in metastatic cells, all or a portion of a metastatic marker gene
or expression product can be introduced into a cell. Optionally,
the gene or expression product can be a component of a therapeutic
composition comprising a pharmaceutically acceptable carrier (see
below). The entire coding sequence can be introduced, as described
above. Alternatively, a portion of the metastatic marker protein
or a nucleotide sequence encoding it can be introduced into the
cell.
Expression of an endogenous metastatic marker genes in a cell can
also be altered by introducing in frame with the endogenous metastatic
marker genes a DNA construct comprising a metastatic marker protein
targeting sequence, a regulatory sequence, an exon, and an unpaired
splice donor site by homologous recombination, such that a homologously
recombinant cell comprising the DNA construct is formed. The new
transcription unit can be used to turn the metastatic marker genes
on or off as desired. This method of affecting endogenous gene expression
is taught in U.S. Pat. No. 5,641,670.
The targeting sequence is a segment of at least 10, 12, 15, 20,
or 50 contiguous nucleotides selected from the nucleotide sequence
shown in SEQ ID NO:1-18. The transcription unit is located upstream
of a coding sequence of the endogenous metastatic marker protein
gene. The exogenous regulatory sequence directs transcription of
the coding sequence of the metastatic marker genes.
Expression of the metastatic marker proteins of the present invention
can be used to screen for drugs which have a therapeutic anti-metastatic
effect. The effect of a test compound on metastatic marker protein
synthesis can also be used to identify test compounds which modulate
metastasis. Synthesis of metastatic marker proteins in a biological
sample, such as a cell culture, tissue sample, or cell-free homogenate,
can be measured by any means for measuring protein synthesis known
in the art, such as incorporation of labeled amino acids into proteins
and detection of labeled metastatic marker proteins in a polyacrylamide
gel. The amount of metastatic marker proteins can be detected, for
example, using metastatic marker protein-specific antibodies of
the invention in Western blots. The amount of the metastatic marker
proteins synthesized in the presence or absence of a test compound
can be determined by any means known in the art, such as comparison
of the amount of metastatic marker protein synthesized with the
amount of the metastatic marker proteins present in a standard curve.
The effect of a test compound on metastatic marker protein synthesis
can also be measured by Northern blot analysis, by measuring the
amount of metastatic marker protein mRNA expression in response
to the test compound using metastatic marker protein specific nucleotide
probes of the invention, as is known in the art. A test compound
which decreases synthesis of a metastatic marker protein encoded
by a polynucleotide comprising SEQ ID NOS:1, 4, 11, 16, 17, or 18
or which increases synthesis of a metastatic marker protein encoded
by a polynucleotide comprising SEQ ID NOS:2, 3, 6, 7, 8, 9, 10,
12, 13, or 15 is identified as a possible therapeutic agent.
Typically, a biological sample, such as a breast or colon sample,
is contacted with a range of concentrations of the test compound,
such as 1.0 nM, 5.0 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 mM, 10 mM,
50 mM, and 100 mM. Preferably, the test compound increases or decreases
expression of a metastatic marker protein by 60%, 75%, or 80%. More
preferably, an increase or decrease of 85%, 90%, 95%, or 98% is
achieved.
The invention provides therapeutic compositions for increasing
or decreasing expression of metastatic marker protein as is appropriate.
Therapeutic compositions for increasing metastatic marker gene expression
are desirable for metastatic markers down-regulated in metastatic
cells. These comprise polynucleotides encoding all or a portion
of a metastatic marker protein gene expression product. Preferably,
the therapeutic composition contains an expression construct comprising
a promoter and a polynucleotide segment encoding at least six contiguous
amino acids of the metastatic marker protein. Within the expression
construct, the polynucleotide segment is located downstream from
the promoter, and transcription of the polynucleotide segment initiates
at the promoter. A more complete description of gene transfer vectors,
especially retroviral vectors is contained in U.S. Ser. No. 08/869,309.
Decreased metastatic marker gene expression is desired in conditions
in which the metastatic marker gene is upregulated in metastatic
cancer. Therapeutic compositions for treating these disorders comprise
a polynucleotide encoding a reagent which specifically binds to
a metastatic marker protein expression product, as disclosed herein.
Metastatic marker therapeutic compositions of the invention also
comprise a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are well known to those in the art. Such carriers
include, but are not limited to, large, slowly metabolized macromolecules,
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers, and inactive
virus particles. Pharmaceutically acceptable salts can also be used
in the composition, for example, mineral salts such as hydrochlorides,
hydrobromides, phosphates, or sulfates, as well as the salts of
organic acids such as acetates, proprionates, malonates, or benzoates.
Therapeutic compositions can also contain liquids, such as water,
saline, glycerol, and ethanol, as well as substances such as wetting
agents, emulsifying agents, or pH buffering agents. Liposomes, such
as those described in U.S. Pat. No. 5,422,120, WO 95/13796, WO 91/14445,
or EP 524,968 B1, can also be used as a carrier for the therapeutic
composition.
Typically, a therapeutic metastatic marker composition is prepared
as an injectable, either as a liquid solution or suspension; however,
solid forms suitable for solution in, or suspension in, liquid vehicles
prior to injection can also be prepared. A metastatic marker composition
can also be formulated into an enteric coated tablet or gel capsule
according to known methods in the art, such as those described in
U.S. Pat. No. 4,853,230, EP 225,189, AU 9,224,296, and AU 9,230,801.
Administration of the metastatic marker therapeutic agents of the
invention can include local or systemic administration, including
injection, oral administration, particle gun, or catheterized administration,
and topical administration. Various methods can be used to administer
a therapeutic metastatic marker composition directly to a specific
site in the body.
For treatment of tumors, for example, a small tumor or metastatic
lesion can be located and a therapeutic metastatic marker composition
injected several times in several different locations within the
body of tumor. Alternatively, arteries which serve a tumor can be
identified, and a therapeutic composition injected into such an
artery, in order to deliver the composition directly into the tumor.
A tumor which has a necrotic center can be aspirated and the composition
injected directly into the now empty center of the tumor. A therapeutic
metastatic marker composition can be directly administered to the
surface of a tumor, for example, by topical application of the composition.
X-ray imaging can be used to assist in certain of the above delivery
methods. Combination therapeutic agents, including an the metastatic
marker protein, polypeptide, or subgenomic polynucleotide and other
therapeutic agents, can be administered simultaneously or sequentially.
Receptor-mediated targeted delivery can be used to deliver therapeutic
compositions containing subgenomic polynucleotides, proteins, or
reagents such as antibodies, ribozymes, or antisense oligonucleotides
to specific tissues. Receptor-mediated delivery techniques are described
in, for example, Findeis et al. (1993), Trends in Biotechnol. 11,
202-05; Chiou et al. (1994), GENE THERAPEUTICS: METHODS AND APPLICATIONS
OF DIRECT GENE TRANSFER (J. A. Wolff, ed.); Wu & Wu (1988),
J. Biol. Chem. 263, 621-24; Wu et al. (1994), J. Biol. Chem. 269,
542-46; Zenke et al. (1990), Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59;
Wu et al. (1991), J. Biol. Chem. 266, 338-42.
Alternatively, a metastatic marker therapeutic composition can
be introduced into human cells ex vivo, and the cells then replaced
into the human. Cells can be removed from a variety of locations
including, for example, from a selected tumor or from an affected
organ. In addition, a therapeutic composition can be inserted into
non-affected, for example, dermal fibroblasts or peripheral blood
leukocytes. If desired, particular fractions of cells such as a
T cell subset or stem cells can also be specifically removed from
the blood (see, for example, PCT WO 91/16116). The removed cells
can then be contacted with a metastatic marker therapeutic composition
utilizing any of the above-described techniques, followed by the
return of the cells to the human, preferably to or within the vicinity
of a tumor or other site to be treated. The methods described above
can additionally comprise the steps of depleting fibroblasts or
other non-contaminating tumor cells subsequent to removing tumor
cells from a human, and/or the step of inactivating the cells, for
example, by irradiation.
Both the dose of a metastatic marker composition and the means
of administration can be determined based on the specific qualities
of the therapeutic composition, the condition, age, and weight of
the patient, the progression of the disease, and other relevant
factors. Preferably, a therapeutic composition of the invention
increases or decreases expression of the metastatic marker genes
by 50%, 60%, 70%, or 80%. Most preferably, expression of the metastatic
marker genes is increased or decreased by 90%, 95%, 99%, or 100%.
The effectiveness of the mechanism chosen to alter expression of
the metastatic marker genes can be assessed using methods well known
in the art, such as hybridization of nucleotide probes to mRNA of
the metastatic marker genes, quantitative RT-PCR, or detection of
metastatic marker proteins using specific antibodies.
If the composition contains the metastatic marker proteins, polypeptide,
or antibody, effective dosages of the composition are in the range
of about 5 .mu.g to about 50 .mu.g/kg of patient body weight, about
50 .mu.g to about 5 mg/kg, about 100 .mu.g to about 500 .mu.g/kg
of patient body weight, and about 200 to about 250 .mu.g/kg.
Therapeutic compositions containing metastatic marker subgenomic
polynucleotides can be administered in a range of about 100 ng to
about 200 mg of DNA for local administration in a gene therapy protocol.
Concentration ranges of about 500 ng to about 50 mg, about 1 .mu.g
to about 2 mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g
to about 100 .mu.g of DNA can also be used during a gene therapy
protocol. Factors such as method of action and efficacy of transformation
and expression are considerations that will affect the dosage required
for ultimate efficacy of the metastatic marker protein subgenomic
polynucleotides. Where greater expression is desired over a larger
area of tissue, larger amounts of metastatic marker protein subgenomic
polynucleotides or the same amounts readministered in a successive
protocol of administrations, or several administrations to different
adjacent or close tissue portions of, for example, a tumor site,
may be required to effect a positive therapeutic outcome. In all
cases, routine experimentation in clinical trials will determine
specific ranges for optimal therapeutic effect.
Metastatic marker subgenomic polynucleotides of the invention can
also be used on polynucleotide arrays. Polynucleotide arrays provide
a high throughput technique that can assay a large number of polynucleotide
sequences in a single sample. This technology can be used, for example,
as a diagnostic tool to identify metastatic lesions or to assess
the metastatic potential of a tumor.
To create arrays, single-stranded polynucleotide probes can be
spotted onto a substrate in a two-dimensional matrix or array. Each
single-stranded polynucleotide probe can comprise at least 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 or more
contiguous nucleotides selected from the nucleotide sequences shown
in SEQ ID NOS:1-18. Preferred arrays comprise at least one single-stranded
polynucleotide probe comprising at least 6, 7, 8, 9, 10, 11, 12,
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