Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR THE
IDENTIFICATION OF LUNG TUMOR CELLS
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority under 35 U.S.C. ~ 119(e) to U.S.
Provisional Application no. 60/080,037, filed March 30, 1999, the contents of
which are hereby incorporated by reference into the present disclosure.
TECHNICAL FIELD
This invention relates to the isolation and characterization of novel
transcripts expressed in lung tumor or cancer cells.
BACKGROUND
Despite numerous advances in medical research, cancer remains the
second leading cause of death in the United States. In the industrialized
nations,
roughly one in five persons will die of cancer. Traditional modes of clinical
care,
such as surgical resection, radiotherapy and chemotherapy, have a significant
failure rate, especially for solid tumors. Failure occurs either because the
initial
tumor is unresponsive, or because of recurrence due to regrowth at the
original
site and/or metastases.
Lung cancer is one of the most common malignancies worldwide and is
the leading cause of cancer death in man. See, American Cancer Society, Cancer
facts and figures, 1996, Atlanta. Approximately 178,100 new cases of lung
cancer were to be diagnosed in 1997, accounting for 13% of cancer diagnoses.
An estimated 160,400 deaths due to lung cancer would occur in 1997 accounting
for 29% of all cancer deaths. The one-year survival rates for lung cancer have
increased from 32% in 1973 to 41 % in 1993, largely due to improvements in
surgical techniques. The 5 year survival rate for all stages combined is only
14%.
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The survival rate is 48% for cases detected when the disease is still
localized, but
only 15% of lung cancers are discovered that early. Among various forms of
lung
cancer, non-small cell lung cancer (NSCLC) accounts for nearly 80% of all new
lung cancer cases each year. For patients diagnosed with NSCLC, surgical
resection offers the only chance of meaningful survival. On the other hand,
small
cell lung cancer is the most malignant and fastest growing form of lung cancer
and accounts for the rest of approximately 20% of new cases of lung cancer.
The
primary tumor is generally responsive to chemotherapy, but is followed by wide-
spread metastasis. The median survival time at diagnosis is approximately 1
year,
with a 5 year survival rate of 5%.
In spite of major advances in cancer therapy including improvements in
surgical resection, radiation treatment and chemotherapy, successful
intervention
for lung cancer in particular, relies on early detection of the cancerous
cells.
Neoplasia resulting in benign tumors may be completely cured by removing the
mass surgically. If a tumor becomes malignant, as manifested by invasion of
surrounding tissue, it becomes much more difficult to eradicate. Therefore,
there
remains a considerable need in the art for the development of methods for
detecting the disease at the early stage. There also exits a pressing need in
the art
for developing diagnostic method to monitor or progenies the progression of
the
disease as well as methods to treat various conditions. However, the vast
variability in the nature of the disease has rendered the search for cellular
markers,
such as genes that are preferably overexposed in primary lung cancer cells and
useful for diagnostic and therapeutic methods, difficult.
This invention provides compositions and methods that are useful for the
early diagnosis of a lung neoplasm.
DISCLOSURE OF THE INVENTION
The present invention provides methods for aiding in the diagnoses of the
neoplastic condition of a lung cell, and methods for screening for a potential
therapeutic agent for the reversal of the neoplastic condition. It also
provides
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genes that are overexpressed in lung cancer cells, as well as novel genes that
are
expressed in primary lung cancer cells.
Accordingly, one embodiment of this invention is a method of diagnosing
the neoplastic condition of a lung cell by screening for the presence of a
transcript
which is expressed in a neoplastic lung cell but not normal lung cells or
tissue. In
one aspect of this invention, the transcript is from a known gene, which until
the
subject invention, was unknown to be differentially expressed in lung cancer
or
lung tumor tissue, and not expressed in normal lung tissue, or expressed to a
lesser
extent than in lung cancer tissue or cells. In a separate embodiment, only a
fragment of the transcript is known as a prior Expressed Sequence Tag ("EST").
However, the presence, absence or differential expression of the EST in lung
cancer tissue versus normal lung tissue remained unknown until Applicants'
invention. The corresponding full length open reading frame of the EST and
corresponding genomic DNA may or may not be known to those of skill in the
art.
In a further embodiment, the polynucleotide identifies a previously
unidentified or uncharacterized fragment or full length coding region (open
reading frame) of a gene or polynucleotide. This invention also provides the
method to identify the gene, fragment or full length coding region
corresponding
to the isolated polynucleotide of this invention. The genes, fragments, and
full
length coding regions obtainable by these methods are further claimed herein.
Another embodiment of the invention is a screen for a potential
therapeutic agent for the reversal of the neoplastic condition of a lung cell,
wherein the cell is characterized by the presence of at least one of a
polynucleotide identified herein by SEQ ID NOS. 1 through 40. The method
comprises contacting a neoplastic lung cell or tissue with an effective amount
of a
potential agent and assaying for reversal of the neoplastic condition. In
another
embodiment, the polynucleotide used in the method is obtained by
identification
of larger fragment or full-length coding sequence corresponding to the
sequence
of SEQ ID NOS: 1-40.
In another embodiment, the invention includes a method of diagnosing the
condition of a lung cell to determine whether it is predisposed to or is in a
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neoplastic condition. The method requires screening for the presence of a
peptide
transcribed from a polynucleotide which is expressed in a neoplastic lung cell
but
not normal lung cells or tissue. In one aspect of this invention, the peptide
is from
a known gene, which until the subject invention, was unknown to be
differentially
expressed in lung cancer or lung tumor tissue, and not expressed in normal
lung
tissue. In a separate embodiment, only a fragment of the polynucleotide
encoding
the peptide is known as a prior Expressed Sequence Tag ("EST"). The full
length
open reading frame of the gene corresponding to the EST may or may not be
characterized. However, the presence or absence of the EST gene product in
lung
cancer tissue versus normal lung tissue previously had not been characterized.
The novel peptides are also included within this invention.
The invention also includes kits for use in the detection and screening
methods described herein. The kits contain, in a suitable packaging, the
agents
and reagents necessary to practice the claimed invention as well as
instructions to
I 5 conduct the screen.
Also described are non-human transgenic animals that are genetically
modified so that a polynucleotide sequence associated with lung cancer has
been
disrupted. The disrupted polynucleotide sequence may be those shown in SEQ ID
NOS: I-40, a polynucleotide corresponding to these sequences, or sequences
obtained by identification of larger fragment or full-length coding sequence
corresponding to SEQ ID NOS: 1-40.
MODES) FOR CARRYING OUT THE INVENTION
Throughout this disclosure, various publications, patents and published
patent specifications are referenced by an identifying citation. The
disclosures of
these publications, patents and published patent specifications are hereby
incorporated by reference into the present disclosure to more fully describe
the
state of the art to which this invention pertains.
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Definitions
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of immunology, molecular biology,
microbiology, cell biology and recombinant DNA, which are within the skill of
the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A
LABORATORY MANUAL, 2°d edition (1989); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN
ENZYMULOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J.
MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds.
(1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I.
Freshney, ed. ( 1987)).
As used in the specification and claims, the singular form "a", "an" and
"the" include plural references unless the context clearly dictates otherwise.
For
example, the term "a cell" includes a plurality of cells, including mixtures
thereof.
As used herein, the term "comprising" is intended to mean that the
compositions and methods include the recited elements, but not excluding
others.
"Consisting essentially of when used to define compositions and methods, shall
mean excluding other elements of any essential significance to the
combination.
Thus, a composition consisting essentially of the elements as defined herein
would not exclude trace contaminants from the isolation and purification
method
and pharmaceutically acceptable carriers, such as phosphate buffered saline,
preservatives, and the like. "Consisting of" shall mean excluding more than
trace
elements of other ingredients and substantial method steps for administering
the
compositions of this invention. Embodiments defined by each of these
transition
terms are within the scope of this invention.
The term "isolated" means separated from constituents, cellular and
otherwise, in which the polynucleotide, peptide, polypeptide, protein,
antibody, or
fragments thereof, are normally associated with in nature. In one aspect of
this
invention, an isolated polynucleotide is separated from the 3' and 5'
contiguous
nucleotides with which it is normally associated with in its native or natural
environment, e.g., on the chromosome. As is apparent to those of skill in the
art, a
S
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non-naturally occurring polynucleotide, peptide, polypeptide, protein,
antibody, or
fragments thereof, does not require "isolation" to distinguish it from its
naturally
occurnng counterpart. In addition, a "concentrated", "separated" or "diluted"
polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof,
is
distinguishable from its naturally occurnng counterpart in that the
concentration
or number of molecules per volume is greater than "concentrated" or less than
"separated" than that of its naturally occurring counterpart. A
polynucleotide,
peptide, polypeptide, protein, antibody, or fragments thereof, which differs
from
the naturally occurring counterpart in its primary sequence or for example, by
its
glycosylation pattern, need not be present in its isolated form since it is
distinguishable from its naturally occurring counterpart by its primary
sequence,
or alternatively, by another characteristic such as glycosylation pattern.
Although
not explicitly stated for each of the inventions disclosed herein, it is to be
understood that all of the above embodiments for each of the compositions
disclosed below and under the appropriate conditions, are provided by this
invention. Thus, a non-naturally occurring polynucleotide is provided as a
separate embodiment from the isolated naturally occurnng polynucleotide. A
protein produced in a bacterial cell is provided as a separate embodiment from
the
naturally occurring protein isolated from a eucaryotic cell in which it is
produced
in nature.
The terms "polynucleotide" and "oligonucleotide" are used interchangeably,
and refer to a polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides
may
have any three-dimensional structure, and may perform any function, known or
unknown. The following are non-limiting examples of polynucleotides: a gene or
gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal
RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence,
nucleic acid probes, and primers. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs. If
present,
modifications to the nucleotide structure may be imparted before or after
assembly
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of the polymer. The sequence of nucleotides may be interrupted by non-
nucleotide
components. A polynucleotide may be further modified after polymerization,
such
as by conjugation with a labeling component. The term also refers to both
double-
and single-stranded molecules. Unless otherwise specified or required, any
embodiment of this invention that is a polynucleotide encompasses both the
double-stranded form and each of two complementary single-stranded forms
known or predicted to make up the double-stranded form.
A polynucleotide is composed of a specific sequence of four nucleotide
bases: adenine (A); cytosine (C); guanine (G); thymine {T); and uracil (LT)
for
guanine when the polynucleotide is RNA. Thus, the term "polynucleotide
sequence" is the alphabetical representation of a polynucleotide molecule.
This
alphabetical representation can be input into databases in a computer having a
central processing unit and used for bioinformatics applications such as
functional
genomics and homology searching.
A "gene" refers to a polynucleotide containing at least one open reading
frame that is capable of encoding a particular polypeptide or protein after
being
transcribed and translated. Any of the polynucleotides sequences described
herein
may be used to identify larger fragments or full-length coding sequences of
the
gene with which they are associated. Methods of isolating larger fragment
sequences are known to those of skill in the art, some of which are described
herein. A "oncogene" refers to a polynucletide containing at least one open
reading frame, that is capable of transforming a normal cell to a cancerous
(or
neoplastic or tumor) cell when introduced into a host cell. Oncogenes are
often
altered forms of the cellular counterpart, namely the "proto-oncogenes" that
are
incapable of cell transformation when expressed at the level present in a non-
cancer cell.
A "gene product" refers to the amino acid (e.g., peptide or polypeptide)
generated when a gene is transcribed and translated.
As used herein a second polynucleotide "corresponds to" another {a first)
polynucleotide if it is related to the first polynucleotide by any of the
following
relationships:
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1 ) The second polynucleotide comprises the first polynucleotide and the
second polynucleotide encodes a gene product.
2) The second polynucleotide is S' or 3' to the first polynucleotide in
cDNA, RNA, genomic DNA, or fragment of any of these
polynucleotides. For example, a second polynucleotide may be a
fragment of a gene that includes the first and second polynucleotides.
The first and second polynucleotides are related in that they are
components of the gene coding for a gene product, such as a protein or
antibody. However, it is not necessary that the second polynucleotide
comprises or overlaps with the first polynucleotide to be encompassed
within the definition of "corresponding to" as used herein. For
example, the first polynucleotide may be a fragment of a 3'
untranslated region of the second polynucleotide. The first and second
polynucleotide may be fragment of a gene coding for a gene product.
The second polynucleotide may be an exon of the gene while the first
polynucleotide may be an intron of the gene.
3) The second polynucleotide is the complement of the first
polynucleotide.
A "probe" when used in the context of polynucleotide manipulation refers
to an oligonucleotide that is provided as a reagent to detect a target
potentially
present in a sample of interest by hybridizing with the target. Usually, a
probe
will comprise a label or a means by which a label can be attached, either
before or
subsequent to the hybridization reaction. Suitable labels include, but are not
limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and
proteins, including enzymes.
A "primer" is a short polynucleotide, generally with a free 3' -OH group
that binds to a target or "template" potentially present in a sample of
interest by
hybridizing with the target, and thereafter promoting polymerization of a
polynucleotide complementary to the target. A "polymerase chain reaction"
("PCR") is a reaction in which replicate copies are made of a target
polynucleotide using a "pair of primers" or a "set of primers" consisting of
an
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"upstream" and a "downstream" primer, and a catalyst of polymerization, such
as
a DNA polymerase, and typically a thermally-stable polymerase enzyme.
Methods for PCR are well known in the art, and taught, for example in "PCR: A
PRACTICAL APPROACH" (M. MacPherson et al., IRL Press at Oxford University
Press ( 1991 )). All processes of producing replicate copies of a
polynucleotide,
such as PCR or gene cloning, are collectively referred to herein as
"replication."
A primer can also be used as a probe in hybridization reactions, such as
Southern
or Northern blot analyses. Sambrook et al., supra.
A "sequence tag" or "SAGE tag" is a short sequence, generally under
about 20 nucleotides, that occurs in a certain position in messenger RNA. The
tag
can be used to identify the corresponding transcript and gene from which it
was
transcribed. A "ditag" is a dimer of two sequence tags.
The term "cDNAs" refers to complementary DNA, that is mRNA
molecules present in a cell or organism made in to cDNA with an enzyme such as
reverse transcriptase. A "cDNA library" is a collection of all of the mRNA
molecules present in a cell or organism, all turned into cDNA molecules with
the
enzyme reverse transcriptase, then inserted into "vectors" (other DNA
molecules
that can continue to replicate after addition of foreign DNA). Exemplary
vectors
for libraries include bacteriophage (also known as "phage"), viruses that
infect
bacteria, for example, lambda phage. The library can then be probed for the
specific cDNA (and thus mRNA) of interest.
A "gene delivery vehicle" is defined as any molecule that can carry
inserted one or more polynucleotides into a host cell. Examples of gene
delivery
vehicles are liposomes, biocompatible polymers, including natural polymers and
synthetic polymers; lipoproteins; polypeptides; polysaccharides;
lipopolysaccharides; artificial viral envelopes; metal particles; and
bacteria,
viruses and viral vectors, such as baculovirus, adenovirus and retrovirus,
bacteriophage, cosmid, plasmid, fungal vector and other recombination vehicles
typically used in the art which have been described for replication and/or
expression in a variety of eucaryotic and procaryotic hosts. The gene delivery
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vehicles may be used for replication of the inserted polynucleotide, gene
therapy
as well as for simple polypeptide and protein expression.
"Vector" means a self replicating nucleic acid molecule that transfers an
inserted polynucleotide into and/or between host cells. The term is intended
to
include vectors that function primarily for insertion of a nucleic acid
molecule
into a cell, replication vectors that function primarily for the replication
of nucleic
acid and expression vectors that function for transcription and/or translation
of the
DNA or RNA. Also intended are vectors that provide more than one of the above
functions.
"Host cell" is intended to include any individual cell or cell culture which
can be or has been a recipient for vectors or for the incorporation of
exogenous
nucleic acid molecules, polynucleotides and/or proteins. It also is intended
to
include progeny of a single cell. The progeny may not necessarily be
completely
identical (in morphology or in genomic or total DNA complement) to the
original
parent cell due to natural, accidental, or deliberate mutation. The cells may
be
procaryotic or eucaryotic, and include but are not limited to bacterial cells,
yeast
cells, insect cells, animal cells, and mammalian cells, e.g., marine, rat,
simian or
human.
The term "genetically modified" means containing and/or expressing a
foreign gene or nucleic acid sequence which in turn, modifies the genotype or
phenotype of the cell or its progeny. In other words, it refers to any
addition,
deletion or disruption to a cell's endogenous nucleotides.
As used herein, "expression" refers to the process by which
polynucleotides are transcribed into mRNA and translated into peptides,
polypeptides, or proteins. If the polynucleotide is derived from genomic DNA,
expression may include splicing of the mRNA, if an appropriate eucaryotic host
is
selected. Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation sequences for
ribosome binding. For example, a bacterial expression vector includes a
promoter
such as the lac promoter and for transcription initiation the Shine-Dalgarno
sequence and the start codon AUG (Sambrook et al. (1989) supra ). Similarly,
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eucaryotic expression vector includes a heterologous or homologous promoter
for
RNA polymerase II, a downstream polyadenylation signal., the start codon AUG,
and a termination codon for detachment of the ribosome. Such vectors can be
obtained commercially or assembled by the sequences described in methods well
known in the art, for example, the methods described below for constructing
vectors in general.
"Differentially expressed" as applied to a gene, refers to the differential
production of the mRNA transcribed from the gene or the protein product
encoded
by the gene. A differentially expressed gene may be overexpressed or
underexpressed as compared to the expression level of a normal or control
cell. In
one aspect, it refers to a differential that is 2.5 times, preferably 5 times,
or
preferably 10 times higher or lower than the expression level detected in a
control
sample. The term "differentially expressed" also refers to nucleotide
sequences in
a cell or tissue which are expressed where silent in a control cell or not
expressed
where expressed in a control cell.
The term "polypeptide" is used in its broadest sense to refer to a
compound of two or more subunit amino acids, amino acid analogs, or
peptidomimetics. The subunits may be linked by peptide bonds. In another
embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc.
As
used herein the term "amino acid" refers to either natural and/or unnatural or
synthetic amino acids, including glycine and both the D or L optical isomers,
and
amino acid analogs and peptidomimetics. A peptide of three or more amino acids
is commonly called an oligopeptide if the peptide chain is short. If the
peptide
chain is long, the peptide is commonly called a polypeptide or a protein.
"Hybridization" refers to a reaction in which one or more polynucleotides
react to form a complex that is stabilized via hydrogen bonding between the
bases
of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick
base pairing, Hoogstein binding, or in any other sequence-specific manner. The
complex may comprise two strands forming a duplex structure, three or more
strands forming a multi-stranded complex, a single self hybridizing strand, or
any
combination of these. A hybridization reaction may constitute a step in a more
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extensive process, such as the initiation of a PCR reaction, or the enzymatic
cleavage of a polynucleotide by a ribozyme.
Hybridization reactions can be performed under conditions of different
"stringency". In general, a low stringency hybridization reaction is carried
out at
about 40 °C in 10 X SSC or a solution of equivalent ionic
strength/temperature. A
moderate stringency hybridization is typically performed at about 50 °C
in 6 X
SSC, and a high stringency hybridization reaction is generally performed at
about
60 °C in 1 X SSC.
When hybridization occurs in an antiparallel configuration between two
single-stranded polynucleotides, the reaction is called "annealing" and those
polynucleotides are described as "complementary". A double-stranded
polynucleotide can be "complementary" or "homologous" to another
polynucleotide, if hybridization can occur between one of the strands of the
first
polynucleotide and the second. "Complementarity" or "homology" (the degree
that one polynucleotide is complementary with another) is quantifiable in
terms of
the proportion of bases in opposing strands that are expected to form hydrogen
bonding with each other, according to generally accepted base-pairing rules.
A polynucleotide or polynucleotide region (or a polypeptide or
polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or
95%) of "sequence identity" to another sequence means that, when aligned, that
percentage of bases (or amino acids) are the same in comparing the two
sequences. This alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example those
described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al.,
eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default
parameters are used for alignment. A preferred alignment program is BLAST,
using default parameters. In particular, preferred programs are BLASTN and
BLASTP, using the following default parameters: Genetic code = standard;
filter
= none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62;
Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-
redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations +
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SwissProtein + SPupdate + PIR. Details of these programs can be found at the
following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
As used herein, "solid phase support" or "solid support", used
interchangeably, is not limited to a specific type of support. Rather a large
number of supports are available and are known to one of ordinary skill in the
art.
Solid phase supports include silica gels, resins, derivatized plastic films,
glass
beads, cotton, plastic beads, alumina gels. As used herein, "solid support"
also
includes synthetic antigen-presenting matrices, cells, and iiposomes. A
suitable
solid phase support may be selected on the basis of desired end use and
suitability
for various protocols. For example, for peptide synthesis, solid phase support
may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem
Inc., Peninsula Laboratories, etc.), POLYHIPE~ resin (obtained from Aminotech,
Canada), polyamide resin {obtained from Peninsula Laboratories), polystyrene
resin grafted with polyethylene glycol (TentaGel~, Rapp Polymere, Tubingen,
1 S Germany) or polydimethylacrylamide resin (obtained from
Milligen/Biosearch,
California).
A polynucleotide of the invention also can be attached to a solid support
for use in high throughput screening assays. PCT WO 97/10365, for example,
discloses the construction of high density oligonucleotide chips. See also,
U.S.
Patent Nos. 5,405,783; 5,412,087; and 5,445,934. Using this method, the probes
are synthesized on a derivatized glass surface. Photoprotected nucleoside
phosphoramidites are coupled to the glass surface, selectively deprotected by
photolysis through a photolithographic mask, and reacted with a second
protected
nucleoside phosphoramidite. The coupling/deprotection process is repeated
until
the desired probe is complete.
An "antibody" is an immunoglobulin molecule capable of binding an
epitope present on an antigen. As used herein, the term encompasses not only
intact immunoglobulin molecules such as monoclonal and polyclonal antibodies,
but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, bi-
specific
antibodies, humanized proteins and modifications of the immunoglobulin
molecule that comprise an antigen recognition site of the required
specificity.
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An "antibody complex" is the combination of antibody (as defined above)
and its binding partner or ligand.
The term "recognized" intends that an antibody of the present invention
binds with either or both of higher affinity or avidity to an epitope present
on a
polypeptide of this invention than for an unrelated polypeptide. Assays for
avidity and affinity of an antibody complex are known in the art.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor
cells", "cancer" and "cancer cells", (used interchangeably) refer to cells
which
exhibit relatively autonomous growth, so that they exhibit an aberrant growth
phenotype characterized by a significant loss of control of cell proliferation
(i.e.,
de-regulated cell division). Neoplastic cells can be malignant or benign. A
metastatic cell or tissue means that the cell can invade and destroy
neighboring
body structures.
"Suppressing" tumor growth indicates a growth state that is curtailed when
1 S compared to growth without contact with educated, antigen-specific immune
effector cells described herein. Tumor cell growth can be assessed by any
means
known in the art, including, but not limited to, measuring tumor size,
determining
whether tumor cells are proliferating using a 3H-thyinidine incorporation
assay, or
counting tumor cells. "Suppressing" tumor cell growth means any or all of the
following states: slowing, delaying, and stopping tumor growth, as well as
tumor
shrinkage.
Hyperplasia is a form of controlled cell proliferation involving an increase
in cell number in a tissue or organ, without significant alteration in
structure or
function. Metaplasia is a form of controlled cell growth in which one type of
fully
differentiated cell substitutes for another type of differentiated cell.
Metaplasia
can occur in epithelial or connective tissue cells. Atypical metaplasia
involves a
somewhat disorderly metaplastic epithelium.
As used herein, the term "reversing the neoplastic state of the cell" is
intended to include apoptosis, necrosis or any other means of preventing cell
division, reduced tumorigenicity, loss of pharmaceutical resistance,
maturation,
differentiation or reversion of the neoplastic phenotypes as described herein.
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A "composition" is intended to mean a combination of active agent and
another compound or composition, inert (for example, a detectable agent or
label)
or active, such as an adjuvant.
A "pharmaceutical composition" is intended to include the combination of
an active agent with a carrier, inert or active, making the composition
suitable for
diagnostic or therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water, and emulsions, such as an oil/water or
water/oil
emulsion, and various types of wetting agents. The compositions also can
include
stabilizers and preservatives. For examples of carriers, stabilizers and
adjuvants,
see Martin, REMINGTON'S PHARM. sCl., 15th Ed. (Mack Publ. Co., Easton (1975)).
An "effective amount" is an amount sufficient to effect beneficial or
desired results. An effective amount can be administered in one or more
administrations, applications or dosages.
A "subject," "individual" or "patient" is used interchangeably herein,
which refers to a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, marines, simians, humans, farm
animals,
sport animals, and pets.
A "control" is an alternative subject or sample used in an experiment for
comparison purpose. A control can be "positive" or "negative". For example,
where the purpose of the experiment is to determine a correlation of an
altered
expression level of a proto-oncogene with a particular type of cancer, it is
generally preferable to use a positive control (a subject or a sample from a
subject,
carrying such alteration and exhibiting syndromes characteristic of that
disease),
and a negative control (a subject or a sample from a subject lacking the
altered
expression and clinical syndrome of that disease).
A "transgenic animal" refers to a genetically engineered animal or
offspring of genetically engineered animals. The transgenic animal may contain
genetic material from at least one unrelated organism (such as from a
bacteria,
CA 02323833 2000-09-18
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virus, plant, or other animal) or may contain a mutation which interferes with
expression of a gene product.
A systematic analysis of transcripts present in non-small cell lung cancer
(NSCLC) was performed by Serial Analysis of Gene Expression ("SAGE")
(U.S. Patent No. 5,695,937). SAGE analysis involves identifying nucleotide
sequences expressed in cells. Briefly, SAGE analysis began with providing
complementary deoxyribonucleic acid (cDNA) from (1) the neoplastic population
and (2) normal cells. Both cDNAs were linked to primer sites. Sequence tags
were then created, for example, using the appropriate primers to amplify the
DNA. By measuring the differences in these tags between the two cell types,
sequences that are aberrantly expressed in the neoplastic cell population were
identified.
Polynucleotides and Claimed Utilities
TJsing SAGE, the sequence tags represented by SEQ ID NOS. 1 through
40 were identified and further characterized. Thus, this invention provides a
population of polynucleotides represented by SEQ ID NOS. 1 through 40, or
their
respective complements. Compositions (described in detail below) and a
database
(also described below) containing these polynucleotides also are provided by
this
invention.
Polynucleotides corresponding to SEQ ID NOS. 24 to 26, 29, 32 to 35,
and 38, represent previously unidentified polynucleotides or genes. Thus, this
invention also provides a population of polynucleotides comprising at least
one
polynucleotide having a sequence selected from the group consisting of SEQ ID
NOS. 24 to 26, 29, 32 to 35, and 38, or their respective complements. Also
provided by this invention are polynucleotides that correspond to
polynucleotides
having a sequence of SEQ ID NOS. 24 to 26, 29, 32 to 35, and 38. In one
embodiment, these polynucleotides are obtained by identification of a larger
fragment or full-length coding sequence of these polynucleotides. Gene
delivery
vehicles, host cells, compositions (described in detail below) and databases
(also
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described below) containing these polynucleotides also are provided by this
invention.
The invention also encompasses polynucleotides which differ from that of
the polynucleotides described above, but which produce the same phenotypic
effect, such as the allele. These altered, but phenotypically equivalent
polynucleotides are referred to "equivalent nucleic acids." This invention
also
encompasses polynucleotides characterized by changes in non-coding regions
that
do not alter the phenotype of the polypeptide produced therefrom when compared
to the polynucleotide herein. This invention further encompasses
polynucleotides,
which hybridize to the polynucleotides of the subject invention under
conditions
of moderate or high stringency. Alternatively, the polynucleotides are at
least
85%, or at least 90%, or more preferably, greater or equal to 95% identical as
determined by a sequence alignment program when run under default parameters.
Also provided in the present invention are polypeptides encoded by an
EST or by a known gene, which until the instant invention, was unknown to be
differentially expressed in lung cancer. Further embodied in the polypeptides
of
the present invention are novel sequences including fragments thereof or
complements thereof that hybridize to the same coding sequence, to which the
polypeptide encoded by the nucleotides depicted in SEQ ID NOS. 24 to 26, 29,
32
to 35 or 38 hybridizes. These sequences are unique in their over-
representation in
lung cancer cells and not in normal lung cells, thus being particularly useful
in
detecting a lung cancer cell.
The process of identification of larger fragment or the fixll-length coding
sequence to which the partial sequence depicted in SEQ ID NOS. 24-26, 29, 32-
35 or 38 hybridizes preferably involves the use of the methods and reagents
provided in this invention, either singularly or in combination. Any
conventional
recombinant DNA techniques applicable for isolating polynucleotides can also
be
used.
One such method involves the S'-RACE-PCR technique, in which the poly-
A mRN A that contains the coding sequence of particular interest is first
reverse
transcribed with a 3'-primer comprising the sequence disclosed herein. The
newly
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synthesized cDNA strand is then tagged with an anchor primer with a known
sequence, which preferably contains a convenient cloning restriction site
attached at
the 5'end. The tagged cDNA is then amplified with the 3'-primer (or a nested
primer sharing sequence homology to the internal sequences of the coding
region)
and the S'-anchor primer. The amplification may be conducted under conditions
of
various levels of stringency to optimize the amplification specificity. S'-
RACE-PCR
can be readily performed using commercial kits (available from, e.g., BRL Life
Technologies Inc, Clotech) according to the manufacturer's instructions.
Isolating the complete coding sequence of a gene can also be carried out in a
hybridization assay using a suitable probe. The probe preferably comprises at
least
10 nucleotides, and more preferably exhibits sequence homology to the
polynucleotide depicted in SEQ ID NOS. 24-26, 29, 32-35 or 38. Other high
throughput screens for cDNAs, such as those involving gene chip technology,
can
also be employed in obtaining the complete cDNA sequence.
In addition, databases exist that reduce the complexity of ESTs by
assembling contiguous EST sequences into tentative genes. For example, TIGR
has assembled human ESTs into a datable called THC for tentative human
consensus sequences. The THC database allows for a more definitive assignment
compared to ESTs alone. Software programs exist (TIGR assembler and TIGEM
EST assembly machine and contig assembly program (see Huang, X. ( 1996)
Genomics 33 :21-23)) that allow for assembling ESTs into contiguous sequences
from any organism.
Alternatively, mRNA from a sample preparation is used to construct
cDNA library in the ZAP Express vector following the procedure described in
Velculescu et al. (1997) Science 270:484. The ZAP Express cDNA synthesis kit
(Stratagene) is used accordingly to the manufacturer's protocol. Plates
containing
250 to 2000 plaques are hybridized as described in Rupert et al. (1988) Mol.
Cell.
Bio. 8:3104 to oligonucleotide probes with the same conditions previously
described for standard probes except that the hybridization temperature is
reduced
to room temperature. Washes are performed in 6X standard-saline-citrate 0.1
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SDS for 30 minutes at room temperature. The probes are labeled with 32P-ATP
through use of T4 polynucleotide kinase.
A partial cDNA (3' fragment) can be isolated by 3' directed PCR reaction.
This procedure is a modification of the protocol described in Polyak et al.
(1997)
Nature 389:300. Briefly, the procedure uses SAGE tags in PCR reaction such
that
the resultant PCR product contains the SAGE tag of interest as well as
additional
cDNA, the length of which is defined by the position of the tag with respect
to the
3' end of the cDNA. The cDNA product derived from such a transcript driven
PCR reaction can be used for many applications.
RNA from a source believed to express the cDNA corresponding to a
given tag is first converted to double-stranded cDNA using any standard cDNA
protocol. Similar conditions used to generate cDNA for SAGE library
construction can be employed except that a modified oligo-dT primer is used to
derive the first strand synthesis. For example, the oligonucleotide of
composition
5'-B-TCC GGC GCG CCG TTT T CC CAG TCA CGA(30)-3', contains a poly-T
stretch at the 3' end for hybridization and priming from poly-A tails, an M13
priming site for use in subsequent PCR steps, a 5' Biotin label (B) for
capture to
strepavidin-coated magnetic beads, and an AscI restriction endonuclease site
for
releasing the cDNA from the streptavidin-coated magnetic beads. Theoretically,
any sufficiently-sized DNA region capable of hybridizing to a PCR primer can
be
used as well as any other 8 base pair recognizing endonuclease.
cDNA constructed utilizing this or similar modified oligo-dT primer is
then processed exactly as described in U.S. Patent No. 5,695,937 up until
adapter
ligation where only one adapter is ligated to the cDNA pool. After adapter
ligation, the cDNA is released from the streptavidin-coated magnetic beads and
is
then used as a template for cDNA amplification.
Various PCR protocols can be employed using PCR priming sites within
the 3' modified oligo-dT primer and the SAGE tag. The SAGE tag-derived PCR
primer employed can be of varying length dictated by 5' extension of the tag
into
the adaptor sequence. cDNA products are now available for a variety of
applications.
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This technique can be further modified by: ( 1 ) altering the length and/or
content of the modified oligo-dT primer; (2) ligating adaptors other than that
previously employed within the SAGE protocol; (3) performing PCR from
template retained on the streptavidin-coated magnetic beads; and (4) priming
first
strand cDNA synthesis with non-oligo-dT based primers.
Gene trapper technology can also be used. The reagents and
manufacturer's instructions for this technology are commercially available
from
Life Technologies, Inc., Gaithersburg, Maryland. Briefly, a complex population
of single-stranded phagemid DNA containing directional cDNA inserts is
enriched for the target sequence by hybridization in solution to a
biotinylated
oligonucleotide probe complementary to the target sequence. The hybrids are
captured on streptavidin-coated paramagnetic beads. A magnet retrieves the
paramagnetic beads from the solution, leaving nonhybridized single-stranded
DNAs behind. Subsequently, the captured single-stranded DNA target is released
from the biotinylated oligonucleotide. After release, the cDNA clone is
further
enriched by using a nonbiotinylated target oligonucleotide to specifically
prime
conversion of the single-stranded target to double-stranded DNA. Following
transformation and plating, typically 20% to 100% of the colonies represent
the
cDNA clone of interest. To identify the desired cDNA clone, the colonies may
be
screened by colony hybridization using the 32P-labeled oligonucleotide as
described above for solution hybridization, or alternatively by DNA sequencing
and alignment of all sequences obtained from numerous clones to determine a
consensus sequence.
The polynucleotides of this invention can be replicated using PCR. PCR
technology is the subject matter of United States Patent Nos. 4,683,195,
4,800,159, 4,754,065, and 4,683,202 and described in PCR: THE POLYMERASE
CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) and
references cited therein.
Alternatively, one of skill in the art can use the sequences provided herein
and a commercial DNA synthesizer to replicate the DNA. Accordingly, this
invention also provides a process for obtaining the polynucleotides of this
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invention by providing the linear sequence of the polynucleotide, appropriate
primer molecules, chemicals such as enzymes and instructions for their
replication
and chemically replicating or linking the nucleotides in the proper
orientation to
obtain the polynucleotides. In a separate embodiment, these polynucleotides
are
further isolated. Still further, one of skill in the art can insert the
polynucleotide
into a suitable replication vector and insert the vector into a suitable host
cell
(procaryotic or eucaryotic) for replication and amplification. The DNA so
amplified can be isolated from the cell by methods well known to those of
skill in
the art. A process for obtaining polynucleotides by this method is further
provided herein as well as the polynucleotides so obtained.
RNA can be obtained by first inserting a DNA polynucleotide into a
suitable host cell. The DNA can be inserted by any appropriate method, e.g.,
by
the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or
vector)
or by electroporation. When the cell replicates and the DNA is transcribed
into
RNA; the RNA can then be isolated using methods well known to those of skill
in
the art, for example, as set forth in Sambrook et al. ( 1989) Supra. For
instance,
mRNA can be isolated using various lytic enzymes or chemical solutions
according to the procedures set forth in Sambrook et al. ( 1989) Supra or
extracted
by nucleic-acid-binding resins following the accompanying instructions
provided
by manufactures.
As noted above, this invention further provides various methods for aiding
in the diagnosis of the neoplastic state of a lung cell that is or is
predisposed to be
characterized by abnormal cell growth in the form of, e.g., malignancy,
hyperplasia or metaplasia. The neoplastic state of a cell generally is
determined
by noting whether the growth of the cell is not governed by the usual
limitation of
normal growth. For the purposes of this invention, the term also is to include
genotypic changes that occur prior to detection of this growth in the form of
a
tumor and that are causative of these phenotypic changes. The phenotypic
changes associated with the neoplastic state of a cell (a set of in vitro
characteristics associated with a tumorigenic ability in vivo) include a more
rounded cell morphology, looser substratum attachment, loss of contact
inhibition,
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loss of anchorage dependence, release of proteases such as plasminogen
activator,
increased sugar transport, decreased serum requirement, expression of fetal
antigens and etc. (See Luria, et al. (1978) GENERAL VIROLOGY 3d Ed., 436-446,
(John Wiley & Sons, New York)).
The methods of this invention screen for the presence of polynucleotides
which can be identified by at least one of the sequences provided in SEQ ID
NOS.
1 through 40. Also provided are the polynucleotides of SEQ ID NOS 1 through
40 and the gene products of these polynucleotides. In a separate embodiment,
the
presence of a polypeptide or protein which is transcribed and translated (or
is a
subfragment of the gene product corresponding to the polynucleotide
transcript) is
indicative of the presence of the neoplastic condition of the cell. The
transcript is
identified by screening for mRNA that hybridizes to a probe comprising a
polynucleotide of any of SEQ ID NOS. 1 through 40, or their complements, or by
amplifying nucleic acid using a primer comprising a polynucleotide of any of
1 S SEQ ID NOS. 1 through 40, or their complement. PCR is the preferred method
of
amplifying sequences, although traditional cloning techniques also will
amplify a
known sequence and therefore fall within the scope of this invention.
Sequences
of polynucleotides isolated from samples suspected of containing lung cancer
cells can be compared against a database that comprises SEQ. ID NOS. 1 through
40 using an algorithm that identified sequence homologies. The presence of
high
sequence identity between the sequence sample and at least one of the
sequences
in the database is a positive indication that the sample contains a lung
cancer cell.
These methods can be used for aiding in the diagnosis of a lung cancer
such as squamous cell lung cancer by detecting a genotype that is correlated
with
a phenotype characteristic of primary lung tumor cells. Thus, by detecting
this
genotype prior to tumor growth, one can predict a predisposition to cancer or
provide early diagnosis.
Cell or tissue samples used for this invention encompass body fluid, solid
tissue samples, tissue cultures or cells derived therefrom and the progeny
thereof,
and sections or smears prepared from any of these sources, or any other
samples
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that may contain a lung cell: Thus, a preferred sample is one that is prepared
from
a subject's lung tissue.
In assaying for mRNA that hybridizes to the transcript, mRNA contained
in the aforementioned samples is first extracted according to standard methods
in
the art. For instance, in RNA can be isolated using various lytic enzymes or
chemical solutions according to the procedures set forth in Sambrook et al.
(1989), supra or extracted by nucleic-acid-binding resins following the
accompanying instructions provided by manufactures. The mRNA contained in
the extracted nucleic acid sample is then detected by hybridization (e.g.
Northern
blot analysis) and/or amplification procedures according to methods widely
known in the art or based on the methods exemplified herein.
Nucleic acid molecules having at least 10 nucleotides and exhibiting
sequence complementarity or homology to SEQ ID NOS. 1 through 40 find utility
as hybridization probes. In some aspects, the full coding sequence of the
transcript, i.e., for SEQ ID NOS. 1-20, are known. Accordingly, any portion of
the known sequences available in GenBank, or homologous sequences, can be
used in the methods of this invention. It is known in the art that a
"perfectly
matched" probe is not needed for a specific hybridization. Minor changes in
probe sequence achieved by substitution, deletion or insertion of a small
number
of bases do not affect the hybridization specificity. In general, as much as
20%
base-pair mismatch (when optimally aligned) can be tolerated. Preferably, a
probe useful for detecting the aforementioned mRNA is at least about 80%
identical to the homologous region of comparable size contained in the
previously
identified sequences identified by SEQ ID NOS. 1 through 20, which correspond
to previously characterized genes or SEQ ID NOS. 21-23, 27, 28, 30, 31, 37, 39
and 40, which correspond to known ESTs. More preferably, the probe is 85%
identical to the corresponding gene sequence after alignment of the homologous
region; even more preferably, it exhibits 90% identity. Percent identity is
determined as described above.
These probes can be used in radioassays (e.g. Southern and Northern blot
analysis) to detect, prognose, diagnose or monitor various neoplastic states
in lung
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cells or tissue. The probes also can be attached to a solid support such as a
chip
for use in high throughput screening assays for the detection and monitoring
of
lung neoplasm. Accordingly, this invention also provides at least one of the
transcripts identified as SEQ ID NOS. 1 through 40, or its complement,
attached
to a solid support for use in high throughput screens.
A polynucleotide of the invention also can be attached to a solid support
for use in high throughput screening assays. Using this method, the probes are
synthesized on a derivatized glass surface. Photoprotected nucleoside
phosphoramidites are coupled to the glass surface, selectively deprotected by
photolysis through a photolithographic mask, and reacted with a second
protected
nucleoside phosphoramidite. The coupling/deprotection process is repeated
until
the desired probe is complete.
The expression level of a gene is determined through exposure of a nucleic
acid sample to the probe-modified chip. Extracted nucleic acid is labeled, for
example, with a fluorescent tag, preferably during an amplification step.
Hybridization of the labeled sample is performed at an appropriate stringency
level. The degree of probe-nucleic acid hybridization is quantitatively
measured
using a detection device, such as a confocal microscope. See U.S. Patent
Nos. 5,578,832; and 5,631,734. The obtained measurement is directly correlated
with gene expression level.
Results from the chip assay are typically analyzed using a computer
software program. See, for example, EP 71 ?,113 A2 and WO 95/20681. The
hybridization data is read into the program, which calculates the expression
level
of the targeted gene(s). This figure is compared against existing data sets of
gene
expression levels for that cell type.
For example, the database and methods of using the database provides a
means to differentiate a cell expressing a peptide epitope which is the
natural
counterpart of a synthetic antigenic peptide epitope of the invention from a
cell
which does not express the epitope or expresses it at a higher or lower level
from
the cell in question. Expression of polynucleotides encoding the peptide is
measured. One cell would serve as a "reference cell" and the cell whose
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expression of a polynucleotide encoding a peptide corresponding to a synthetic
antigenic peptide epitope of the invention is to be measured could be referred
to as
the "test cell". As an example, the method can be used to distinguish a normal
cell (in this case, the reference cell) from a neoplastic cell (i.e., the test
cell). It
also allows one to differentiate between neoplastic cells biopsied from
different
regions from a patient or different subjects or gene expression before or
after
treatment with a potential therapeutic agent. It can be used to analyze drug
toxicity and efficacy, as well as to selectively look at protein categories
which are
expected to be affected by a drug or which may be overexpressed as a result of
treatment with a drug, such as the various mufti-drug resistant genes.
Additional
utilities of the database include, but are not limited to analysis of the
developmental state of a test cell, the influence of viral or bacterial
infection,
control of cell cycle, effect of a tumor suppressor gene or lack thereof,
polymorphism within the cell type, apoptosis, and the effect of regulatory
genes.
The total size of fragment, as well as the size of the complementary
stretches, will depend on the intended use or application of the particular
nucleic
acid segment. Smaller.fragments will generally find use in hybridization
embodiments, wherein the length of the complementary region may be varied,
such as between about 10 and about 100 nucleotides, or even full length
according
to the complementary sequences one wishes to detect.
Nucleotide probes having complementary sequences over stretches greater
than 10 nucleotides in length are generally preferred, so as to increase
stability
and selectivity of the hybrid, and thereby improving the specificity of
particular
hybrid molecules obtained. More preferably, one can design nucleic acid
molecules having gene-complementary stretches of about 25 nucleotides or more.
In some instances, molecules have complementary stretches of about 50
nucleotides in length, or even longer may be desired. Such fragments may be
readily prepared by, for example, directly synthesizing the fragment by
chemical
means, by application of nucleic acid reproduction technology, such as the
PCRTM
technology with two priming oligonucleotides as described in U.S. Pat. No.
4,603,102 or by introducing selected sequences into recombinant vectors for
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recombinant production. A preferred probe is about 50-75 or more preferably,
50-
100, nucleotides in length.
In certain embodiments, it will be advantageous to employ nucleic acid
sequences of the present invention in combination with an appropriate means,
such as a label, for detecting hybridization and therefore complementary
sequences. A wide variety of appropriate indicator means are known in the art,
including fluorescent, radioactive, enzymatic or other ligands, such as
avidin/biotin, which are capable of giving a detectable signal. In preferred
embodiments, one will likely desire to employ a fluorescent label or an enzyme
tag, such as crease, alkaline phosphatase or peroxidase, instead of
radioactive or
other environmental undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known which can be employed to provide a
means visible to the human eye or spectrophotometrically, to identify specific
hybridization with complementary nucleic acid-containing samples.
Hybridization reactions can be performed under conditions of different
"stringency". Relevant conditions include temperature, ionic strength, time of
incubation, the presence of additional solutes in the reaction mixture such as
formamide, and the washing procedure. Higher stringency conditions are those
conditions, such as higher temperature and lower sodium ion concentration,
which
require higher minimum complementarity between hybridizing elements for a
stable hybridization complex to form. Conditions that increase the stringency
of a
hybridization reaction are widely known and published in the art. See, for
example, (Sambrook, et al., (1989), supra) and the definitions, supra .
The nucleotide probes of the present invention can also be used as primers
for the detection of genes or gene transcripts that are expressed in
neoplastic lung
cells but not normal lung tissue. For the purpose of this invention,
amplification
means any method employing a primer-dependent polymerise capable of
replicating a target sequence with reasonable fidelity. Amplification may be
carried out by natural or recombinant DNA-polymerises such as T7 DNA
polymerise, Klenow fragment of E.coli DNA polymerise, and reverse
transcriptase.
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A preferred amplification method is PCR. General procedures for PCR
are taught in MacPherson, et al. PCR: A PRACTICAL APPROACH (IRL Press at
Oxford University Press { 1991 )). However, PCR conditions used for each
application reaction are empirically determined. A number of parameters
influence the success of a reaction. Among them are annealing temperature and
time, extension time, Mg2+ ATP concentration, pH, and the relative
concentration
of primers, templates, and deoxyribonucleotides.
After amplification, the resulting DNA fragments can be detected by
agarose gel electrophoresis followed by visualization with ethidium bromide
staining and ultraviolet illumination. A specific amplification
polynucleotides
having complementary sequences to the polynucleotides identified in SEQ ID
NOS. 1 through 40, demonstrating that the amplified DNA fragment has the
predicted size, exhibits the predicated restriction digestion pattern, and/or
hybridizes to the correct cloned DNA sequence.
1 S Expression of novel transcript can also be determined by assaying for the
presence of the protein product. Determining the protein level involves a)
providing a biological sample suspected of containing polypeptides; and (b)
measuring the amount of any immunospecific binding that occurs between an
antibody reactive to the protein product and detecting the presence of any
antibody: protein complex formed. The presence of a complex indicates that the
protein product was present in the sample and therefore, the sample contained
a
neoplastic lung cell.
Gene Delivery Vehicles and Host Cells
This invention also provides a polynucleotide, as described above,
incorporated into a gene delivery vehicle for expression an/or or insertion
vector
for incorporation into cells. Vectors that contain both a promoter and a
cloning
site into which a polynucleotide can be operatively linked are well known in
the
art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are
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WO 99/SOZ78 PCT/US99/06938
commercially available from sources such as Stratagene (La Jolla, CA) and
Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro
transcription, it may be necessary to remove, add or alter 5' and/or 3'
untranslated
portions of the clones to eliminate extra, potential inappropriate alternative
translation initiation codons or other sequences that may interfere with or
reduce
expression, either at the level of transcription or translation.
Alternatively,
consensus ribosome binding sites can be inserted immediately 5' of the start
codon to enhance expression. Examples of vectors are viruses, such as
baculovirus and retrovirus, bacteriophage, adenovirus, adeno-associated virus,
cosmid, plasmid, fungal vectors and other recombination vehicles typically
used
in the art which have been described for expression in a variety of eucaryotic
and
procaryotic hosts, and may be used for gene therapy as well as for simple
protein
expression.
Among these are several non-viral vectors, including DNA/liposome
complexes, and targeted viral protein DNA complexes. To enhance delivery to a
cell, the nucleic acid or proteins of this invention can be conjugated to
antibodies
or binding fragments thereof which bind cell surface antigens. Liposomes that
also comprise a targeting antibody or fragment thereof can be used in the
methods
of this invention. This invention also provides the targeting complexes for
use in
the methods disclosed herein.
Polynucleotides are inserted into vector genomes using methods well
known in the art. For example, insert and vector DNA can be contacted, under
suitable conditions, with a restriction enzyme to create complementary ends on
each molecule that can pair with each other and be joined together with a
ligase.
Alternatively, synthetic nucleic acid linkers can be ligated to the termini of
restricted polynucleotide. These synthetic linkers contain nucleic acid
sequences
that correspond to a particular restriction site in the vector DNA.
Additionally, an
oligonucleotide containing a termination codon and an appropriate restriction
site
can be ligated for insertion into a vector containing, for example, some or
all of
the following: a selectable marker gene, such as the neomycin gene for
selection
of stable or transient transfectants in mammalian cells; enhancer/promoter
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sequences from the immediate early gene of human CMV for high levels of
transcription; transcription termination and RNA processing signals from SV40
for mRNA stability; SV40 polyoma origins of replication and ColEl for proper
episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA
promoters for in vitro transcription of sense and antisense RNA. Other means
are
well known and available in the art.
This invention further provides host cells, as defined above, comprising a
polynucleotide of this invention.
Polypeptides and Their Utilities
The peptides used in accordance with the methods of the present invention
can be obtained in any one of a number of conventional ways. Because they will
generally be short sequences, they can be prepared by chemical synthesis using
standard techniques. Particularly convenient are the solid phase peptide
synthesis
techniques. Automated peptide synthesizers are commercially available, as are
the reagents required for their use. Alternatively, the peptides can be
prepared by
enzymatic digestion or cleavage of naturally occurring proteins. The peptides
can
also be prepared using recombinant techniques known to those of skill in the
art.
In one embodiment, isolated peptides of the present invention can be
synthesized using an appropriate solid state synthetic procedure. Steward and
Young (1968) SOLID PHASE PEPTIDE SYNTHESIS, Freemantle, San Francisco,
Calif. A preferred method is the Merrifield process. Merrifield (1967) Recent
Progress in Hormone Res. 23:451. The antigenic activity of these peptides may
conveniently be tested using, for example, the assays as described herein.
Once an isolated peptide of the invention is obtained, it may be purified by
standard methods including chromatography (e.g., ion exchange, affinity, and
sizing column chromatography), centrifugation, differential solubility, or by
any
other standard technique for protein purification. For immunoaffinity
chromatography, an epitope may be isolated by binding it to an affinity column
comprising antibodies that were raised against that peptide, or a related
peptide of
the invention, and were affixed to a stationary support.
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Alternatively, affinity tags such as hexa-His (Invitrogen), Maltose binding
domain (New England Biolabs), influenza coat sequence (Kolodziej et al. ( 1991
)
Methods Enzymol. 194:508-509), and glutathione-S-transferase can be attached
to
the peptides of the invention to allow easy purification by passage over an
appropriate affinity column. Isolated peptides can also be physically
characterized using such techniques as proteolysis, nuclear magnetic
resonance,
and x-ray crystallography.
Also included within the scope of the invention are peptides encoded by
the polynucleotides of this invention that are differentially modified during
or
after translation, e.g., by phosphorylation, glycosylation, crosslinking,
acylation,
proteolytic cleavage, linkage to an antibody molecule, membrane molecule or
other ligand. Ferguson et al. (i988) Ann. Rev. Biochem. 57:285-320. 'This is
achieved using various chemical methods or by expressing the polynucleotides
in
different host cells, e.g., bacterial, mammalian, yeast, or insect cells.
A polypeptide of the invention can be used in a variety of formulations,
which may vary depending on the intended use. A polypeptide can be covalently
or non-covalently linked (complexed) to various other molecules, the nature of
which may vary depending on the particular purpose. For example, a peptide of
the invention can be covalently or non-covalently complexed to a
macromolecular
carnet, including, but not limited to, natural and synthetic polymers,
proteins,
polysaccharides, poly(aminoacid), polyvinyl alcohol, polyvinyl pyrrolidone,
and
lipids. A peptide can be conjugated to a fatty acid, for introduction into a
liposome. U.S. Patent No. 5,837,249. A synthetic peptide of the invention can
be complexed covalently or non-covalently with a solid support, a variety of
which are known in the art. A synthetic antigenic peptide epitope of the
invention
can be associated with an antigen-presenting matrix with or without co
stimulatory molecules, as described in more detail below.
Examples of protein carriers include, but are not limited to, superantigens,
serum albumin, tetanus toxoid, ovalbumin, thyroglobulin, myoglobulin, and
immunoglobulin.
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Peptide-protein carrier polymers may be formed using conventional
crosslinking agents such as carbodiimides. Examples of carbodiimides are 1-
cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1-ethyl-3-(3-
dimethyaminopropyl) carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44-
dimethylpentyl) carbodiimide.
Examples of other suitable crosslinking agents are cyanogen bromide,
glutaraldehyde and succinic anhydride. In general, any of a number of
homobifunctional agents including a homobifunctional aldehyde, a
homobifunctional epoxide, a homobifunctional imidoester, a homobifunctional N-
hydroxysuccinimide ester, a homobifunctional maleimide, a homobifunctional
alkyl halide, a homobifunctional pyridyl disulfide, a homobifunctional aryl
halide,
a homobifunctional hydrazide, a homobifunctional diazonium derivative and a
homobifunctional photoreactive compound may be used. Also included are
heterobifunctional compounds, for example, compounds having an amine-reactive
and a sulfhydryl-reactive group, compounds with an amine-reactive and a
photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-
reactive group.
Specific examples of such homobifunctional crosslinking agents include
the bifunctional N-hydroxysuccinimide esters
dithiobis(succinimidylpropionate),
disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional
imidoesters
dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the
bifunctional sulfliydryl-reactive crosslinkers 1,4-di-[3'-(2'-pyridyldithio)
propion-
amido]butane, bismaleimidohexane, and bis-N-maleimido-1, 8-octane; the
bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-
3,3'-
dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4-
azidosalicylamido)ethyl]disulfide; the bifunctional aldehydes formaldehyde,
malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a
bifunctional epoxide such as 1,4-butaneodiol diglycidyl ether, the
bifunctional
hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid
dihydrazide; the bifunctional diazoniums o-toiidine, diazotized and bis-
diazotized
benzidine; the bifunctional alkylhalides N1N'-ethylene-bis(iodoacetamide),
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N1N'-hexamethylene-bis(iodoacetamide), N1N'-undecamethylene-
bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala'-
diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively.
Examples of other common heterobifunctional cross-linking agents that
may be used to effect the conjugation of proteins to peptides include, but are
not
limited to, SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-
carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB
(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p-
maleimidophenyl)butyrate), GMBS (N-(Y-maleimidobutyryloxy)succinimide
ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-
(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT
(succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP (N-
succinimidyl 3-(2-pyridyldithio)propionate).
Crosslinking may be accomplished by coupling a carbonyl group to an
amine group or to a hydrazide group by reductive amination.
Peptides of the invention also may be formulated as non-covalent
attachment of monomers through ionic, adsorptive, or biospecific interactions.
Complexes of peptides with highly positively or negatively charged molecules
may be done through salt bridge formation under low ionic strength
environments,
such as in deionized water. Large complexes can be created using charged
polymers such as poly-(L-glutamic acid) or poly-(L-lysine) which contain
numerous negative and positive charges, respectively. Adsorption of peptides
may be done to surfaces such as microparticle latex beads or to other
hydrophobic
polymers, forming non-covalently associated peptide-superantigen complexes
effectively mimicking crosslinked or chemically polymerized protein. Finally,
peptides may be non-covalently linked through the use of biospecific
interactions
between other molecules. For instance, utilization of the strong affinity of
biotin
for proteins such as avidin or streptavidin or their derivatives could be used
to
form peptide complexes. These biotin-binding proteins contain four binding
sites
that can interact with biotin in solution or be covalently attached to another
molecule. Wilchek (1988) Anal Biochem. 171:1-32. Peptides can be modified to
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possess biotin groups using common biotinylation reagents such as the N-
hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts with available
amine groups on the protein. Biotinylated peptides then can be incubated with
avidin or streptavidin to create large complexes. The molecular mass of such
polymers can be regulated through careful control of the molar ratio of
biotinylated peptide to avidin or streptavidin.
Also provided by this application are the peptides and polypeptides
described herein conjugated to a detectable agent for use in the diagnostic
methods. For example, detectably labeled peptides and polypeptides can be
bound to a column and used for the detection and purification of antibodies.
They
also are useful as immunogens for the production of antibodies, as described
below.
The peptides of this invention also can be combined with various liquid
phase carriers, such as sterile or aqueous solutions, pharmaceutically
acceptable
carriers, suspensions and emulsions. Examples of non-aqueous solvents include
propyl ethylene glycol, polyethylene glycol and vegetable oils. When used to
prepare antibodies, the carriers also can include an adjuvant that is useful
to non-
specifically augment a specific immune response. A skilled artisan can easily
determine whether an adjuvant is required and select one. However, for the
purpose of illustration only, suitable adjuvants include, but are not limited
to,
Freund's Complete and Incomplete, mineral salts and polynucleotides.
In one embodiment, the peptides of this invention are used to raise
antibodies that specifically bind epitopes present on the peptides. The
antibodies
are useful to detect the gene products isolated from a cell sample.
A variety of techniques are available in the art for protein analysis. They
include but are not limited to radioimmunoassays, ELISA (enzyme linked
immunoradiometric assays), "sandwich" immunoassays, immunoradiometric
assays, in situ immunoassays (using e.g., colloidal gold, enzyme or
radioisotope
labels), western blot analysis, immunoprecipitation assays, immunoflourescent
assays, and PAGE-SDS. See Harlow and Lane (1988) supra. and Sambrook et al.
(1989) supra.
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To detect and quantify the immunospecific binding, or signals generated
during hybridization or amplification procedures, digital image analysis
systems
including but not limited to those that detect radioactivity of the probes or
chemiluminescence can be employed.
In diagnosing malignancy, hyperplasia or metaplasia characterized by a
expression of a transcript identified herein by SEQ ID NOS. 1 through 40, or
its
complement, one typically conducts a comparative analysis of the subject and
appropriate controls. Preferably, a diagnostic test includes a control sample
derived from a subject (hereinafter positive control), that exhibits
expression of
the transcript and clinical characteristics of the malignancy or metaplasia
lung
cancer. More preferably, a diagnosis also includes a control sample derived
from
a subject (hereinafter negative control), that lacks the clinical
characteristics of the
neoplastic state and whose expression of the transcript in question is
negative or
de minimus. A positive correlation between the subject and the positive
control
with respect to the identified alterations indicates the presence or a
predisposition
to the development of a neoplastic lung condition. A lack of correlation
between
the subject and the negative control confirms the diagnosis.
Drug Screening
The present invention also provides a screen for various agents and
methods for reversing the neoplastic condition of the cells or selectively
inhibiting
growth or proliferation of the cells described above. In one aspect, the
screen
assays for agents which are useful for the treatment of malignancy,
hyperplasia or
metaplasia characterized by expression of the transcript described herein.
Thus, to practice the method in vitro, suitable cell cultures or tissue
cultures are first provided. The cell can be a cultured cell or a genetically
modified cell in which a transcript from SEQ ID NOS. 1 through 40, or its
complement, is expressed. Alternatively, the cells can be from a tissue
biopsy.
The cells are cultured under conditions (temperature, growth or culture medium
and gas (C02)) and for an appropriate amount of time to attain exponential
proliferation without density dependent constraints. It also is desirable to
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maintain an additional separate cell culture; one which does not receive the
agent
being tested as a control.
As is apparent to one of skill in the art, suitable cells may be cultured in
microtiter plates and several agents may be assayed at the same time by noting
genotypic changes, phenotypic changes or cell death.
When the agent is a composition other than a DNA or RNA nucleic acid
molecule, the suitable conditions may be by directly added to the cell culture
or
added to culture medium for addition. As is apparent to those skilled in the
art, an
"effective" amount must be added which can be empirically determined.
For the purposes of this invention, an "agent" is intended to include, but
not be limited to a biological or chemical compound such as a simple or
complex
organic or inorganic molecule, a peptide, a protein (e.g. antibody) or an
oligonucleotide (e.g. anti-sense). A vast array of compounds can be
synthesized,
for example oligomers, such as oligopeptides and oligonucleotides, and
synthetic
organic compounds based on various core structures, and these are also
included
in the term "agent". In addition, various natural sources can provide
compounds
for screening, such as plant or animal extracts, and the like. It should be
understood, although not always explicitly stated that the agent is used alone
or in
combination with another agent, having the same or different biological
activity as
the agents identified by the inventive screen. The agents and methods also are
intended to be combined with other therapies.
As noted above, lung cells having overexpression of a proto-oncogene that
results in the neoplastic state are suitably treated by this method. These
cells can
be identified by any method known in the art that allows for the
identification of
overexpression of the proto-oncogene. One such method is exemplified below.
When the agent is a nucleic acid, it can be added to the cell cultures by
methods well known in the art, which includes, but is not limited to calcium
phosphate precipitation, microinjection or electroporation.
One can determine if the object of the method, i.e., reversal of the
neoplastic state of the cell, has been achieved by a reduction of cell
division,
differentiation of the cell or assaying for a reduction or loss of transcript
CA 02323833 2000-09-18
WO 99/50278 PCT/US99/06938
expression. Cellular differentiation can be monitored by histological methods
or
by monitoring for the presence or loss of certain cell surface markers, which
may
be associated with an undifferentiated phenotype, e.g. CD34 on primative
hematopoietic stem cells.
Kits containing the agents and instructions necessary to perform the screen
and in vitro method as described herein also are claimed.
When the subject is an animal such as a rat or mouse, the method provides
a convenient animal model system which can be used prior to clinical testing
of
the therapeutic agent. In this system, a candidate agent is a potential drug
if
transcript expression is reduced in a neoplastic lung cell or if symptoms
associated
or correlated to the presence of cells containing transcript expression are
ameliorated, each as compared to untreated, animal having the pathological
cells.
It also can be useful to have a separate negative control group of cells or
animals
which are healthy and not treated, which provides a basis for comparison.
1 S These agents of this invention and the above noted compounds and their
derivatives may be used for the preparation of medicaments for use in the
methods described herein.
In a preferred embodiment, an agent of the invention is administered to
treat lung cancer. In a further preferred embodiment, an agent of the
invention is
administered to treat squamous cell lung cancer. Therapeutics of the invention
can also be used to prevent progression from a pre-neoplastic or non-malignant
state into a neoplastic or a malignant state.
Various delivery systems are known and can be used to administer a
therapeutic agent of the invention, e.g., encapsulation in liposomes,
microparticles, microcapsules, expression by recombinant cells, receptor-
mediated endocytosis (see, e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-
4432), construction of a therapeutic nucleic acid as part of a retroviral or
other
vector, etc. Methods of delivery include but are not limited to intra-
arterial, intra-
muscular, intravenous, intranasal, and oral routes. In a specific embodiment,
it
may be desirable to administer the pharmaceutical compositions of the
invention
locally to the area in need of treatment; this may be achieved by, for
example, and
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WO 99/50278 PCT/US99/06938
not by way of limitation, local infusion during surgery, by injection, or by
means
of a catheter.
The agents identified herein as effective for their intended purpose can be
administered to subjects or individuals susceptible to or at risk of
developing a
disease correlated to the overexpression of these proto-oncogenes. When the
agent is administered to a subject such as a mouse, a rat or a human patient,
the
agent can be added to a pharmaceutically acceptable carrier and systemically
or
topically administered to the subject. To determine patients that can be
beneficially treated, a tumor sample is removed from the patient and the cells
are
assayed for the overexpression of the proto-oncogene. Therapeutic amounts can
be empirically determined and will vary with the pathology being treated, the
subject being treated and the e~cacy and toxicity of the agent. When delivered
to
an animal, the method is useful to further confirm efficacy of the agent. As
an
example of an animal model, groups of nude mice (Balb/c NCR nu/nu female,
Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 105 to
about 109 hyperproliferative, cancer or target cells as defined herein. When
the
tumor is established, the agent is administered, for example, by subcutaneous
injection around the tumor. Tumor measurements to determine reduction of
tumor size are made in two dimensions using venier calipers twice a week.
Other
animal models may also be employed as appropriate.
Administration in vivo can be effected in one dose, continuously or
intermittently throughout the course of treatment. Methods of determining the
most effective means and dosage of administration are well known to those of
skill in the art and will vary with the composition used for therapy, the
purpose of
the therapy, the target cell being treated, and the subject being treated.
Single or
multiple administrations can be carried out with the dose level and pattern
being
selected by the treating physician. Suitable dosage formulations and methods
of
administering the agents can be found below.
The agents and compositions of the present invention can be used in the
manufacture of medicaments and for the treatment of humans and other animals
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WO 99/50278 PCT/US99/06938
by administration in accordance with conventional procedures, such as an
active
ingredient in pharmaceutical compositions.
The pharmaceutical compositions can be administered orally, intranasally,
parenterally or by inhalation therapy, and may take the form of tablets,
lozenges,
granules, capsules, pills, ampoules, suppositories or aerosol form. They may
also
take the form of suspensions, solutions and emulsions of the active ingredient
in
aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to
an
agent of the present invention, the pharmaceutical compositions can also
contain
other pharmaceutically active compounds or a plurality of compounds of the
invention.
More particularly, an agent of the present invention also referred to herein
as the active ingredient, may be administered for therapy by any suitable
route
including oral, rectal, nasal, topical (including transdermal, aerosol, buccal
and
sublingual), vaginal, parental (including subcutaneous, intramuscular,
intravenous
and intradermal) and pulmonary. It will also be appreciated that the preferred
route will vary with the condition and age of the recipient, and the disease
being
treated.
Ideally, the agent should be administered to achieve peak concentrations
of the active compound at sites of disease. This may be achieved, for example,
by
the intravenous injection of the agent, optionally in saline, or orally
administered,
for example, as a tablet, capsule or syrup containing the active ingredient.
Desirable blood levels of the agent may be maintained by a continuous infusion
to
provide a therapeutic amount of the active ingredient within disease tissue.
The
use of operative combinations is contemplated to provide therapeutic
combinations requiring a lower total dosage of each component antiviral agent
than may be required when each individual therapeutic compound or drug is used
alone, thereby reducing adverse effects.
While it is possible for the agent to be administered alone, it is preferable
to present it as a pharmaceutical formulation comprising at least one active
ingredient, as defined above, together with one or more pharmaceutically
acceptable carriers therefor and optionally other therapeutic agents. Each
carrier
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must be "acceptable" in the sense of being compatible with the other
ingredients
of the formulation and not injurious to the patient.
Formulations include those suitable for oral, rectal, nasal, topical
(including transdermal, buccal and sublingual), vaginal, parenteral (including
subcutaneous, intramuscular, intravenous and intradermal) and pulmonary
administration. The formulations may conveniently be presented in unit dosage
form and may be prepared by any methods well known in the art of pharmacy.
Such methods include the step of bringing into association the active
ingredient
with the carrier which constitutes one or more accessory ingredients. In
general,
the formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely divided solid
carriers or both, and then if necessary shaping the product.
Formulations of the present invention suitable for oral administration may
be presented as discrete units such as capsules, cachets or tablets, each
containing
a predetermined amount of the active ingredient; as a powder or granules; as a
solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-
water
liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may
also
be presented a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or
more accessory ingredients. Compressed tablets may be prepared by compressing
in a suitable machine the active ingredient in a free-flowing form such as a
powder or granules, optionally mixed with a binder (e.g., povidone, gelatin,
hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant
(e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium
carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets
may
be made by molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may optionally be coated
or
scored and may be formulated so as to provide slow or controlled release of
the
active ingredient therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile. Tablets may
optionally
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be provided with an enteric coating, to provide release in parts of the gut
other
than the stomach.
Formulations suitable for topical administration in the mouth include
lozenges comprising the active ingredient in a flavored basis, usually sucrose
and
acacia or tragacanth; pastilles comprising the active ingredient in an inert
basis
such as gelatin and glycerin, or sucrose and acacia; and mouthwashes
comprising
the active ingredient in a suitable liquid carrier.
Pharmaceutical compositions for topical administration according to the
present invention may be formulated as an ointment, cream, suspension, lotion,
powder, solution, past, gel, spray, aerosol or oil. Alternatively, a
formulation may
comprise a patch or a dressing such as a bandage or adhesive plaster
impregnated
with active ingredients and optionally one or more excipients or diluents.
If desired, the aqueous phase of the cream base may include, for example,
at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or
more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol,
sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical
formulations may desirably include a compound which enhances absorption or
penetration of the agent through the skin or other affected areas. Examples of
such dermal penetration enhancers include dimethylsulfoxide and related
analogues.
The oily phase of the emulsions of this invention may be constituted from
known ingredients in an known manner. While this phase may comprise merely
an emulsifier (otherwise known as an emulgent), it desirably comprises a
mixture
of at lease one emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with a lipophilic
emulsifier which acts as a stabilizer. It is also preferred to include both an
oil and
a fat. Together, the emulsifiers) with or without stabilizers) make up the so-
called emulsifying wax, and the wax together with the oil and/or fat make up
the
so-called emulsifying ointment base which forms the oily dispersed phase of
the
cream formulations.
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WO 99/50278 PCT/US99/06938
Emulgents and emulsion stabilizers suitable for use in the formulation of
the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl
alcohol, glyceryl monostearate and sodium lauryl sulphate.
The choice of suitable oils or fats for the formulation is based on achieving
the desired cosmetic properties, since the solubility of the active compound
in
most oils likely to be used in pharmaceutical emulsion formulations is very
low.
Thus the cream should preferably be a non-greasy, non-staining and washable
product with suitable consistency to avoid leakage from tubes or other
containers.
Straight or branched chain, mono- or dibasic alkyl esters such as di-
isoadipate,
isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl
myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl
palmitate
or a blend of branched chain esters known as Crodamol CAP may be used, the
last
three being preferred esters. These may be used alone or in combination
depending on the properties required. Alternatively, high melting point lipids
such as white soft paraffin and/or liquid para~n or other mineral oils can be
used.
Formulations suitable for topical administration to the eye also include eye
drops wherein the active ingredient is dissolved or suspended in a suitable
earner,
especially an aqueous solvent for the agent.
Formulations for rectal administration may be presented as a suppository
with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable fox vaginal administration may be presented as
pessaries, tampons, creams, gels, pastes, foams or spray formulations
containing
in addition to the agent, such carriers as are known in the art to be
appropriate.
Formulations suitable for nasal administration, wherein the carrier is a
solid, include a coarse powder having a particle size, for example, in the
range of
about 20 to about 500 microns which is administered in the manner in which
snuff
is taken, i.e., by rapid inhalation through the nasal passage from a container
of the
powder held close up to the nose. Suitable formulations wherein the carrier is
a
liquid for administration as, for example, nasal spray, nasal drops, or by
aerosol
administration by nebulizer, include aqueous or oily solutions of the agent.
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Formulations suitable for parenteral administration include aqueous and
non-aqueous isotonic sterile injection solutions which may contain anti-
oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic with
the
blood of the intended recipient; and aqueous and non-aqueous sterile
suspensions
which may include suspending agents and thickening agents, and liposomes or
other microparticulate systems which are designed to target the compound to
blood components or one or more organs. The formulations may be presented in
unit-dose or mufti-dose sealed containers, for example, ampoules and vials,
and
may be stored in a freeze-dried (lyophilized) condition requiring only the
addition
of the sterile liquid carrier, for example water for injections, immediately
prior to
use. Extemporaneous injection solutions and suspensions may be prepared from
sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or
unit, daily subdose, as herein above-recited, or an appropriate fraction
thereof, of
1 S a agent.
It should be understood that in addition to the ingredients particularly
mentioned above, the formulations of this invention may include other agents
conventional in the art having regard to the type of formulation in question,
for
example, those suitable for oral administration may include such further
agents as
sweeteners, thickeners and flavoring agents. It also is intended that the
agents,
compositions and methods of this invention be combined with other suitable
compositions and therapies.
Antibodies
Also provided by this invention is an antibody capable of specifically
forming a complex with the proteins or polypeptides as described above. The
term "antibody" includes polyclonal antibodies and monoclonal antibodies. The
antibodies include, but are not limited to mouse, rat, and rabbit or human
antibodies.
Laboratory methods for producing poiyclonal antibodies and monoclonal
antibodies, as well as deducing their corresponding nucleic acid sequences,
are
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known in the art, see Harlow and Lane (1988) supra and Sambrook et al. (1989)
supra. The monoclonal antibodies of this invention can be biologically
produced
by introducing protein or a fragment thereof into an animal, e.g., a mouse or
a
rabbit. The antibody producing cells in the animal are isolated and fused with
myeloma cells or heteromyeloma cells to produce hybrid cells or hybridomas.
Accordingly, the hybridoma cells producing the monoclonal antibodies of this
invention also are provided.
Thus, using the protein or fragment thereof, and well known methods, one
of skill in the art can produce and screen the hybridoma cells and antibodies
of
this invention for antibodies having the ability to bind the proteins or
polypeptides.
If a monoclonal antibody being tested binds with the protein or
polypeptide, then the antibody being tested and the antibodies provided by the
hybridomas of this invention are equivalent. It also is possible to determine
without undue experimentation, whether an antibody has the same specificity as
the monoclonal antibody of this invention by determining whether the antibody
being tested prevents a monoclonal antibody of this invention from binding the
protein or polypeptide with which the monoclonal antibody is normally
reactive.
If the antibody being tested competes with the monoclonal antibody of the
invention as shown by a decrease in binding by the monoclonal antibody of this
invention, then it is likely that the two antibodies bind to the same or a
closely
related epitope. Alternatively, one can pre-incubate the monoclonal antibody
of
this invention with a protein with which it is normally reactive, and
determine if
the monoclonal antibody being tested is inhibited in its ability to bind the
antigen.
If the monoclonal antibody being tested is inhibited then, in all likelihood,
it has
the same, or a closely related, epitopic specificity as the monoclonal
antibody of
this invention.
The term "antibody" also is intended to include antibodies of all isotypes.
Particular isotypes of a monoclonal antibody can be prepared either directly
by
selecting from the initial fusion, or prepared secondarily, from a parental
hybridoma secreting a monoclonal antibody of different isotype by using the
sib
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selection technique to isolate class switch variants using the procedure
described
in Steplewski et al. (1985) Proc. Natl. Acad. Sci. 82:8653 or Spira et al.
(1984) J.
Immunol. Methods 74:307.
This invention also provides biological active fragments of the polyclonal
and monoclonal antibodies described above. These "antibody fragments" retain
some ability to selectively bind with its antigen or immunogen. Such antibody
fragments can include, but are not limited to:
(1) Fab,
(2) Fab',
(3 ) F(ab')2,
(4) Fv, and
(S) SCA
A specific example of "a biologically active antibody fragment" is a CDR
region of the antibody. Methods of making these fragments are known in the
art,
see for example, Harlow and Lane (1988) supra.
The antibodies of this invention also can be modified to create chimeric
antibodies and humanized antibodies (Oi, et al. (1986) BioTechniques
4(3):214).
Chimeric antibodies are those in which the various domains of the antibodies'
heavy and light chains are coded for by DNA from more than one species.
The isolation of other hybridomas secreting monoclonal antibodies with
the specificity of the monoclonal antibodies of the invention can also be
accomplished by one of ordinary skill in the art by producing anti-idiotypic
antibodies (Herlyn et al. ( 1986) Science 232:100). An anti-idiotypic antibody
is
an antibody which recognizes unique determinants present on the monoclonal
antibody produced by the hybridoma of interest.
Idiotypic identity between monoclonal antibodies of two hybridomas
demonstrates that the two monoclonal antibodies are the same with respect to
their
recognition of the same epitopic determinant. Thus, by using antibodies to the
epitopic determinants on a monoclonal antibody it is possible to identify
other
hybridomas expressing monoclonal antibodies of the same epitopic specificity.
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It is also possible to use the anti-idiotype technology to produce
monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic
monoclonal antibody made to a first monoclonal antibody will have a binding
domain in the hypervariable region which is the mirror image of the epitope
bound by the first monoclonal antibody. Thus, in this instance, the anti-
idiotypic
monoclonal antibody could be used for immunization for production of these
antibodies.
As used in this invention, the term "epitope" is meant to include any
determinant having specific affinity for the monoclonal antibodies of the
invention. Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and usually
have
specific three dimensional structural characteristics, as well as specific
charge
characteristics.
The antibodies of this invention can be linked to a detectable agent or
label. There are many different labels and methods of labeling known to those
of
ordinary skill in the art.
The antibody-label complex is useful to detect the protein or fragments in
a sample, using standard immunochemical techniques such as
immunohistochemistry as described by Harlow and Lane (1988) supra.
Competitiv a and non-competitive immunoassays in either a direct or indirect
format are examples of such assays, e.g., enzyme linked immunoassay (ELISA)
radioimmunoassay (RIA) and the sandwich (immunometric) assay. Those of skill
in the art will know, or can readily discern, other immunoassay formats
without
undue experimentation.
The coupling of antibodies to low molecular weight haptens can increase
the sensitivity of the assay. The haptens can then be specifically detected by
means of a second reaction. For example, it is common to use haptens such as
biotin, which reacts avidin, or dinitropherryl, pyridoxal, and fluorescein,
which
can react with specific anti-hapten antibodies. See Harlow and Lane (1988)
supra.
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The monoclonal antibodies of the invention also can be bound to many
different Garners. Thus, this invention also provides compositions containing
the
antibodies and another substance, active or inert. Examples of well-known
carriers include glass, polystyrene, polypropylene, polyethylene, dextran,
nylon,
amylases, natural and modified celluloses, polyacrylamides, agaroses and
magnetite. The nature of the Garner can be either soluble or insoluble for
purposes of the invention. Those skilled in the art will know of other
suitable
carriers for binding monoclonal antibodies, or will be able to ascertain such,
using
routine experimentation.
Compositions
Also provided by this invention are compositions containing or comprising
one or more of the polynucleotides, genes, polypeptides, proteins, gene
delivery
vehicles, vectors, host cells or antibodies and a carrier, such as a solid
support. In
one embodiment, the carrier is a pharmaceutically acceptable carrier, as
defined
above. Medicaments useful for the diagnosis and treatment of lung cancer are
further provided by this invention.
Non-Human Transgenic Animals
In another aspect, the novel polynucleotide sequences associated with lung
cancer can be used to generate transgenic animal models. In recent years,
geneticists have succeeded in creating transgenic animals, for example mice,
by
manipulating the genes of developing embryos and introducing foreign genes
into
these embryos. Once these genes have integrated into the genome of the
recipient
2S embryo, the resulting embryos or adult animals can be analyzed to determine
the
function of the gene. The mutant animals are produced to understand the
function of known genes in vivo and to create animal models of human diseases.
(see, e.g., Chisaka et al. (1992) 355:516-520; Joyner et al. (1992) in
POSTIMPLANTATION DEVELOPMENT IN THE MOUSE (Chadwick and Marsh, eds.,
John Wiley & Sons, United Kingdom) pp:277-297; Dorin et al. (1992) Nature
359:211-21 S).
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U.S. Patent Nos. 5,464,764 and 5,487,992 describe one type of transgenic
animal in which the gene of interest is deleted or mutated sufficiently to
disrupt its
function. (See, also U.S. Patent Nos. 5,631,153 and 5,627,059). These "knock-
out" animals, made by taking advantage of the phenomena of homologous
recombination, can be used to study the function of a particular gene sequence
in
vivo. The polynucleotide sequences described herein are useful in preparing
animal models of lung cancer.
Computational Analysis and Genomics Applications
This invention also provides a process for preparing a database comprising
at least one of the sequences identified in SEQ ID NO. 1 to 40 or its
respective
complement. Alternatively, the database comprises at least one of the
sequences
selected from the group consisting of 24 to 26, 29, 32, or 38, or its
respective
complement. The polynucleotide sequences are stored in a digital storage
medium such that a data processing system for standardized representation of
the
genes that identify a lung cancer cell is compiled. The data processing system
is
useful to analyze gene expression between two cells by first selecting a cell
suspected of being of a neoplastic phenotype or genotype and then isolating
polynucleotides from the cell. The isolated polynucleotides are sequenced. The
sequences from the sample are compared with the sequences) present in the
database using homology search techniques. Greater than 90%, more preferably
greater than 95% and more preferably, greater than or equal to 97% sequence
identity between the test sequence and at least one sequence identified by SEQ
ID
NO. 1 through 40 or its complement is a positive indication that the
polynucleotide has been isolated from a lung cancer cell as defined above.
In an alternative embodiment, the polynucleotides of this invention are
sequenced and the information regarding sequence and in some embodiments,
relative expression, is stored in any functionally relevant program, e.g., in
Compare Report using the SAGE software (available through Dr. Ken Kinzler at
Johns Hopkins University). The Compare Report provides a tabulation of the
polynucleotide sequences and their abundance for the samples (say A, B, C and
D
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above) normalized to a defined number of polynucleotides per library (say
25,000). This is then imported into MS-ACCESS either directly or via copying
the data into an Excel spreadsheet first and then from there into MS-ACCESS
for
additional manipulations. Other programs such as SYBASE or Oracle that permit
the comparison of polynucleotide numbers could be used as alternatives to MS-
ACCESS. Enhancements to the software can be designed to incorporate these
additional functions. These functions consist in standard Boolean, algebraic,
and
text search operations, applied in various combinations to reduce a large
input set
of polynucleotides to a manageable subset of polynucleotides of specifically
defined interest.
The researcher may create groups containing one or more projects) by
combining the counts of specific polynucleotides within a group (e.g.,
GroupNormal = Normal l + Normal2, GroupTumor = PrimaryTumorl +
TumorCellLine). Additional characteristic values are also calculated for each
tag
in the group (e.g., average count, minimum count, maximum count). The
researcher may calculate individual tag count ratios between groups, for
example
the ratio of the average GroupNormal count to the average GroupTumor count for
each polynucleotide. The researcher may calculate a statistical measure of the
significance of observed differences in tag counts between groups.
The following examples are intended to illustrate, but not limit this
invention.
EXAMPLES
Example l:
A systematic analysis of transcripts present in non-small cell lung cancer
(NSCLC) was performed by Serial Analysis of Gene Expression ("SAGE")
(U.S. Patent No. 5,695,937). Primary squamous cell lung cancers containing
over
95% neoplastic components from two unrelated patients were selected for SAGE
analysis. Patient A was 58-year old and diagnosed with moderately
differentiated
cancer at the lower right lobe of the lung at the time of surgery. Patient B
was 68-
year old and diagnosed with poorly differentiated cancer of the lower right
lobe.
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Normal small airway epithelial cells obtained from two independent individuals
were used as the negative controls.
The SAGE libraries were constructed essentially as described in
Velculescu V et al. (1995) Science 270:484-487. PolyA RNAs isolated from lung
tumors of patients A and B, and from normal small airway epithelial cells were
converted to double-stranded cDNA. The cDNA was then cleaved vrith an
anchoring enzyme NIaIII and divided into two pools. Linkers containing
recognition sites for the tagging enzyme BsmFI was ligated to each pool: After
BsmFl restriction, SAGE tag overhangs were filled-in with Klenow, and tags
from the two pools were combined and ligated to each other. The ligation
product
was diluted and then amplified by PCR. The resulting PCR product was then
analyzed by polyacrylamide gel electrophoresis (PAGE), and the PCR product
containing two tags ligated tail to tail (ditag) was excised and then cleaved
with
NIaIII. After NIaIII restriction, the ditags was excised and self ligated. The
concatenated products were separated by PAGE and those containing 500 to
2000 nucleotide base pairs were excised and cloned for subsequent sequence
analysis.
Approximately 2000-4000 individual colonies were isolated and
sequenced from each SAGE library. The sequences of over 50,000 tags that
represent about 15,000 unique transcripts in each library were analyzed in
order to
generate a comprehensive profile of gene expression patterns in lung cancer.
Tables I and II summarize the comparative SAGE analyses of cDNA clones
derived from the lung cancers of two individuals and the lungs of two normal
individuals.
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Table I provides the transcripts of those sequences which correspond to
previously characterized genes.
TABLE I
Sequence Known Gene SEQ ID NO GenBank
Accession
No.
AAGGAGCAAG carboxylesterase1 X52973;
M5509
CTCCTGGGCG NB 1 2 M58026;
AA0555
GATAGCACAG 1 GFbpS 3 L27560
TGCTGCCTGT HCG4, BST2 4 X81005
CCATTTTTAC U2snrnp aux fac S M96982
GTCCCTGCCT GST sub 4 or 6 X08020;
GST 1
or GSTM 2 J03817
CAACTAATTC apolipe J or 7 X14723;
SP40 or
trpm-2, or sulfated J02908;
gP2 M74816
GTTATAAGAT DSSI 8 U41515;
U61847
TATTTTTGTT thioredoxin 9 X91247;
reductase 579851
CAGATAACAT myeloblast 10 D 13641
mitochondria)
outer
memb protein
TGTACCTGTA a-tubulin 11 K00558
CCAGGGGAGA p27 12 X67325
GAGAAAACCC sox 2 or HMG I3 231560
box
ATGTACCTGA epithelial memb 14 X94770;
proT 2/XMP U52100
TTCTAACATA Na/K ATPase (3 15 X03747;
subunit U 16799;
M25160
GGTGGTGTCT glutathione perox-16 X68314;
like protein X53463
TACTAGTCCT HSP90 - 17 D87666;
X15183
ATGCAGCCAT ODC-1 18 X55362
TGCTGCCCTG _ 19 X13293;
B-myb
T28987
TGGCCCGACG 8-oxo-D-GTPase 20 D16581
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Table II summarizes those trancripts that do not correspond to a previously
characterized gene. Several of the sequences correspond to reported Expressed
Sequence Tags (ESTs). Based on the inventors search of publicly available
databases, the remaining transcripts identify novel, uncharacterized genes.
TABLE II
Sequence EST (Yes/No)SEQ ID NO GenBank
Accession No.
TGCCGTTTTG Yes 21 AA 1272
GATGAGGAGA Yes 22 AA0228; AA0372
TGGAAATGAC Yes 23 AA0451
TAATACTTTT No 24 None
CAATAA.AATT No 25 None
AAGGCTGGAA No 26 None
CGGCCACAGA Yes 27 AA1142; AA1215
GCGCAGACTT Yes 28 887082
TATACGCTCA No 29 None
TAGTAAGTCA Yes 30 AA 1220
GCTTGAATAA Yes 31 H88507
TCCCCGTTAC No 32 None
ACCTTTACTG No 33 None
TCCCCGTAAC No 34 None
ATGATCCCTG No 3 5 None
TATCTGTCTA Yes 36 AA0635
TCTGCTAAAG Yes 37 AA0294; AA0456
TCCCTAATTA No 38 None
GAATCTGGAG Yes 39 553724; AA0019
GACGACTGAC ~ Yes 40 N29050; W24362
Cloning and Sequencing of Genes Corresponding to Novel Transcripts
This invention also provides polynucleotides which are fragments of
novel, uncharacterized genes. These transcripts are provided in SEQ ID NOS. 24-
26, 29, 32-35 and 38. Using the methods disclosed herein, the open reading
frames of the genes corresponding to these transcripts can be isolated and
1 S sequenced. Accordingly, polynucleotides comprising these transcripts or
their
respective complements also are provided by this invention. The complete open
reading frames or fragments thereof can be inserted into a vector or host cell
and
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used to reproduce the sequences or produce polypeptides. Antibodies, and in
particular, monoclonal antibodies, that specifically bind to the polypeptides
may
be generated based on well known methods. The antibodies can be used to screen
for expression of the gene corresponding to the transcript.
It is to be understood that while the invention has been described in
conjunction with the above embodiments, that the foregoing description and
examples are intended to illustrate and not limit the scope of the invention.
Other
aspects, advantages and modifications within the scope of the invention will
be
apparent to those skilled in the art to which the invention pertains.
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SEQUENCE LISTING
<110> Genzyme Corporation
Beaudry, Gary A.
Madden, Stephen L.
Bertelsen, Arthur H.
<120> COMPOSITIONS AND METHODS FOR THE
IDENTIFICATION OF LUNG TUMOR CELLS
<130> 159792001740
<140> Unassigned
<141> Herewith
<150> 60/080,037
<151> 1998-03-31
<160> 90
<170> FastSEQ for Windows Version 3.0
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aaggagcaag 10
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<211> 10
<212> DNA
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tgctgcctgt 10
<210> 5
<211> 10
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<212> DNA
<213> Artificial Sequence
<400> 5
ccatttttac
<210> 6
<211> 10
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gtccctgcct 10
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<212> DNA
<213> Artificial Sequence
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caactaattc 10
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<400> 8
gttataagat 10
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<400> 9
tatttttgtt 10
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<900> 10
cagataacat 10
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<212> DNA
<213> Artificial Sequence
<400> 11
tgtacctgta 10
<210> 12
<211> 10
<212> DNA
<213> Artificial Sequence
2
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<400> 12
ccaggggaga
<210> 13
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 13
gagaaaaccc 10
<210> 19
<211> 10
<212> DNA
<213> Artificial Sequence
<900> 14
atgtacctga 10
<210> 15
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 15
ttctaacata 10
<210> 16
<211> 10
<212> DNA
<213> Artificial Sequence
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ggtggtgtct
<210> 17
<211> 10
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<213> Artificial Sequence
<400> 17
tactagtcct
<210> 18
<211> 10
<212> DNA
<213> Artificial Sequence
<900> 18
atgcagccat 10
<210> 19
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 19
3
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tgctgccctg
10
<210> 20
<211> 10
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<213> Artificial Sequence
<900> 20
tggcccgacg 10
<210> 21
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 21
tgccgttttg
10
<210> 22
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 22
gatgaggaga
10
<210> 23
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 23
tggaaatgac 10
<210> 24
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 24
taatactttt 10
<210> 25
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 25
caataaaatt 10
<210> 26
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 26
aaggctggaa 10
4
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<210> 27
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 27
cggccacaga 10
<210> 28
<211> 10
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gcgcagactt 10
<210> 29
<211> 10
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tatacgctca 10
<210> 30
<211> 10
<212> DNA
<213> ArtificialSequence
<400> 30
tagtaagtca 10
<210> 31
<211> 10
<212> DNA
<213> ArtificialSequence
<900> 31
gcttgaataa 10
<210> 32
<211> 10
<212> DNA
<213> Artificial Sequence
<900> 32
tccccgttac 10
<210> 33
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 33
acctttactg 10
<210> 34
<2i1> 10
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<212> DNA
<213> Artificial Sequence
<400> 34
tccccgtaac 10
<210> 35
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 35
atgatccctg
10
<210> 36
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 36
tatctgtcta
10
<210> 37
<211> 10
<212> DNA
<213> Artificial Sequence
<900> 37
tctgctaaag
10
<210> 38
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 38
tccctaatta
10
<210> 39
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 39
gaatctggag
10
<210> 40
<211> 10
<212> DNA
<213> Artificial Sequence
<400> 90
gacgactgac
10
6
