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CA 02592330 2007-06-27
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GITR ANTIBODIES FOR THE DIAGNOSIS OF NSCLC
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. 119(e) from
U.S. Provisional Patent Application Serial No. 60/645,349, filed January 19,
2005,
which application is incorporated herein by reference in its entirety.
TECHNICAL FIELD
The invention relates to compositions and methods useful in therapeutic,
diagnostic and screening methods for human cancers and related malignancies.
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
second leading cause of ca.ncer 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, making lung cancer more deadly than the combination of breast,
prostrate and colorectal cancers. Jemal, A. et al. (2004) Cancer Statistics
2004, CA:
A Cancer Journal for Clinicians 53:5-26. The one-year survival rates for lung
cancer
have increased from 32% in 1973 to 41 % in 1993, largely due to improvements
in
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surgical techniques. The 5 year survival rate for all stages combined is only
14%.
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. Small cell
lung
cancer is the most malignant and fastest growing form 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%. For patients diagnosed with NSCLC, surgical
resection
offers the only chance of meaningful survival._
There are five types of non-small cell lung cancer: squamous cell carcinoma,
adenocarcinoma, large cell carcinoma, adenosquamous carcinoma and
undifferentiated carcinoma. Adenosquamous carcinomas begin in cells that
appear
flattened when viewed under a microscope. Undifferentiated carcinoma cells do
not
appear like normal cells and multiply uncontrollably. Squamous cell cancer is
the
most common type of lung cancer. It develops from the cells that line the
airways.
Adenocarcinoma develops from a glandular or secretory cells that produce mucus
(phlegm). Large cell lung cancer has been thus named because the cells look
large
and rounded when they are viewed under a microscope.
Non-small cell cancer also is characterized by four clinical stages. Stage I
is
very localized cancer with no cancer in the lymph nodes. Stage II cancer has
spread to
the lymph nodes at the top of the affected lung. Stage III cancer has spread
near to
where the cancer started. This can be to the chest wall, the covering of the
lung
(pleura), the middle of the chest (mediastinum) or other lymph nodes. Stage IV
cancer
has spread to another part of the body.
Several antibody therapies are in development to treat lung cancer. Cetuximab
and gifitinib are approved by the U.S. Food and Drug Administration for these
cancers. Cetuximab in combination with chemotherapy has provided some benefit
to
NSCLC patients but further trials are still neecled. Kelly, K. et al. (2003)
Proc. Am.
Soc. Clin. Oncol. 22:644.
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Therefore, an effective treatment for NSCLC is still required. This invention
satisfies this need and provides related advantages as well.
DISCLOSURE OF THE INVENTION
The present invention provides compositions and methods for aiding in the
diagnoses of the condition of a cell, for identifying and/or distinguisliing
normal and
neoplastic cells and for identifying potential therapeutic agents to reverse
neoplasia
and/or ameliorate the symptoms associated with the presence of neoplastic
cells in a
subject.
Accordingly, embodiments of the invention are directed to rnethods of
diagnosing the condition of a cell by screening for the presence of a
elifferentially
expressed gene identified in Table 1. In one aspect, the differential
expression of the
gene is indicative of the neoplastic state of a cell of epithelial origin,
e.g., non-small
cell lung cancer (NSCLC), ovarian, breast, prostate and colon cancers.
Expression
can be detected by any appropriate method, including for example, by detecting
the
quantity of mRNA transcribed from the gene or the quantity of cDNA, produced
from
the reverse transcription of the mRNA transcribed from the gene or the
quantity of the
polypeptide or protein encoded by the gene. These methods can be performed on
a
sample by sample basis or modified for high throughput analysis. Additionally,
databases containing quantitative full or partial transcripts or protein
sequences
isolated from a cell sample can be searched and analyzed for the presence and
amount
of transcript or expressed gene product.
Another aspect of the invention is a screen to identify therapeutic agents
that
reverse or treat neoplasia and tumors, wherein the cell and/or tumor is
characterized
by the differential expression of a polypeptide or protein identified in Table
1. The
method comprises contacting the cell previously identified as possessing this
genotype with an effective amount of a potential agent and assaying far
reversal of the
neoplastic condition.
Further provided are polynucleotides encoding the proteins, fragment(s)
thereof or polypeptides shown in Table 1, (also referred to herein as gene
expression
product), gene delivery vehicles comprising these polynucleotides and host
cells
comprising these polynucleotides. The proteins, polypeptides or fragment(s)
thereof
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are also useful to generate antibodies that specifically recognize and bind to
these
molecules. The antibodies can be polyclonal or monoclonal. These antibodies
can be
used to isolate protein or polypeptides expressed from the genes encoding the
polypeptides and to detect neoplastic cells or tumors.
The invention also provides isolated host cells and recoinbinant host cells
that
contain a polynucleotide encoding the peptides identified in Table 1 and/or
fragment(s) thereof. The cells can be prokaryotic or eukaryotic and by way of
example only, can be any one or more of bacterial, yeast, animal, mammalian,
human
and particular subtypes thereof, e.g., stein cells, antigen presenting cells
(APCs:) such
as dendritic cells (DCs) or T cells.
Table 1
Gene Unigene & Locus Normal Cancer cell Seq. ID Nos _
GenBank Link cell Expression
Numbers ID* expression
GITR Hs.212680 8784 Adeno and 1,2
(a!k/a. squamous
"TNFRSFIB") AF117297.1 cancers;
AF241229.1 NSCLC;
AF125304.1 ovarian,
AY358877.1 breast;
NM_148901.1 prostate and
NM148902.1 colon cancers
NM004195.2
NP004186
NP683699
NT 077913
*web address is = ncbi.nlm.nih.gov/LocusLink/list.cgi.
Further provided by this invention is a method for monitoring a cancer in a
subject by assaying, at different times, the expression level of the gene of
interest and
comparing the expression levels of the gene or to determine if expression has
increased or decreased, thereby monitoring the cancer in the subject. A kit
for use in
a diagnostic method or drug screen is further provided herein. The kit
comprises at
least one agent (e.g., probe, primer or antibody) that detects expression of
the ge:ne
and instructions for use.
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BRIEF DESCRIPTION OF THE SEQUENCE TINGS
As used herein, the term "GITR gene" refers to at least the ORF of a
contiguous polynucleotide sequence and that encodes a protein or polypeptide
having
the biological activity as set forth herein. LocusLiiik, supra, reports that
the protein
encoded by this gene is a member of the TNF-receptor superfamily. This
receptor has
been reported to have increased expression upon T-cell activation, and it is
thought to
play a key role in dominant immunological self-tolerance maintained by
CD25(+)CD4(+) regulatory T cells. Knockout studies in mice also suggest the
role of
this receptor is in the regulation of CD3-driven T-cell activation and
programmed cell
death. Three alternatively spliced transcript variants of this gene encoding
distinct
isoforms have been reported.
Sequence ID NO.: 1 is one example of a GITR polynucleotide sequence, and
others are known in the art, examples of which include, but are not limited to
the
sequences set forth in Table 1, and the sequences that encode GITR gene
expression
products as defined herein. Also included within this definition are
biologically
equivalent sequences such as those sequences that code for the polypeptide of
SEQ ID
NO:2 and those having at least 90% or alternatively, at least 95% sequence
homology
to an exemplary sequence, such as SEQ ID NO.: 1, and as determined by percent
identity sequence analysis run under default parameters. Also within this
definition
are biologically equivalent genes or polynucleotides that are identified by
the ability
to hybridize under conditions of high stringency to the minus strand. It may
be
desirable to use non-human genes, the polynucleotide sequences of which are
known
in the art. See for example, UniGene Cluster Hs.212680. Polynucleotide
fragments
are also known in the art, and include but are not limited to GenBank
Accession
numbers: BI911657.1; AI499936.1; AI214481.1; and AI923712.1. These are
particularly useful as probes or primers.
As used herein, the term "GITR gene expression product, protein or
polypeptide" includes the amino acid sequence of SEQ ID NO.: 2 as well as the
amino acid sequences transcribed and translated from the GITR genes identified
above, without regard to the gene expression system, e.g., bacterial or other
prokaryotic cell, yeast cell, mammalian cell such as a simian, bovine or human
cell.
The term includes isolated, naturally occurring polypeptides isolated from
tissue
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samples as well as recombinantly produced proteins and polypeptides. The term
also
includes polypeptides having the amino acid sequences that are at least 90 %
or
alternatively at least 95% homologous to SEQ ID NO.:2 and which have the
biological activity as described herein. Examples of homologous amino acid
sequences include, but are not limited to polypeptides have the amino acid
sequence
of SEQ ID NO.: 2 or other GITR gene expression product that has been modified
by
conservative amino acid substitutions.
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.
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
ENZYMOLOGY (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 herein, certain terms have the following defined meanings.
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.
All numerical designations, e.g., pH, temperature, time, concentration, and
molecular weight, including ranges, are approximations which are varied (+) or
(-)
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by increments of 0.1. It is to be understood, although not always explicitly
stated that
all numerical designations are preceded by the term "about". It also is to be
understood, although not always explicitly stated, that the reagents described
herein
are merely exemplary and that equivalents of such are known in the art.
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 can 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 (for
example, a
probe, primer, EST or SAGE tag), exons, introns, messenger RNA (niRNA),
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 can comprise
modified
nucleotides, such as methylated nucleotides and nucleotide analogs. If
present,
modifications to the nucleotide structure can be imparted before or aiter
assembly of the
polymer. The sequence of nucleotides can be interrupted by non-nucleotide
components. A polynucleotide can 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 (U) 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
(ORF) that is capable of encoding a particular polypeptide or protein after
being
transcribed and translated. Any of the polynucleotides sequences described
herein
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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.
A "gene product" or alternatively a "gene expression product" refers to the
amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed
and
translated.
The term "polypeptide" is used interchangeably with the tenn "protein" and in
its broadest sense refers 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 and L optical isomers,
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.
"Under transcriptional control" is a term well understood in the art and
indicates that transcription of a polynucleotide sequence, usually a DNA
sequence,
depends on its being operatively linked to an element which contributes to the
initiation of, or promotes, transcription. "Operatively linked" refers to a
juxtaposition
wherein the elements are in an arrangement allowing them to function.
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.
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The term "isolated" means separated from constituents, cellular and otherwise,
in which the polynucleotide, peptide, polypeptide, protein, antibody or
fragment(s)
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 natu.ral environment,
e.g., on the
chromosome. As is apparent to those of skill in the art, a non-naturally
occurring
polynucleotide, peptide, polypeptide, protein, antibody or fragment(s)
thereof, does
not require "isolation" to distinguish it from its naturally occurring
counterpart. In
addition, a "concentrated", "separated" or "diluted" polynucleotide, peptide,
polypeptide, protein, antibody or fragment(s) thereof, is distinguishable from
its
naturally occurring 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 fragment(s) 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. Thus, a non-naturally occurring
polynucleotide is provided as a separate embodiment from the isolated
naturally
occurring polynucleotide. A protein produced in a bacterial cell is provided
as a
separate embodiment from the naturally occurring protein isolated from a
eukaryotic
cell in which it is produced in nature.
"Gene delivery," "gene transfer," and the like as used herein, are terms
referring to the introduction of an exogenous polynucleotide (sometimes
referred to as
a "transgene") into a host cell, irrespective of the method used for the
introduction.
Such methods include a variety of well-known techniques such as vector-
mediated
gene transfer (by, e.g., viral infection/transfection or various other protein-
based or
lipid-based gene delivery complexes) as well as techniques facilitating the
delivery of
"naked" polynucleotides (such as electroporation, "gene gun" delivery and
various
other techniques used for the introduction of polynucleotides). The introduced
polynucleotide may be stably or transiently maintained in the host cell.
Stable
maintenance typically requires that the introduced polynucleotide either
contains an
origin of replication compatible with the host cell or integrates into a
replicon of the
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host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear
or
mitochondrial chromosome. A number of vectors are known in the art to be
capable
of rnediating transfer of genes to mammalian cells.
A "gene delivery vehicle" is defined as any molecule that can carry inserted
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; recombinant yeast cells, metal particles; and bacteria or viruses,
such as
baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal
vectors and other recombination vehicles typically used in the art which have
been
described for expression in a variety of eukaryotic and prokaryotic hosts and
may be
used for gene therapy as well as for simple protein expression.
A "viral vector" is defined as a recombinantly produced virus or viral
particle
that comprises a polynucleotide to be delivered into a host cell, either iiz
vivo, ex vivo
or in vitro. Examples of viral vectors include retroviral vectors, adenovirus
vectors,
adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus
vectors,
such as Semliki Forest virus-based vectors and Sindbis virus-based vectors,
have also
been developed for use in gene therapy and immunotherapy. See, Schlesinger and
Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat.
Med.
5(7):823-827. In aspects where gene transfer is mediated by a retroviral
vector, a
vector construct refers to the polynucleotide comprising the retroviral genome
or part
thereof and a therapeutic gene. As used herein, "retroviral mediated gene
transfer" or
"retroviral transduction" carries the same meaning and refers to the process
by which
a gene or nucleic acid sequences are stably transferred into the host cell by
virtue of
the virus entering the cell and integrating its genome into the host cell
genome. The
virus can enter the host cell via its normal mechanism of infection or be
modified
such that it binds to a different host cell surface receptor or ligand to
enter the cell.
As used herein, "retroviral vector" refers to a viral particle capable of
introducing
exogenous nucleic acid into a cell through a viral or viral-like entry
mechanism.
Retroviruses carry their genetic information in the form of RNA; however,
once the virus infects a cell, the RNA is reverse-transcribed into the DNA
form which
CA 02592330 2007-06-27
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integrates into the genomic DNA of the infected cell. The integrated DNA form
is
called a provirus.
In aspects where gene transfer is mediated by a DNA viral vector, such as an
adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to
the
polynucleotide comprising the viral genome or part thereof and a transgene.
Adenoviruses (Ads) are a relatively well characterized, homogenous group of
viruses,
including over 50 serotypes. See e.g., WO 95/27071. Ads are easy to grow and
do not
require integration into the host cell genome. Recombinant Ad derived vectors,
particularly those that reduce the potential for recombination and generation
of wild-
type virus, have also been constructed. See e.g., WO 95/00655 and WO 95/11984.
Wild-type AAV has high infectivity and specificity integrating into the host
cell's
genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-
6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.
Vectors that contain both a promoter and a cloning site into which a
polynucleotide can be operatively linked are well known in the ai L. Such
vectors are
capable of transcribing RNA in vitro or in vivo and are 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 a_t the level of transcription
or
translation. Alternatively, consensus ribosome binding sites can be inserted
immediately 5' of the start codon to enhance expression.
Gene delivery vehicles also include several non-viral vectors, including
DNA/liposome complexes, recombinant yeast cells and targeted viral protein-DNA
complexes. Liposomes that also comprise a targeting antibody or fragment
thereof
can be used in the methods of this invention. To enhance delivery to a cell,
the
nucleic acid or proteins of this invention can be conjugated to antibodies or
binding
fragment(s) thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
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
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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 "upstream" and a
"downstream" primer and a catalyst of polymerization, such as a DNA polymerase
and, typically, a tliermally-stable polymerase enzyzne. 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 Unive:xsity 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.
An expression "database" denotes a set of stored data that represent a
collection of sequences, which in turn represent a collection of biological
reference
materials.
The term "cDNAs" refers to complementary DNA that is mRNA molecules
present in a cell or organism made into cDNA witlh 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). Exemplaxy 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.
As used herein, "expression" refers to the process by which polynucleotides
are transcribed into mRNA and/or the process by vvhich the transcribed mRNA is
subsequently being translated into peptides, polypeptides or proteins. If the
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polynucleotide is derived from genomic DNA, expression may include splicing of
the
mRNA in a eukaryotic cell. "Differentially expressed" as applied to a gene,
refers to
the differential production of the mRNA transcribed and/or translated from the
gene
or the protein product encoded by the gene. A differentially expressed gene
rnay be
overexpressed or underexpressed as compared to the expression level of a
normal or
control cell. However, as used herein overpression as at least 1.25 fold or,
alternatively, at least 1.5 fold or, alternatively, at least 2 fold expression
over that
detected in a normal or healthy counterpart cell or tissue. 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.
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, microarrays and chips. As used herein, "solid
support"
also includes synthetic antigen-presenting matrices, cells and liposomes. A
stuitable
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, Germany) ox
polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).
A polynucleotide also can be attached to a solid support for use in high
25, 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 also known as chip arrays.
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.
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"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-Criclc 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 extensive process,
such as
the initiation of a PCR reaction or the enzymatic cleavage of a
polynucleoticie by a
ribozyme.
Hybridization reactions can be performed under conditions of different
"stringency". In general, a low stringency hybridization reaction is carried o-
ut at
about 40 C in lOx SSC or a solution of equivalent ionic strength/temperature.
A
moderate stringency hybridization is typically performed at about 50 C in 6x
SSC,
and a high stringency hybridization reaction is generally performed at about
60 C in
lx 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
ba_ses (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.
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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 + SwissProtein + SPupdate + PIR. Details of these programs can be
found at the following Internet address: www.ncbi.nlm.nih.gov/cgi-bin/BLASrT.
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 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
cvr tissue
means that the cell can invade and destroy neighboring body structures.
"Suppressing" tumor growth indicates a growth state that is curtailed when
compared to growth without therapeutic intervention. Tumor cell growth can be
assessed by any means known in the art, including, but not limited to,
measurirng
tumor size, determining whether tumor cells are proliferating using a 3H-
thymidine
incorporation assay or counting tumor cells. "Suppressing" tumor cell growth
rneans
any or all of the following states: slowing, delaying and stopping tumor
growtla, as
well as tumor shrinkage.
The term "antigen" is well understood in the art and includes substances which
are immunogenic. The term as used herein also includes substances which induce
immunological unresponsiveness or anergy.
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A "native" or "natural" or "wild-type" antigen is a polypeptide, protein or a
fragment which contains an epitope and which has been isolated from a natural
biological source. It also can specifically bind to an antigen receptor.
As used herein, an "antibody" includes whole antibodies and any antigen
binding fragment or a single chain thereof. Thus the term "antibody" includes
any
protein or peptide containing molecule that comprises at least a portion of an
immunoglobulin molecule. Examples of such include, but are not limited to a
complementarity determining region (CDR) of a heavy or light chain or a ligand
binding portion thereof, a heavy chain or light chain variable region, a heavy
chain or
light chain constant region, a framework (FR) region, or any portion thereof,
or at
least one portion of a binding protein, any of which can be incorporated into
an
antibody of the present invention.
The antibodies can be polyclonal or monoclonal and can be isolated from any
suitable biological source, e.g., murine, rat, sheep and canine. Additional
sources are
identified infra.
The term "antibody" is further intended to encompass digestion fragments,
specified portions, derivatives and variants thereof, including antibody
mimetics or
comprising portions of antibodies that mimic the structure and/or function of
an
antibody or specified fragment or portion thereof, including single chain
antibodies
and fragments thereof. Examples of binding fragments encompassed within the
term
"antigen binding portion" of an antibody include a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH, domains; a F(ab')2 fragment, a
bivalent fragment comprising two Fab fragments linked by a disulfide bridge at
the
hinge region; a Fd fragment consisting of the VH and CH, domains; a Fv
fragment
consisting of the VL and VH domains of a single arm of an antibody, a dAb
fragment
(Ward et al. (1989) Nature 341:544-546), which consists of a VH domain; and an
isolated complementarity determining region (CDR). Furthermore, although the
two
domains of the Fv fragment, VL and VH, are coded for by separate genes, they
can be
joined, using recombinant methods, by a synthetic linker that enables them to
be made
as a single protein chain in which the VL and VH regions pair to form
monovalent
molecules (known as single chain Fv (scFv)). Bird et al. (1988) Science
242:423-426
and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Single chain
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antibodies are also intended to be encompassed within the term "fragment of an
antibody." Any of the above-noted antibody fragments are obtained using
conventional techniques known to those of skill in the art, and the fragments
are
screened for binding specificity and neutralization activity in the same
maimer as are
intact antibodies.
The term "epitope" means a protein determinant capable of specific binding to
an antibody. Epitopes 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.
Conformational and nonconformational epitopes are distinguished in that the
binding
to the former but not the latter is lost in the presence of denaturing
solvents.
The term "antibody derivative" is intended to encompass molecules that bind
an epitope as defined above and which are modifications or derivatives of a
native
monoclonal antibody of this invention. Derivatives include, but are not
limited to, for
example, bispecific, multispecific, heterospecific, trispecific,
tetraspecific,
multispecific antibodies, diabodies, chimeric, recombinant and humanized.
The term "bispecific molecule" is intended to include any agent, e.g., a
protein, peptide, or protein or peptide complex, which has two different
binding
specificities. The term "multispecific molecule" or "heterospecific molecule"
is
intended to include any agent, e.g. a protein, peptide, or protein or peptide
complex,
which has more than two different binding specificities.
The term "heteroantibodies" refers to two or more antibodies, antibody
binding fragments (e.g.', Fab), derivatives thereof, or antigen binding
regions linked
together, at least two of which have different specificities.
The term "human antibody" as used herein, is intended to include antibodies
having variable and constant regions derived from human germline
immunoglobulin
sequences. The human antibodies of the invention may include amino acid
residues
not encoded by human germline immunoglobulin sequences (e.g., mutations
introduced by random or site-specific mutagenesis in vitro or by somatic
mutation in
vivo). However, the term "human antibody" as used herein, is not intended to
include
antibodies in which CDR sequences derived from the germline of another
mammalian
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species, such as a mouse, have been grafted onto human framework sequences.
Thus,
as used herein, the term "human antibody" refers to an antibody in which
substantially
every part of the protein (e.g., CDR, frameworlc, CL, CH domains (e.g., CHI,
CHZ, CH3),
hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor
sequence changes or variations. Similarly, antibodies designated primate
(monkey,
baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster,
and the
like) and other mammals designate such species, sub-genus, genus, sub-family,
family
specific antibodies. Further, chimeric antibodies include any combination of
the
above. Such changes or variations optionally and preferably retain or reduce
the
immunogenicity in humans or other species relative to non-modified antibodies.
Thus, a human antibody is distinct from a chimeric or humanized antibody. It
is
pointed out that a human antibody can be produced by a non-hunian animal or
prokaryotic or eukaryotic cell that is capable of expressing functionally
rearranged
human iYnmunoglobulin (e.g., heavy chain and/or light chain) genes. Further,
when a
human antibody is a single chain antibody, it can comprise a linker peptide
that is not
found in native human antibodies. For example, an Fv can comprise a linker
peptide,
such as two to about eight glycine or other amino acid residues, which
connects the
variable region of the heavy chain and the variable region of the light chain.
Such
linker peptides are considered to be of human origin.
As used herein, a human antibody is "derived from" a particular germline
sequence if the antibody is obtained from a system using human immunoglobulin
sequences, e.g., by immunizing a transgenic mouse carrying human
immunoglobulin
genes or by screening a human immunoglobulin gene library. A human antibody
that
is "derived from" a human germline immunoglobulin sequence can be identified
as
such by comparing the amino acid sequence of the human antibody to the amino
acid
sequence of human germline immunoglobulins. A selected human antibody
typically
is at least 90% identical in amino acids sequence to an amino acid sequence
encoded
by a human germline immunoglobulin gene and contains amino acid residues that
identify the human antibody as being human when compared to the germline
immunoglobulin amino acid sequences of other species (e.g., murine germline
sequences). In certain cases, a human antibody may be at least 95%, or even at
least
96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid
sequence
encoded by the germline immunoglobulin gene. Typically, a human antibody
derived
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from a particular human germline sequence will display no more than 10 amino
acid
differences from the amino acid sequence encoded by the human germline
immunoglobulin gene. In certain cases, the human antibody may display no more
than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the
amino acid
sequence encoded by the gerinline immunoglobulin gene.
The terms "monoclonal antibody" or "monoclonal antibody composition" as
used herein refer to a preparation of antibody molecules of single molecular
composition. A rnonoclonal antibody composition displays a single binding
specificity and affinity for a particular epitope.
A "human monoclonal antibody" refers to antibodies displaying a single
binding specificity which have variable and constant regions derived from
human
germline immunoglobulin sequences.
The term "recombinant human antibody", as used herein, includes all human
antibodies that are prepared, expressed, created or isolated by recombinant
means,
such as antibodies isolated from an animal (e.g., a mouse) that is transgenic
or
transchromosomal for human immunoglobulin genes or a hybridoma prepared
therefrom, antibodies isolated from a host cell transformed to express the
antibody,
e.g., from a transfectoma, antibodies isolated from a recombinant,
combinatorial
human antibody library, and antibodies prepared, expressed, created or
isolated by
any other means that involve splicing of human immunoglobulin gene sequences
to
other DNA sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin sequences. In
certain
embodiments, however, such recombinant human antibodies can be subjected to in
vitro mutagenesis (or, when an animal transgenic for human Ig sequences is
used, in
vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while derived from
and
related to human germline VH and VL sequences, may not naturally exist within
the
human antibody germline repertoire in vivo.
As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl)
that
is encoded by heavy chain constant region genes.
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The terms "transgenic, nonliuman animal" refers to a nonhuman animal
having a genome comprising one or more human heavy and/or light chain
transgenes
or transchromosomes (either integrated or non-integrated into the animal's
natural
genomic DNA) and which is capable of expressing fully human antibodies. For
example, a transgenic rat can have a huxnan light chain transgene and either a
human
heavy chain transgene or human heavy chain transchromosome, such that the rat
produces human anti-INF-a antibodies. The human heavy chain transgene can be
integrated into the chromosomal DNA of the rat, or the human heavy chain
transgene
can be maintained extrachromosomally. Transgenic and transchromosomal animals
are capable of producing multiple isotypes of human monoclonal antibodies to
Alpha
V (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype
switching.
A"composition" is also intended to encompass a combination of active agent
and another carrier, e.g., compound or composition, inert (for example, a
detectable
agent or label) or active, such as an adjuvant, diluent, binder, stabilizer,
buffers, salts,
lipophilic solvents, preservative, adjuvant or the like. Carriers also include
pharmaceutical excipients and additives proteins, peptides, amino acids,
lipids, and
carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and
oligosaccharides; derivatized sugars such as alditols, aldonic acids,
esterified sugars
and the like; and polysaccharides or sugar polymers), which can be present
singly or
in combination, comprising alone or in combination 1-99.99% by weight or
volume.
Exemplary protein excipients include serum albumin such as human serum albumin
(HSA), recombinant human albumin (rFiA), gelatin, casein, and the like.
Representative amino acid/antibody cornponents, which can also function in a
buffering capacity, include alanine, glycine, arginine, betaine, histidine,
glutamic acid,
aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine,
phenylalanine,
aspartame, and the like. Carbohydrate excipients are also intended within the
scope
of this invention, examples of which include but are not limited to
monosaccharides
such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the
like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;
polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,
starches, and
the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol
sorbitol
(glucitol) and myoinositol.
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The term carrier further includes a buffer or a pH adjusting agent; typically,
the buffer is a salt prepared from an organic acid or base. Representative
buffers
include organic acid salts such as salts of citric acid, ascorbic acid,
gluconic acid,
carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid;
Tris,
trometharnine hydrochloride, or phosphate buffers. Additional carriers include
polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a
polymeric
sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-.quadrature.-
cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents,
sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates
such as
"TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids),
steroids
(e.g., cholesterol), and chelating agents (e.g., EDTA).
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 and any of the above noted carriers with the additional provisio
that they
be acceptable for use in vivo. For examples of carriers, stabilizers and
adjuvants, see
Martin REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975) and
Williams & Williams, (1995), and in the "PHYSICIAN'S DESK REFERENCE", 52"d
ed., Medical Economics, Montvale, N.J. (1998).
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.
The invention provides an antibody or a variant, derivative or fragment
thereof
that specifically recognizes and binds an epitope on the polgreplide or
protein
expressed by a gene identified in Table 1. In one aspect, the antibodies are
isolated.
In another aspect, they are combined with a suitable carrier. The antibodies
can be
polyclonal or monoclonal and can be isolated from any species, murine, rat,
simian, or
recobminantly produced and isolated. Also provided by this invention are the
hybridoma cell lines that produce these monoclonal antibodies, alone in
combination
with a carrier or in culture.
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Also provided by this invention is a polypeptide that comprises an antibody,
variant, derivative or fragment thereof, including but not limited to
iinmunoglobulin
chains and CDRs.
The present invention further provides an anti-idiotypic antibody. An anti-
idiotype antibody includes any protein or peptide containing molecule that
comprises
at least a portion of an immunoglobulin molecule, such as but not limited to
at least
one complementarity determining region (CDR) of a heavy or light chain or a
ligand
binding portion thereof, a heavy chain or light chain variable region, a heavy
chain or
light chain constant region, a framework region, or any portion thereof, that
can be
incorporated into an antibody of the present invention. An anti-idiotype
antibody of
the invention can include or be derived from any mammal, such as but not
limited to a
human, a mouse, a rabbit, a rat, a rodent, a primate, and the like.
This invention further provides an isolated polynucleotide that encodes an
antibody of this invention, alone in combination with vectors, carriers,
pharmaceutically acceptable carriers, diluents, and host cells.
The present invention further provides, in one aspect, isolated nucleic acid
molecules comprising, complementary, or hybridizing to, a polynucleotide
encoding
at least one antibody or anti-idiotype antibody of this invention, comprising
at least
one specified sequence, domain, portion or variant thereof. The present
invention
further provides recombinant vectors comprising the nucleotides, host cells
containing
the nucleic acids and/or recombinant vectors, as well as methods of making
and/or
using such nucleic acids, vectors and/or host cells. Methods for isolating,
replicating
and expressing polynucleotides are known in the art and described infra.
One or more of the above can be further combined with a carrier, a
pharmaceutically acceptable carrier or medical device which is suitable for
use of the
antibody or related composition in diagnostic or therapeutic methods.
The carrier can be a liquid phase carrier or solid phase carrier, e.g., bead,
gel
or carrier rnolecule such as a liposome. The composition can optionally
fiuther
comprise at least one further compound, protein or composition.
An additional example of "carriers" includes therapeutically active agents
such as another peptide or protein (e.g., an Fab' fragment). For example, an
antibody
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of this invention, variant, derivative or fragment thereof can be functionally
linked
(e.g., by chemical coupling, genetic fusion, noncovalent association or
otherwise) to
one or more other molecular entities, such as another asitibody (e.g., to
produce a
bispecific or a multispecific antibody), a cytotoxin, a cellular ligand or an
antigen.
Accordingly, this invention encompasses a large variety of antibody
conjugates, bi-
and multispecific inolecules, and fusion proteins, whether or not they target
the same
epitope as the antibodies of this invention.
Yet additional examples of carriers are organic molecules (also terrned
modifying agents) or activating agents, that can be covalently attached,
directly or
indirectly, to an antibody of this invention. Attachment of the molecule can
improve
pharmacokinetic properties (e.g., increased in vivo serum half-life). Examples
of
organic molecules include, but are not limited to a hydrophilic polymeric
group, a
fatty acid group or a fatty acid ester group. As used herein, the term "fatty
acid"
encompasses mono-carboxylic acids and di-carboxylic acids. A "hydrophilic
polymeric group," as the term is used herein, refers to an organic polymer
that is more
soluble in water than in octane.
Hydrophilic polymers suitable for modifying antibodies of the invention can
be linear or branched and include, for example, polyalkane glycols (e.g., PEG,
monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates
(e.g.,
dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers
of
hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the
like),
polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like)
and
polyvinyl pyrolidone. A suitable hydrophilic polymer that modifies the
antibody of
the invention has a molecular weight of about 800 to about 150,000 Daltons as
a
separate molecular entity. The hydrophilic polymeric group can be substituted
with
one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic
polymers that
are substituted with a fatty acid or fatty acid ester group can be prepared by
employing suitable methods. For example, a polymer comprising an amine group
can
be coupled to a carboxylate of the fatty acid or fatty acid ester, and an
activated
carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid
or fatty
acid ester can be coupled to a hydroxyl group on a polymer.
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Fatty acids and fatty acid esters suitable for modifying antibodies of the
invention can be saturated or can contain one or more units of unsaturation.
Examples of such include, but are not limited to n-dodecanoate, n-
tetradecanoate, n-
octadecanoate, n-eicosanoate, n-docosanoate, n-triacontanoate, n-
tetracontanoate, cis-
.DELTA.9-octadecanoate, all cis-.DELTA.5,8,11,14-eicosatetraenoate,
octanedioic
acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the
like.
Suitable fatty acid esters include mono-esters of dicarboxylic acids that
comprise a
linear or branched lower alkyl group. The lower alkyl group can comprise from
one
to about twelve, preferably one to about six, carbon atoms.
In yet another aspect, the present invention provides a transgenic nonhuman
animal, such as a transgenic mouse (also referred to herein as a "HuMAb
mouse"),
which expresses a fully human monoclonal antibody that neutralizes at least
one
protein subtype similar to an antibody of this invention as defined above. In
a
particular embodiment, the transgenic nonhuman animal is a transgenic mouse
having
a genome comprising a humarn heavy chain transgene and a human light chain
transgene encoding all or a portion of an anti-alpha V antibody of the
invention.
Preferably, the transgenic non.human animal, e.g., the transgenic mouse, is
capable of
producing multiple isotypes of human monoclonal antibodies to an epitope of
interest
by undergoing V-D-J recombination and isotype switching. Isotype switching may
occur by, e.g., classical or non-classical isotype switching.
Accordingly, in another embodiment, the invention provides isolated cells
derived or isolated from a transgenic nonhuman animal as described above,
e.g., a
transgenic mouse, which express human antibodies. The isolated B-cells can
then be
immortalized by fusion to an immortalized cell to provide a source (e.g., a
hybridorna)
of human antibodies. These hybridomas are also included within the scope of
the
invention.
The present invention further provides at least one antibody method or
composition, for diagnosing a cancer of epithelial origin, e.g., non-small
cell lung
cancer (NSCLC), ovarian, breast, prostate or colon cancer, in a cell, tissue,
organ,
animal or patient and/or, prior to, subsequent to, or during a related
condition, as
known in the art and/or as described herein. They are also used to prognose or
monitor disease progression.
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Also provided is a composition containing at least one antibody of this
invention, variant, derivative or fragment thereof, suitable f>r
administration in an
effective aniount to modulate or ameliorate syrnptoms associated with cancers
of
epithelial origin, e.g., non-small cell lung cancer (NSCLC), ovarian, breast,
prostate
and colon cancer, or treat at least one such cancer. The compositions include,
for
example, phannaceutical and diagnostic compositions/kits, comprising a
pharniaceutically acceptable carrier and at least one antibody of this
invention,
variant, derivative or fragment thereof. As noted above, the composition can
further
comprise additional antibodies or therapeutic agents which irz combination,
provide
multiple therapies tailored to provide the maximum therapeutic benefit.
Alternatively, a composition of this invention can be co-administered with
other therapeutic and cytotoxic agents, whether or not linked to them or
administered
in the same dosing. They can be coadministered simultaneously with such agents
(e.g., in a single composition or separately) or can be administered before or
after
administration of such agents. Such agents can include corticosteroids,
nonsteroidal
immune suppressants, antimalarials, and nonsteroidal anti-inflammatory drugs.
The
compositions can be combined with alternative therapies such as administration
of
corticosteroids, nonsteroidal immune suppressants, antimalarials, and
nonsteroidal
anti-inflamatory drugs.
The methods of this invention can be practiced either in vitro or in vivo.
When
practiced in vitro, the methods require contacting the cells with (e.g.,
administering or
delivering to the cells) one or more antibodies and/or related therapeutic
compositions, derivatives etc. containing the antibodies as described above.
The antibodies and compositions can be delivered by any suitable means and
with any suitable formulation. Accordingly, a formulation coznprising an
antibody of
this invention is further provided herein. The formulation can further
comprise one or
more preservative or stabilizer such as phenol, m-cresol, p-crE;sol, o-cresol,
chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,
formaldehyde,
clilorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl,
ethyl,
propyl, butyl and the like), benzalkonium chloride, benzethonium chloride,
sodium
dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Any
suitable concentration or mixture can be used as known in the art, such as
0.001-5%,
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or any range or value therein, such as, but not limited to 0.0@ 1, 0.003,
0.005, 0.009,
0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0_8, 0.9,
1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3.0, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any
range or value
therein. Non-limiting examples include, no preservative, 0.1 -2% m-cresol
(e.g., 0.2,
0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5,
1.9, 2.0,
2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.00/c-) phenol (e.g.,
0.05, 0.25,
0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009,
0.001,
0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1 , 0.2, 0.3,
0.5, 0.75, 0.9,
and 1.0%).
As noted above, the invention provides an article of rnanufacture, comprising
packaging material and at least one vial comprising a solution of at least
antibody as
of this invention with the prescribed buffers and/or preservatives, optionally
in an
aqueous diluent, wherein said packaging material comprises a label that
indicates that
such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20,
24, 30, 36,40,
48, 54, 60, 66, 72 hours or greater. The invention further corrnprises an
article of
manufacture, comprising packaging material, a first vial comprising at least
one
lyophilized antibody of this invention and a second vial comprising an aqueous
diluent of prescribed buffer or preservative, wherein said packaging material
comprises a label that instructs a patient to reconstitute the antibody in the
aqueous
diluent to form a solution that can be held over a period of twenty-four hours
or
greater.
The range antibody includes amounts yielding upon reconstitution, if in a
wet/dry system, concentrations from about 1.0 g/ml to about 1000 mg/ml,
although
lower and higher concentrations are operable and are dependent on the intended
delivery vehicle, e.g., solution formulations will differ from transdermal
patch,
pulmonary, transmucosal, or osmotic or micro pump methods -
The formulations of the present invention can be prepared by a process which
comprises mixing at least one antibody of this invention and a_ preservative
selected
from the group consisting of phenol, m-cresol, p-cresol, o-cresol,
chlorocresol, benzyl
alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the lilce),
benzalkonium
chloride, benzethonium chloride, sodium dehydroacetate and t.himerosal or
mixtures
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WO 2006/078911 PCT/US2006/002050
thereof in an aqueous diluent. Mixing of the antibody and preservative in an
aqueaus
diluent is carried out using conventional dissolution and mixing procedures.
For
example, a measured amount of at least one antibody in buffered solution is
comb3ned
with the desired preservative in a buffered solution in quantities sufficient
to provide
the antibody and preservative at the desired concentrations. Variations of
this process
would be recognized by one of ordinary skill in the art, e.g., the order the
coinponents
are added, whether additional additives are used, the temperature and pH at
whicli the
formulation is prepared, are all factors that can be optimized for the
concentration and
means of administration used.
The compositions and formulations can be provided to patients as clear
solutions or as dual vials comprising a vial of lyophilized antibody that is
reconstituted with a second vial containing the aqueous diluent. Either a
single
solution vial or dual vial requiring reconstitution can be reused multiple
times and can
suffice for a single or multiple cycles of patient treatment and thus provides
a more
convenient treatment regimen than currently available. Recognized devices
comprising these single vial systems include those pen-injector devices for
delivery of
a solution such as BD Pens, BD Autojectore, Humaject®' NovoPen®, B-
D®Pen, AutoPen®, and OptiPen®, GenotropinPen®,
Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,
Biojector®, iject®, J-tip Needle-Free Injector®, Intraject®,
Medi-
Ject®, e.g., as made or developed by Becton Dickensen (Franklin Lakes,
N.J.
available at bectondickenson.com), Disetronic (Burgdorf, Switzerland,
available at
disetronic.com; Bioject, Portland, Oregon (available at bioject.com); National
Medical Products, Weston Medical (Peterborough, UK, available at weston-
medical.com), Medi-Ject Corp (Minneapolis, Minn., available at mediject.com).
ANTIBODIES
The antibodies of this invention are monoclonal antibodies, although in
certain
aspects, polyclonal antibodies can be utilized. They also can be functional
fragments,
antibody derivatives or antibody variants. They can be chimeric, humanized, or
totally human. A functional fragment of an antibody includes but is not
limited to
Fab, Fab', Fab2, Fab'2, and single chain variable regions. Antibodies can be
produced in cell culture, in phage, or in various animals, including but not
limited to
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WO 2006/078911 PCT/US2006/002050
cows, rabbits, goats, mice, rats, hanlsters, guinea pigs, sheep, dogs, cats,
monkeys,
chimpanzees, apes, etc. So long as the fragment or derivative retains
specificity of
binding or neutralization ability as the antibodies of this invention it can
be used.
Antibodies can be tested for specificity of binding by comparing binding to
appropriate antigen to binding to irrelevant antigen or antigen mixture under
a given
set of conditions. If the antibody binds to the appropriate antigen at least
2, 5, 7, and
preferably 10 times more than to irrelevant antigen or antigen mixture then it
is
considered to be specific. Specific assays, e.g., ELISA, for determining
specificity
are known in the art.
The antibodies also are characterized by their ability to specifically
recognize
and bind an epitope of interest.
The monoclonal antibodies of the invention can be generated using
conventional hybridoma techniques known in the art and well-described in the
literature. For example, a hybridoma is produced by fusing a suitable immortal
cell
line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-
AG14, NSO,
NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5,
U397, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI,
NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the
like, or heteromyelomas, fusion products thereof, or any cell or fusion cell
derived
therefrom, or any other suitable cell line as known in the art (see, e.g.,
www.atcc.org,
www.lifetech.com., and the like), with antibody producing cells, such as, but
not
limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or
other immune
or B cell containing cells, or any other cells expressing heavy or light chain
constant
or variable or framework or CDR sequences, either as endogenous or
heterologous
nucleic acid, as recombinant or endogenous, viral, bacterial, algal,
prokaryotic,
amphibian, insect, reptilian, fish, matnmalian, rodent, equine, ovine, goat,
sheep,
primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,
chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded,
hybridized, and the like or any combination thereof. Antibody producing cells
can
also be obtained from the peripheral blood or, preferably the spleen or lyrnph
nodes,
of humans or other suitable animals that have been immunized with the antigen
of
interest. Any other suitable host cell can also be used for expressing-
heterologous or
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CA 02592330 2007-06-27
WO 2006/078911 PCT/US2006/002050
endogenous nucleic acid encoding an antibody, specified fragment or variant
thereof,
of the present invention. The fused cells (hybridomas) or recombinant cells
can be
isolated using selective culture conditions or other suitable known methods,
and
cloned by limiting dilution or cell sorting, or other known methods.
Other suitable methods of producing or isolating antibodies of the requisite
specificity can be used, including, but not limited to, methods that select
recombinant
antibody from a peptide or protein library (e.g., but not limited to, a
bacteriophage,
ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as
available
from various commercial vendors such as Cambridge Antibody Technologies
(Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg, Del.), Biovation
(Aberdeen,
Scotland, UK) Biolnvent (Lund, Sweden), using methods known in the art. See
U.S.
Pat. Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514;
5,976,862. Alternative methods rely upon immunization of transgenic animals
(e.g.,
SCID mice, Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu
et
al., (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.
93:154-161
that are capable of producing a repertoire of human antibodies, as known in
the art
and/or as described herein. Such techniques, include, but are not limited to,
ribosome
display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes
et al.,
(1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody
producing
technologies (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Pat.
No.
5,627,052, Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al., Proc.
Natl,
Acad. Sci. USA (1996) 93:7843-7848); gel microdroplet and flow cytometry
(Powell
et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).;
Gray et
al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol: 13:787-
790);
B-cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134
(1994).
Antibody variants of the present invention can also be prepared using
delivering a polynucleotide encoding an antibody of this invention to a
suitable host
such as to provide transgenic animals or mammals, such as goats, cows, horses,
sheep,
and the like, that produce such antibodies in their milk. These methods are
known in
the art and are described for example in U.S. Pat. Nos. 5,827,690; 5,849,992;
4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.
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WO 2006/078911 PCT/US2006/002050
The term "antibody variant" includes post-translational modification to linear
polypeptide sequence of the antibody or fragment. For example, U.S. Patent
No. 6,602,684 B 1 describes a method for the generation of modified glycol-
forms of
antibodies, including whole antibody molecules, antibody fragments, or fusion
proteins that include a region equivalent to the Fc region of an
immunoglobulin,
having enhanced Fc-mediated cellular toxicity, and glycoproteins so generated.
Antibody variants also can be prepared by delivering a polynucleotide of this
invention to provide transgenic plants and cultured plant cells (e.g., but not
limited to
tobacco, maize, and duckweed) that produce such antibodies, specified portions
or
variants in the plant parts or in cells cultured therefrom. For example,
Cramer et al.
(1999) Curr. Top. Microbol. Immunol. 240:95-118 and references cited therein,
describe the production of transgenic tobacco leaves expressing large amounts
of
recombinant proteins, e.g., using an inducible promoter. Transgenic maize have
been
used to express mammalian proteins at commercial production levels, with
biological
activities equivalent to those produced in other recombinant systems or
purified from
natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-
147 and
references cited therein. Antibody variants have also been produced in large
amounts
from transgenic plant seeds including antibody fragments, such as single chain
antibodies (scFv's), including tobacco seeds and potato tubers. See, e.g.,
Conrad et
al.(1998) Plant Mol. Biol. 38:101-109 and reference cited therein. Thus,
antibodies of
the present invention can also be produced using transgenic plants, according
to know
methods.
Antibody derivatives can be produced, for example, by adding exogenous
sequences to modify immunogenicity or reduce, enhance or modify binding,
affinity,
on-rate, off-rate, avidity, specificity, half-life, or any other suitable
characteristic.
Generally part or all of the non-human or human CDR sequences are maintained
while the non-human sequences of the variable and constant regions are
replaced with
human or other amino acids.
In general, the CDR residues are directly and most substantially involved in
influencing antigen binding. Humanization or engineering of antibodies of the
present invention can be performed using any known method, such as but not
limited
to those described in U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514,
5,817,483,
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WO 2006/078911 PCT/US2006/002050
5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370,
5,693,762, 5,530,101, 5,585,089, 5,225,539; and 4,816,567.
Techniques for making partially to fully human antibodies are laiown in the
art
and any such techniques can be used. According to one embodiment, fully human
antibody sequences are made in a transgenic mouse which has been engineered to
express human heavy and light chain antibody genes. Multiple strains of such
transgenic mice have been made which can produce different classes of
antibodies. B
cells from transgenic mice which are producing a desirable antibody can be
fused to
make hybridoma cell lines for continuous production of the desired antibody.
(See for
example, Russel, N.D. et al. (2000) Infection and Immunity Apri12000:1820-
1826;
Gallo, M. L. et al. (2000) European J. of Immun. 30:534-540; Green, L. L.
(1999) J.
of Immun. Methods 231:11-23; Yang, X-D et al. (1999A) J. of Leukocyte Biology
66:401-410; Yang, X-D (1999B) Cancer Research 59(6):1236-1243; Jakobovits, A.
(1998) Advanced Drug Delivery Reviews 31:33-42; Green, L. and Jakobovits, A.
(1998) J. Exp. Med. 188(3):483-495; Jakobovits, A. (1998) Exp. Opin. Invest.
Drugs
7(4):607-614; Tsuda, H. et al. (1997) Genomics 42:413-421; Sherman-Gold, R.
(1997). Genetic Engineering News 17(14); Mendez, M. et al. (1997) Nature
Genetics
15:146-156; Jakobovits, A. (1996) WEIR's HANDBOOK OF EXPERIMENTAL
IMMUNOLOGY, THE INTEGRATED IMMUNE SYSTEM VOL. IV, 194.1-194.7; Jakobovits,
A. (1995) Current Opinion in Biotechnology 6:561-566; Mendez, M. et al. (1995)
Genomics 26:294-307; Jakobovits, A. (1994) Current Biology 4(8):761-763;
Arbones,
M. et al. (1994) Immunity 1(4):247-260; Jakobovits, A. (1993) Nature
362(6417):255-258; Jakobovits, A. et al. (1993) Proc. Natl. Acad. Sci. USA
90(6):2551-2555; Kucherlapati, et al. U.S. Patent No. 6,075,181.)
Human monoclonal antibodies can also be produced by a hybridoma which
includes a B cell obtained from a transgenic nonhuman animal, e.g., a
transgenic
mouse, having a genome comprising a human heavy chain transgene and a light
chain
transgene fused to an immortalized cell.
The antibodies of this invention also can be modified to create chimeric
antibodies. 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.
See, e.g., U.S. Patent No.: 4,816,567.
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The term "antibody derivative" also includes "diabodies" which are small
antibody fragments with two antigen-binding sites, wherein fragments comprise
a
heavy chain variable domain (VH) connected to a light chain variable domain
(VL) in
the same polypeptide chain (VH VL). (See for example, EP 404,097; WO 93/11161;
and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.) By
using a
linker that is too short to allow pairing between the two domains on the same
chain,
the domains are forced to pair with the complementary domains of another chain
and
create two antigen-binding sites. (See also, U.S. Patent No. 6,632,926 to Chen
et al.
which discloses antibody variants that have one or, more amino acids inserted
into a
hypervariable region of the parent antibody and a binding affinity for a
target antigen
which is at least about two fold stronger than the binding affinity of the
parent
antibody for the antigen.)
The term "antibody derivative" further includes "linear antibodies". The
procedure for making the is known in the art and described in Zapata et al.
(1995)
Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of
tandem Fd
segments (VH -CH 1-VH -CH1) which form a pair of antigeri binding regions.
Linear
antibodies can be bispecific or monospecific.
The antibodies of this invention can be recovered and purified from
recombinant cell cultures by known methods including, but not limited to,
protein A
purification, a.inmonium sulfate or ethanol precipitation, acid extraction,
anion or
cation exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. High performance liquid
chromatography ("HPLC") can also be used for purification.
Antibodies of the present invention include naturally purified products,
products of chemical synthetic procedures, and products produced by
recombinant
techniques from a eukaryotic host, including, for example, yeast, higher
plant, insect
and mammalian cells, or alternatively from a prokaryotic cells as described
above.
In some aspects of this invention, it will be useful to detectably or
therapeutically label the antibody. Methods for conjugating antibodies to
these agents
are known in the art. For the purpose of illustration only, antibodies can be
labeled
with a detectable moiety such as a radioactive atom, a chromophore, a
fluorophore, or
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WO 2006/078911 PCT/US2006/002050
the like. Such labeled antibodies can be used for diagnostic techniques,
either in vivo,
or in an isolated test sample. Antibodies can also be conjugated, for example,
to a
pharmaceutical agent, such as chemotherapeutic drug or a toxin. They can be
linked
to a cytokine, to a ligand, to another antibody. Suitable agents for coupling
to
antibodies to achieve an anti-tumor effect include cytokines, such as
interleukin 2 (IL-
2) and Tumor Necrosis Factor (TNF); photosensitizers, for use in photodynamic
therapy, including aluminum (III) phthalocyanine tetrasulfonate,
hematoporphyrin,
and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90
(90Y),
bismuth-212 (a1aBi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium- 186
(186Re), and rhenium-18 S (188Re); antibiotics, such as doxorubicin,
adriamycin,
daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin;
bacterial,
plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A,
staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A
and
native ricin A), TGF-alpha toxin, cytotoxin from chinese cobra (naja naja
atra), and
gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria
and fungi,
such as restrictocin (a ribosome inactivating protein produced by Aspergillus
restrictus), saporin (a ribosome inactivating protein from Saponaria
officinalis), and
RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine
nucleoside);
liposomes containing anti cystic agents (e.g., antisense oligonucleotides,
plasmids
which encode for toxins, methotrexate, etc.); and other antibodies or antibody
fragments, such as F(ab).
With respect to preparations containing antibodies covalently linked to
organic
molecules, they can be prepared using suitable methods, such as by reaction
with one
or more modifying agents. Examples of such include modifying and activating
groups. A "modifying agent" as the term is used herein, refers to a suitable
organic
group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that
comprises an
activating group. Specific examples of these are provided supra. An
"activating
group" is a chemical moiety or functional group that can, under appropriate
conditions, react with a second chemical group thereby forming a covalent bond
between the modifying agent and the second chemical group. Examples of such
are
electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro,
iodo), N-
hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can
react with
thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl
disulfides, 5-
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thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde
functional
group can be coupled to amine- or hydrazide-containing molecules, and an azide
group can react with a trivalent phosphorous group to form phosphoramidate or
phosphorimide linkages. Suitable methods to introduce activating groups into
rnolecules are known in the art (see for example, Hermanson, G. T.,
BIOCONJUGATE TECHNIQUES, Academic Press: San Diego, Calif. (1996)). An
activating group can be bonded directly to the organic group (e.g.,
hydrophilic
polymer, fatty acid, fatty acid ester), or through a linker moiety, for
example a
divalent C1-C12 group wherein one or more carbon atoms can be replaced by a
heteroatom such as oxygen, nitrogen or sulfur. Suitable linker moieties
include, for
example, tetraethylene glycol. Modifying agents that cornprise a linker moiety
can be
produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-
ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of
1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond
between
the free amine and the fatty acid carboxylate. The Boc protecting group can be
removed from the product by treatment with trifluoroacetic acid (TFA) to
expose a
primary amine that can be coupled to another carboxylate as described, or can
be
reacted with maleic anhydride and the resulting product cyclized to produce an
activated maleimido derivative of the fatty acid.
The modified antibodies of the invention can be produced by reacting a human
antibody or antigen-binding fragment with a modifying agent. For example, the
organic moieties can be bonded to the antibody in a non-site specific manner
by
ernploying an amine-reactive modifying agent, for example, an NHS ester of
PEG.
Modified human antibodies or antigen-binding fragments can also be prepared by
reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an antibody or
antigen-
binding fragment. The reduced antibody or antigen-binding fragment can then be
reacted with a thiol-reactive modifying agent to produce the modified antibody
of the
invention. Modified human antibodies and antigen-binding fragments comprising
an
organic moiety that is bonded to specific sites of an antibody of the present
invention
can be prepared using suitable methods, such as reverse proteolysis. See
generally,
I3ermanson, G. T., BIOCONJUGATE TECHNIQUES, Academic Press: San Diego,
Calif. (1996).
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POLYNUCLEOTIDES AND POLYPEPTIDES
Polynucleotides, polypeptides and fragment(s) thereof, can be obtained by
using the sequence information provided in Table 1(and the sequence listing
infra)
and chemical synthesis using a commercially available automated peptide
synthesizer
such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model
430A
or 43 1A, Foster City, CA, USA. The synthesized protein or polypeptide can be
precipitated and further purified, for example by high performance liquid
chromatography (HPLC). Accordingly, this invention also provides a process for
chemically synthesizing the proteins of this invention by providing the
sequence of
the protein and reagents, such as amino acids and enzymes and linking together
the
amino acids in the proper orientation and linear sequence.
Alternatively, the proteins and polypeptides can be obtained by well-known
recombinant methods as described herein using the host cell and vector systems
as
described herein. The host cell can be prokaryotic or eukaryotic. Host cell
systems
are described supra.
DIAGNOSTIC METHODS
As noted above, this invention provides various methods for aiding in the
diagnosis of the state of a cell that is characterized by abnormal cell growth
in the
form of, e.g., malignancy, hyperplasia or metaplasia. The methods are
particularly
useful for aiding in the diagnosis of cancers of epithelial origin, e.g., non-
small cell
lung cancer (NSCLC), ovarian, breast, prostate and colon cancer. The
neoplastic state
of a cell can be 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 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 substraturn attachment, loss of contact inhibition, loss of
anchorage dependence, release of proteases such as plasminogen activator,
increased
sugar transport, decreased serum requirement, expression of fetal antigens and
the
like. (See, Luria et al. (1978) GENERAL VIROLOGY, 3d edition, 436-446 (John
Wiley
& Sons, New York)).
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Accordingly, one embodiment is a method of diagnosing the condition of a
cell by screening for the presence of a differentially expressed
polynucleotide or
polypeptide isolated from a sample containing or suspected of containing
having cells
that express said gene, in which the differential expression of the gene is
indicative of
the neoplastic state of the cell. As shown below, the gene is expressed more
in a
cancer or tumor cell, wherein said cell is one or more of lung, ovarian or
prostate, as
compared to a counterpart normal or healthy cell or tissue.
Detection can be by any appropriate method, including for example, detecting
the quantity of mRNA transcribed from the gene or the quantity of cDNA
produced
from the reverse transcription of the mRNA transcribed from the gene or the
quantity
of the polypeptide or protein encoded by the gene. Probes for each of these
methods
are provided by reverse translating the peptides identified in Table 1 and
using the
polynucleotides encoding the peptides. These methods can be performed on a
sample
by sample basis or modified for high throughput analysis. Additionally,
databases
containing quantitative fu11 or partial transcripts or protein sequences
isolated from a
cell sample can be searched and analyzed for the presence and amount of
transcript or
expressed gene product. In one aspect, the database contains at least one of
the
sequences shown in Table 1 and/or the polynucleotide encoding it.
For the purpose of illustration only, gene expression is determined by noting
the amount (if any, e.g., altered) expression of the gene in the test system
at the level
of an mRNA transcribed from the gene of interest. In a separate embodiment,
augrnentation of the level of the polypeptide or protein encoded by the gene
of interest
is indicative of the presence of the neoplastic condition of the cell. The
method can
be used for aiding in the diagnosis of lung, ovarian or prostate cancer. Thus,
by
detecting this genotype prior to tumor growth, one can predict a
predisposition to
cancer and/or provide early diagnosis and treatment.
Cell or tissue samples used for this invention encompass body fluid, solid
tissue samples, tissue cultures or cells derived there from and the progeny
thereof and
sections or smears prepared from any of these sources or any other samples
that may
contain a cell having a gene described herein. In one embodiment, the sample
comprises cells prepared from a subject's tissue, e.g., lung or tissue which
may
contain a metastatis.
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In assaying for an alteration in mRNA level, nucleic acid contained in the
aforementioned samples is first extracted according to standard methods in the
art.
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. The mRNA of a gene 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 at least one polynucleotide encoding a peptide
identified in Table 1 find utility as hybridization probes. 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 prob e
useful for
detecting inRNA is at least about 80% identical to the homologous region of
comparable size contained in the genes or polynucleotides encoding the
peptides
identified in Table 1. In one aspect, the probe is 85% identical to the
corresponding
polynucleotide sequence after alignment of the homologous region or,
alternatively, it
exhibits 90% identity. These probes can be used in radioassays (e.g., S uthern
and
Northern blot analysis) to detect, prognose, diagnose or monitor various
neoplastic
states resulting from differential expression of a polynucleotide of interest.
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.
Sinaller
fragments derived from the known sequences 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.
In one aspect, nucleotide probes having complementary sequencc:s over
stretches greater than about 10 nucleotides in length are used, so as to
increase
stability and selectivity of the hybrid and, thereby, improving the
specificity of
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particular hybrid molecules obtained. Alternatively, one can design nucleic
acid
molecules having gene-complementary stretches of more than about 25 or
alternatively more than about 50 nucleotides in length or even longer where
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.
Patent No. 4,603,102 or by introducing selected sequences into recombinant
vectors
for recombinant production. In one aspect, a probe is about 50 to about 75,
nucleotides or, alternatively, about 50 to about 100 nucleotides in length.
These
probes can be designed from the sequence of full length genes.
In certain embodiments, it will be advantageous to employ nucleic acid
sequences as described herein 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. One can employ a fluorescent label or an enzyme
tag, such
as urease, 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.
The nucleotide probes of the present invention can also be used as primers and
detection of genes or gene transcripts that are differentially expressed in
certain body
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tissues. Additionally, a primer useful for detecting the aforementioned
differentially
expressed mRNA is at least about 80% identical to the homologous region of
comparable size contained in the previously identified sequences encoding the
peptides identified in Table 1. For the purpose of this invention,
"amplification"
means any method employing a primer-dependent polymerase capable of
replicating a
target sequence with reasonable fidelity. Amplification may be carried out by
natural
or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of
E.coli DNA polymerase and reverse transcriptase.
A known amplification method is PCR, 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 of differentially expressed
genes of
interest can be verified by 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.
The probes also can be attached to a solid support for use in high throughput
screening assays using methods known in the art. PCT WO 97/10365 and U.S.
Patent
Nos. 5,405,783; 5,412,087 and 5,445,934; for example, disclose the
construction of
high density oligonucleotide chips which can contain one or more of the
sequences
disclosed herein. Using the methods disclosed in U.S. Patent Nos. 5,405,783;
5,412,087 and 5,445,934; the probes of this invention 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 can also be determined through exposure of a
nucleic acid sample to a probe-modified chip. Extracted nucleic acid is
labeled, for
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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.
The probes and high density oligonucleotide probe arrays also provide an
effective means of monitoring expression of the gene of interest. They are
also useful
to screen for compositions that upregulate or downregulate the expression of
the gene
of interest.
In another embodiment, the methods of this invention are used to monitor
expression of the gene of interest which specifically hybridize to the probes
of this
invention in response to defined stimuli, such as an exposure of a cell or
subject to a
drug.
In one embodiment, the hybridized nucleic acids are detected by detecting one
or more labels attached to the sample nucleic acids. The labels may be
incorporated
by any of a number of means known to those of skill in the art. However, in
one
aspect, the label is simultaneously incorporated during the amplification step
in the
preparation of the sample nucleic acid. Thus, for example, polymerase chain
reaction
(PCR) with labeled primers or labeled nucleotides will provide a labeled
amplification
product. In a separate embodiment, transcription amplification, as described
above,
using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP)
incorporates a
label into the transcribed nucleic acids.
Alternatively, a label may be added directly to the original nucleic acid
sample
(e.g., mRNA, polyA, mRNA, cDNA, etc.) or to the amplification product after
the
amplification is completed. Means of attaching labels to nucleic acids are
known to
those of skill in the art and include, for example nick translation or end-
labeling (e.g.,
with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment
(ligation) of a nucleic acid linker joining the sample nucleic acid to a label
(e.g., a
fluorophore).
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Detectable labels suitable for use in the present invention include any
composition detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. Useful labels in the
present
invention include biotin for staining with labeled streptavidin conjugate,
magnetic
beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red,
rhodamine,
green fluorescent protein and the like), radiolabels (e.g., 3H, 125I, 35S, 14C
or 32P)
enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others
commonly
used in an ELISA) and colorimetric labels such as colloidal gold or colored
glass or
plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents
teaching the use
of such labels include U.S. Patents Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345;
4,277,437; 4,275,149; and 4,366,241.
Means of detecting such labels are known to those of skill in the art. Thus,
for
example, radiolabels may be detected using photographic film or scintillation
counters, fluorescent markers may be detected using a photodetector to detect
emitted
light. Enzymatic labels are typically detected by providing the enzyme with a
substrate and detecting the reaction product produced by the action of the
enzyme on
the substrate and colorimetric labels are detected by simply visualizing the
colored
label.
As described in more detail in WO 97/10365, the label may be added to the
target (sample) nucleic acid(s) prior to or after the hybridization. These are
detectable
labels that are directly attached to or incorporated into the target (sample)
nucleic acid
prior to hybridization. In contrast, "indirect labels" are joined to the
hybrid duplex
after hybridization. Often, the indirect label is attached to a binding moiety
that has
been attached to the target nucleic acid prior to the hybridization. Thus, for
example,
the target nucleic acid may be biotinylated before the hybridization. After
hybridization, an avidin-conjugated fluorophore will bind the biotin bearing
hybrid
duplexes providing a label that is easily detected. For a detailed review of
methods of
labeling nucleic acids and detecting labeled hybridized nucleic acids; see,
LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR
BIOLOGY, Vol. 24: Hybridization with Nucleic Acid Probes, P. Tijssen, ed.
Elsevier, N.Y. (1993).
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The nucleic acid sample also may be modified prior to hybridization to the
high density probe array in order to reduce sample complexity thereby
decreasing
background signal and improving sensitivity of the measurement using methods
known in the art, e.g., the methods disclosed in WO 97/10365.
Results from the chip assay are typically analyzed using a computer software
program. See, for example, EP 0717 113 A2 and WO 95/2068 1. The hybridization
data is read into the program, which calculates the expression level of the
targeted
gene(s) i.e., the genes identified in Table 1. This figure is compared against
existing
data sets of gene expression levels for diseased and healthy individuals. A
correlation
between the obtained data and that of a set of diseased individuals indicates
the onset
of a disease in the subject patient.
Also within the scope of this application is a database useful for the
detection
of neoplastic lung tissue comprising one or more of the sequences,
polynucleotides
encoding the peptides, or parts thereof, of the peptides listed Table 1.
These 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 then sequenced. The sequences from the sample
are
compared with the sequence(s) present in the database using homology search
techniques described above. In one aspect, greater than 90% is selected or,
alternatively, greater than 95% is selected or, alternatively, greater than or
equal to
97% sequence identity is selected, between the test sequence and at least one
sequence, or polynucleotide encoding it, identified in Table 1 or its
complement, is a
positive indication that the polynucleotide has been isolated from a lung,
prostate or
ovarian cancer cell as defined above.
Alternatively, one can compare a sample against a database. Briefly, multiple
RNAs are isolated from cell or tissue samples using methods known in the art
and
described for example, in Sambrook et al. (1989) supra. Optionally, the gene
transcripts can be converted to cDNA. A sampling of the gene transcripts are
subjected to sequence-specific analysis and quantified. These gene transcript
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sequence abundances are compared against reference database sequence
abundances
including normal data sets for diseased and healthy patients. The patient has
the
disease(s) with which the patient's data set most closely correlates which
includes the
overexpression of the transcripts identified herein.
Differential expression of the gene of interest can also be determined by
examining the protein product. 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,
immunofluorescent assays and PAGE-SDS. One means to determine protein level
involves (a) providing a biological sample containing polypeptides; and (b)
measuring
the amount of any immunospecific binding that occurs between an antibody
reactive
to the expression product of a gene of interest and a component in the sample,
in
which the amount of immunospecific binding indicates the level of the
expressed
proteins.
Antibodies that specifically recognize and bind to the protein products of
these
genes are required for these immunoassays. These rnay be purchased from
commercial vendors or generated and screened using methods well known in the
art.
See, Harlow and Lane (1988) supra and Sambrook et al. (1989) supra.
Alternatively,
polyclonal or monoclonal antibodies that specifically recognize and bind the
protein
product of a gene of interest can be made and isolated using known methods.
In diagnosing malignancy, hyperplasia or metaplasia characterized by a
differential expression of genes, 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
the
predicted change in expression of a gene of interest and clinical
characteristics of the
malignancy or metaplasia of interest. Alternatively, 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 level of
the gene
at question is within a normal range. A positive correlation between the
subject and
the positive control with respect to the identified alterations indicates the
presence of
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or a predisposition to said disease. A lack of correlation between the subject
and the
negative control confirms the diagnosis. In a preferred embodiment, the method
is
used for diagnosing cancers of epithelial origin, e.g., lung, ovarian or
prostate, on the
basis of a differential expression of the gene of interest.
SCREENING ASSAYS
The present invention also provides a screen for identifying leads, drugs,
therapeutic biologics 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 identifies lead compounds or biological agents which are
useful for
the treatment of malignancy, hyperplasia or metaplasia characterized by
differential
expression of the gene of interest.
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 which
differentially expresses the gene of interest associated with a neoplastic
cell.
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 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, the method can be modified for high
throughput analysis and suitable cells may be cultured in microtiter plates
and several
agents may be assayed at the same time by noting genotypic changes, phenotypic
changes and/or cell death.
When the agent is a composition other than a DNA or RNA nucleic acid
molecule, the suitable conditions comprise 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.
The screen involves contacting the agent with a test cell characterized by
differential expression of the gene of interest and then assaying the cell for
the level
of the gene of interest expression. In some aspects, it may be necessary to
determine
the level of the gene of interest expression prior to the assay. This provides
a base
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line to compare expression after administration of the agent to the cell
culture. In
another embodiment, the test cell is a cultured cell from an established cell
line that
differentially expresses a gene of interest. An agent is a possible
therapeutic agent if
gene expression is returned (reduced or increased) to a level that is present
in a cell in
a normal or non-neoplastic state, or the cell selectively dies, or exhibits
reduced rate
of growth.
In yet anotlher aspect, the test cell or tissue sample is isolated from the
subject
to be treated and one or more potential agents are screened to determine the
optimal
therapeutic and/or course of treatment for that individual patient.
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 siinple or complex
organic or
inorganic molecule, a peptide, a protein or an oligonucleotide. 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;
these compounds 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 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. As noted above,
lung
cells having differential expression of a gene of interest 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 differential expression
of the
gene.
When the agent is a nucleic acid, it can be added to the cell cultures by
methods known in the art, which includes, but is not limited to calcium
phosphate
precipitation, microinjection or electroporation. Alternatively or
additionally, the
nucleic acid can be incorporated into an expression or insertion vector for
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incorporation int(> the 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 and
briefly described infra.
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 lirilcers contain nucleic acid sequences that correspond to a
particular
restriction site in the vector DNA. Additionally, an oligonucleotide
containing a
tennination 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 rneomycin gene for selection of stable or transient
transfectants in
mammalian cells; enhancer/promoter 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 RINA promoters for in vitro transcription of sense and
antisense
RNA. Other means are well-known and available in the art.
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 in gene overexpression. Cellular
differentiation
can be monitored b y 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., the expression of the gene of interest.
Kits contairiing 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 rnodel system which can be used prior to clinical testing of
the
therapeutic agent or alternatively, for lead optimization. In this system, a
candidate
agent is a potential drug if gene expression is returned to a normal level or
if
symptoms associated or correlated to the presence of cells containing
differential
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expression of a gene of interest 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.
THERAPEUTIC METHODS
Therapeutic agents provided by this invention, include, but are not limited to
small molecules, polynucleotides, peptides, antibodies, antigen presenting
cells and
include immune effector cells that specifically recognize and lyse cells
expressing the
gene of interest. One can determine if a subject or patient will be
beneficially treated
by the use of agents by screening one or more of the agents against tumor
cells
isolated from the subject or patient using methods known in the art.
Additional
methods are provided infra.
In one embodiment, the therapeutic agent is administered in an amount
effective to treat cancer of epithelial origin, e.g., lung, ovarian, breast,
prostate and
colon cancers. 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 knc)wn 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 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 differential expression of the gene of interest. 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
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administered to the subject. In one aspect, to determine patients that can be
beneficially treated, a tumor sample is removed from the patient and the cells
are
assayed for the differential expression of the gene of interest. Therapeutic
amounts
can be empirically determined and will vary -vvith the pathology being
treated, the
subject being treated and the efficacy and tox'icity of the agent. When
delivered to an
animal, the method is useful to further confirrn efficacy of the agent. As an
example
of an animal model, groups of nude mice (Ba1b/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 treatm.ent. 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 do se 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 inedicaments and for the treatrment of humans and other animals
by
administration in accordance with conventional procedures, such as an active
ingredient in pharmaceutical compositions.
The pharmaceutical compositions canbe administered orally, intranasally,
parenterally or by inhalation therapy, and may take the form of tablets,
lozenges,
granules, capsules, pills, ampoules, suppositorles 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 composi-tions can also contain other
pharmaceutically active compounds or a pluraLity of compositions of the
invention.
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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, intramusculax, 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 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
fonnulations may conveniently be presented in unit do sage 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.
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Formulations of the present invention suitable for oral adininistration 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 rnay 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, cro'ss-linked sodium
carboxymethyl cellulose) surface-active or dispersing agent. Molcled 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 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 moutli 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 fo-
7rmulation 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 3 0% w/w of a polyhydric alcohol, i.e., an alcohol having two or
more
hydroxyl groups such as propylene glycol, butane-l,3-diol, mannitol, sorbitol,
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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 a 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
emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying
wax, anci
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.
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 paraffin 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
carrier,
especially an aqueous solvent for the agent.
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Formulations for rectal administration may be presented as a suppository with
a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for 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.
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, 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 multi-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
an agent.
TRANSGENIC ANIMALS
In another aspect, the gene of interest 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
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the genoine of the recipient embryo, the resulting embryos or adult animals
can be
analyzed to determine the function of the gene. The inutant 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
359:211-215).
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.
EXPERIMENTAL METHODS
Experiment No. 1: Expression Analysis
Non-small cell lung cancer globally represents a huge unmet medical need.
Fresh non-necrotic NSCLC tumor tissue and corresponding normal tissue from
individual patients free of infectious disease were obtained. Fresh tissue
samples
were at least 1.5 grams of tissue less than 24 hrs from surgery shipped on wet
ice.
Prior to analysis, the tissue was assessed by a pathologist for percent tumor
content,
tumor stage and histological type. All of the tissues received were primary
lung
cancer of stage I, II or IIIa where surgery is the first or primary trcatment.
Upon receipt of the tissues, the samples the tissues were minced with crossed
scalpels. The minced tissues with treated with collagenase and elastase until
single
cell suspensions were obtained. The single cell suspensions were washed
several
times and the red blood cells were lysed. The white blood cells were removed
by
exposure of the cell suspension to antibodies to CD64, CD45 and CD141inked to
magnetic beads. The epithelial cells were isolated using an antibody to BerEP4
linked
to a magnetic bead. The endothelial cells were isolated using an antibody to
CD31
linked to a magnetic bead. RNA was prepared from the epithelial cell and
endothelial
cell samples immediately.
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The quality of the RNA from the epithelial and endothelial cells was assessed
overall and by expression of specific markers for the cell types. Cytokeratin
18, von
Willebrand factor, EF 1, P 1 H 12, hevin and cytokeratin 8 were used as
markers to
deterrnine whether the RNA collected represented a relatively pure cell
population of
epithelial cells or endothelial cells. After the quality assessment of the
RNAs, 4
squamous carcinoma samples, 2 adenocarcinoma samples and 3 normal lung tissue
samples were of sufficient quantity and quality for SAGE analysis.
LongSAGETM was performed on the 9 RNA samples to a depth of
approximately 50,000 tags for each library using the methods disclosed in
Nature
Biotechnology (2002) 20:508-512. An in-depth bioinformatics analysis was
undertaken to characterize the SAGE data based upon increased expression of
mRNAs across tumor types and in the squamous and adenocarcinoma compared with
the normal lung cells. The focus for potential antibody therapeutic targets
was on
proteins expressed in the plasma membrane.
GITR, glucocorticoid-induced TNFR-related protein, is known to be expressed
by activated T cells and Treg cells. GITR is also known as activation-
inducible
TNFR family receptor (AITR) and tumor necrosis factor receptor superfamily,
member 18 (TNFRSF-18) (Stephens, G.L. et al. (2004) J. Immunol. 173:5008-5020
and Nocentini, G. et al. (1997) P.N.A.S. 94:6216-6221). GITR ligand binds to
GITR
and triggers NF-kappaB activation through TRAF2. The GITR -GITR ligand
interaction interrupts TCR-CD3 activation - induced apoptosis in T cells and
may be
involved in cell survival. The GITR ligand is also known as AITRL, GITRL, TL6
and hGIRTL. GITR is a 228 amino acid transmembrane protein that is suggested
to
be similar to 4-1BB and CD27. GITR protein has a 19 amino acid signal
sequence,
134 amino acid extracellular region with three cysteine-rich motifs, a 23
amino acid
transmembrane segment and a 52 amino acid cytoplasmic domain. The GITR ligand
is expressed by endothelial cells (including HUVEC), B1 lymphocytes, mature
and
immature dendritic cells, and macrophages (Stephens, G. et al. (2004) supra.).
GITR
is involved in the interactions between T-lymphocytes and endothelial cells
and in the
regulation of T-cell receptor-mediated cell death. GITR mediates NF-kappaB
activation via the TRAF2/NIK pathway. GITR binds to TNF receptor-associated
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factor-1 (TR.AF1), TRAF2 and TRAF3 but not to TR.AFS and TRAF6 (Nocentini, G.
et al. (1997) supra.).
GITR is expressed on CD4+CD25+ T cells and after interaction with GITRL
down-regulates T regulatory suppressor activity. Targeting GITR on tumor cells
and
depletion of CD4+CD25+ T cells could potentiate the efficacy of active tumor
specific therapy (Kohm, A.P. et al. (2004) J. Immunol. 172:4686-4690 and
Shimizu,
J. et al. (2002) Nature Immunol. 3:135-142).
RT-PCR of bulk tissue RNA from 55 lung tumors and 18 normal lung tissues
indicated ~!2-fold increased levels of GITR RNA in 76% of tumors compared with
normal tissues. By RT-PCR GITR has very minimal expression in a variety of .
normal tissues including breast, prostate, brain, heart, kidney, liver,
salivary gland,
spleen stomach, thymus and uterus. RT-PCR of bulk tissues RNA from a variety
of
tumors and corresponding normal tissues indicated ~!:2-fold increased levels
of GITR
RNA in 50% of ovarian cancers (n=40), 25% of melanomas (n=22), 50% of prostate
cancers (n=24), 20% of colon cancers (n=26), and 66% of breast cancers (n=23).
Immunohisochemistry was performed on formalin-fixed paraffin-embedded
human non-small cell lung cancer specimens. The antibodies used were anti-GITR
(Research Systems, BA689), anti-RDC1 (Lifescience, RDCI-LP1439). CD31
(DAKO, M0823), anti-alpha-smooth muscle actin (DAKO, M0851), anti-epthelial
membrane antigen (DAKO, M0804) and wide spectrum cytokeratin (DAKO, Z0622).
Overall, there was strong GITR reactivity on tumor cells of both
adenocarcinoma and squamous carcinoma of the lung. There was also strong
reactivity on infiltrating cells within the tumor stroma of both
adenocarcinoma and
squamous carcinoma of the lung. There was strong RDCI reactivity on tumor
cells of
both adenocarcinoma and squamous carcinoma of the lung. There was moderate
RDC1 reactivity on endothelial cells and pericytes/smooth muscle cells
associated
with the tumor vasculature.
By fluorescence activated cytometry (FACS), the NCI-H358 and NCI-H1436
cell lines have very good expression of GITR, and NCI-H1299, NCI-H522, NCI-H23
and NCI-H647 have good expression of GITR. By FACS the expression of GITR on
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activated PBMC and activated CD3+ T cells is much lower than is the expression
on
non-small cell lung cancer cell lines.
Experiment No. 2: Functional BioAssays
After generation the panel of antibodies are screened using cell based assays
to
identify those which neutralize the function of the target protein and reverse
the
malignant phenotype using methods known in the art. For the purpose of
illustration,
such methods are described in: Stanton, C.A. et al. (2004) Blood 103(2):601-
606 and
Malinda, K.M. et al. (1999) Exp. Cell Res. 250:168-173 (migration assay);
paragraphs [0210] through [0226] of U.S. Patent Publication No. 2004/0253708A1
(apoptosis); paragraphs [0183] through [0194] of U.S. Patent Publication
No. 2004/0258685A1 (inhibition, anti-proliferation, blocking and epitope
binding
assays); Stanton, C.A. et al. (2004) supra (proliferation and cytotoxicity
assays);
Manches, O. et al. (2003) Blood 101(3):949-954 (apoptosis, phagocytosis and
ADCC
assays); and paragraph [0066] of U.S. Patent Publication 2004/0228859A1 (CDC
assay).
Experiment No. 3: In vivo Efficacy
Antibodies that possess the requisite bio-activity as described for example,
in
Experiment No. 2, are then further screened for in vivo efficacy using a
syngeneic
tumor model or human tumor xenographt model. Such assays and models are known
to those of skill in the art. For the purpose of illustration, such methods
and described
in Tumor Models in Cancer Research, Teicher, B.A. ed. in the series Cancer
Drug
Discovery and Development, Humana Press, 2004 and Lev. A. et al. (2004) PNAS
101(24):9051-9056.
Although the above experiments and detailed description are described in
reference to use against NSCLC, it should be apparent to those of skill in the
art that
the methods and compositions of this invention are relevant to the other
cancers
identified in Table 1. Thus, this invention provides methods and compositions
to
diagnose and prognose these malignancies as well.
It is to be understood that while the invention has been described in
conjunction with the above embodiments, that the foregoing description and the
following examples are intended to illustrate and not limit the scope of the
invention.
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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.
57
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