Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR IDENTIFYING THERAPEUTIC TARGETS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. ~ 119(e) of U.S.
Provisional Application Numbers 60/100,436; 60/077,853; and 60/I03,230, filed
January 26, 1998; March 13, 1998; and October 5, 1998, respectively; the
contents of which are hereby incorporated by reference into the present
disclosure.
TECHNICAL FIELD
This invention is in the fields of molecular biology, cell biology and
immunology. More particularly, the invention uses techniques of functional
genomics to correlate the phenotype of a cell with its pattern of gene
expression
and to identify new therapeutic targets.
BACKGROUND. OF THE INVENTION
The imminent acquisition of the sequence of the entire human genome will
provide a wealth of information on gene and genome structure and organization.
In order to use this vast wealth of genetic information in the prediction and
treatment of human disease, the next step is to develop methods for the
analysis of
the data. In particular, methods are required which will allow one to
distinguish
global patterns of differential gene expression between different cells, or
between
different pathological stages of the same cell. Methods of this type are often
denoted functional genomics.
It is well known that many, but not all genes present in a cell are expressed
at any given time. Fundamental questions of biology require knowledge of which
genes are transcribed and the relative abundance of transcripts in different
cells.
Typically, when and to what degree a given gene is expressed has been analyzed
one gene at a time.
Thus, information regarding the identity of all expressed genes in a cell
and the level of expression of these genes would facilitate the study of many
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cellular processes such as activation, differentiation, aging, viral
transformation,
morphogenesis, and mitosis. A comparison of the expressed genes of a
particular
cell or the same cell from various individuals or species, under the same or
different environmental stimuli, provides valuable insight into the molecular
S biology of the cell.
Accordingly, a method that provides a comparison of the expressed genes
of a particular cell as compared to another cell would be of great value. This
invention provides methods for identifying therapeutically-relevant genes
which
are expressed differentially in one cell with respect to another.
SUMMARY OF THE INVENTION
The present invention broadly provides a method for correlating the
phenotype of a cell with its "functional genotype," that is, the constellation
of
expressed sequences in that cell. In addition, the invention provides a means
for
identifying therapeutically-relevant genes and gene products.
This invention also provides computer-related systems and methods.
More specifically, the invention provides a system and method for
automatically
generating a data base of gene tags from cell samples and using the data base
for
filtering the tag counts from the samples into meaningful candidates for
further
testing and analysis.
MODES FOR CARRYING OUT THE INVENTION
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.
The present invention provides a method for identifying a gene associated
with a selected phenotype. Knowledge of the sequence of such a gene will also
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provide the skilled artisan with knowledge of the sequence and structure of
the
protein products) of the gene. In a preferred embodiment, the action of the
gene
and/or its product will be causative or involved in some way with respect to
the
selected phenotype.
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of 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)) and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)}.
Definitions
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.
The terms "polynucleotide" and "nucleic acid molecule" are used
interchangeably to refer to polymeric forms of nucleotides of any length. The
polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or
their
analogs. Nucleotides may have any three-dimensional structure, and may perform
any function, known or unknown. The term "polynucleotide" includes, for
example, single-, double-stranded and triple helical molecules, a gene or gene
fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of
any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
A
nucleic acid molecule may also comprise modified nucleic acid molecules.
The term "differentially expressed" refers to nucleotide sequences in a cell
or tissue which are either more or less expressed than a control cell, or
expressed
where silent in a control cell or not expressed where expressed in a control
cell.
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"Oligonucleotide" refers to polynucleotides of between about 5 and about
100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also
known as oligomers or oligos and may be isolated from genes, or chemically
synthesized by methods known in the art. A "gene" is a hereditary unit that,
in the
classical sense, occupies a specific position (locus) within the genome or
chromosome; a unit that has one or more specific effects upon the phenotype of
the organism; a unit that can mutate to various allelic forms; a unit that
recombines with other such units. Three classes of polynucleotides s are now
recognized: ( 1 ) structural genes that are transcribed into mRNAs, which are
then
translated into polypeptide chains, (2) structural polynucleotides that are
transcribed into rRNA or tRNA molecules which are used directly, and
(3) regulatory sequences that are not transcribed, but serve as recognition
sites for
enzymes and other proteins involved in DNA replication and transcription.
A "primer" refers to an oligonucleotide, usually single-stranded, that
provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid
synthesis. The primer sequence need not reflect the exact sequence of the
template. "PCR primers" refer to primers used in "polymerase chain reaction"
or
"PCR," a method for amplifying a DNA base sequence using a heat-stable
polymerase such as Taq polymerase, and two oligonucleotide primers, one
complementary to the (+)-strand at one end of the sequence to be amplified and
the other complementary to the (-)-strand at the other end. Because the newly
synthesized DNA strands can subsequently serve as additional templates for the
same primer sequences, successive rounds of primer annealing, strand
elongation,
and dissociation produce exponential and highly specific amplification of the
desired sequence. PCR also can be used to detect the existence of the defined
sequence in a DNA sample.
A "sequence tag" or "SAGE tag" is a short sequence, generally under
about 20 nucleotides, that occurs in a certain position in messenger RNA. The
tag
can be used to identify the corresponding transcript and gene from which it
was
transcribed. A "ditag" is a dimer of two sequence tags.
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The term "cDNAs" refers to complementary DNA, that is mRNA
molecules present in a cell or organism made in to cDNA with an enzyme such as
reverse transcriptase. . A "cDNA library" is a collection of all of the mRNA
molecules present in a cell or organism, all turned into cDNA molecules with
the
enzyme reverse transcriptase, then inserted into "vectors" (other DNA
molecules
which can continue to replicate after addition of foreign DNA). Exemplary
vectors for libraries include bacteriophage (also known as "phage"), viruses
that
infect bacteria, for example, lambda phage. The library can then be probed for
the
specific cDNA (and thus mRNA} of interest.
The term "immune effector cells" refers to cells capable of binding an
antigen and which mediate an immune response. These cells include, but not
limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T
lymphocytes (CTLs}, for example CTL lines, CTL clones; and CTLs from tumor,
inflammatory, or other infiltrates. Certain diseased tissue expresses specific
antigens and CTLs specific for these antigens have been identified. For
example,
approximately 80% of melanomas express the antigen known as GP-100.
The term "T-lymphocytes" as used herein denotes lymphocytes that are
phenotypically CD3+, typically detected using an anti-CD3 monoclonal antibody
in combination with a suitable labeling technique. The T-lymphocytes of this
invention are also generally positive for CD4, CDB, or both.
As used herein, the terms "restriction endonucleases" and "restriction
enzymes" refer to bacterial enzymes which bind to a specific double-stranded
DNA sequence termed a recognition site or recognition nucleotide sequence, and
cut double-stranded DNA at or near the specific recognition site. "Type IIS"
restriction endonucleases are those which cleave at a defined distance (up to
20
bases away) from their recognition sites. Endonucleases will be known to those
of skill in the art (see for example, Current Protocols in Molecular Biology,
Vol.
2, 1995, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Unit
3.1.15; New England Biolabs Catalog, 1995). A "naive" cell is a cell that has
never been exposed to an antigen.
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The term "culturing" refers to the in vitro propagation of cells or
organisms on or in media of various kinds. It is understood that the
descendants
of a cell grown in culture may not be completely identical (morphologically,
genetically, or phenotypically) to the parent cell. By "expanded" is meant any
proliferation or division of cells.
A "subject" is a vertebrate, preferably a mammal, more preferably a
human. Mammals include, but are not limited to, marines, simians, humans, farm
animals, sport animals, and pets.
"Host cell" or "recipient cell" is intended to include any individual cell or
cell culture which can be or have been recipients for vectors or the
incorporation
of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also
is
intended to include progeny of a single cell, and the progeny may not
necessarily
be completely identical (in morphology or in genomic or total DNA complement)
to the original parent cell due to natural, accidental, or deliberate
mutation. The
cells may be procaryotic or eucaryotic, and include but are not limited to
bacterial
cells, yeast cells, animal cells, and mammalian cells, e.g., marine, rat,
simian or
human. An "antibody" is an immunoglobulin molecule capable of binding an
antigen. As used herein, the term encompasses not only intact immunoglobulin
molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion
proteins,
humanized proteins and modifications of the immunoglobulin molecule that
comprise an antigen recognition site of the required specificity.
An "antibody complex" is the combination of antibody (as defined above)
and its binding partner or ligand.
A native antigen is a polypeptide, protein or a fragment containing an
epitope, which induces an immune response in the subject.
The term "isolated" means separated from constituents, cellular and
otherwise, in which the polynucleotide, peptide, polypeptide, protein,
antibody, or
fragments thereof, are normally associated with in nature. As is apparent to
those
of skill in the art, a non-naturally occurring polynucleotide, peptide,
polypeptide,
protein, antibody, or fragments thereof, does not require "isolation" to
distinguish
it from its naturally occurring counterpart. In addition, a "concentrated",
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"separated" or "diluted" polynucleotide, peptide, polypeptide, protein,
antibody,
or fragments 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
fragments thereof, which differs from the naturally occurring counterpart in
its
primary sequence or for example, by its glycosylation pattern, need not be
present
in its isolated form since it is distinguishable from its naturally occurring
counterpart by its primary sequence, or alternatively, by another
characteristic
such as glycosylation pattern. Although not explicitly stated for each of the
inventions disclosed herein, it is to be understood that all of the above
embodiments for each of the compositions disclosed below and under the
appropriate conditions, are provided by this invention. Thus, a non-naturally
occurring polynucleotide is provided as a separate embodiment from the
isolated
naturally occurring polynucleotide. A protein produced in a bacterial cell is
provided as a separate embodiment from the naturally occurnng protein isolated
from a eucaryotic cell in which it is produced in nature.
An "isolated" or "enriched" population of cells is "substantially free" of
cells and materials with which it is associated in nature. By "substantially
free" or
"substantially pure" means at least 50% of the population are the desired cell
type,
preferably at least 70%, more preferably at least 80%, and even more
preferably at
least 90%.
A "composition" is intended to mean a combination of active agent and
another compound or composition, inert (for example, a detectable agent ,
solid
support or label) or active, such as an adjuvant.
A "pharmaceutical composition" is intended to include the combination of
an active agent with a carrier, inert or active, making the composition
suitable for
diagnostic or therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water, and emulsions, such as an oil/water or
water/oil
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emulsion, and various types of wetting agents. The compositions also can
include
stabilizers and preservatives. For examples of Garners, stabilizers and
adjuvants,
see Martin, REMINGTON'S PHARM. SCL, 15th Ed. (Mack Publ. Co., Easton (1975)).
An "effective amount" is an amount sufficient to effect beneficial or
desired results. An effective amount can be administered in one or more
administrations, applications or dosages.
As described in more detail below, the present method identifies a
polynucleotide fragment of a gene that confers or is involved in conferring a
selected phenotype to a sample cell, cells, or tissue or presenting a
potential
therapeutic target. The method requires identifying a unique polynucleotide,
the
unique polynucleotide representing a gene that is differentially expressed in
a
sample cell compared to a control cell. In one embodiment, the gene
corresponding to the unique polynucleotideis is identified and cloned, thereby
providing the sequence and identy of the gene conferring the selected
phenotype
to the sample cell or is associated with a selected phenotype but not
necessarily
causative of the selected phenotype. The unique polynucleotide can represent
or
correspond to or be a fragment of a gene that is differentially, overexpressed
or
underexpressed in the sample cell compared to the control cell. More than one
sample cell type can be compared to a single control cell, or alternatively,
more
than one control cell type can be compared to a single sample cell.
Therapeutic
targets can be identified using the methods disclosed herein. The polypeptides
and proteins encoded by these polynucleotides and genes can further produced,
isolated and characterized.
In one embodiment, the method is useful for identifying one or more
secreted biological factors and/or the genes) encoding the factors) or
fragments
thereof. The method involves the steps of providing one or more sample cells
that secrete the factor and one or more control cells that do not secrete the
factor;
obtaining a set of polynucleotides representing gene expression in the sample
cells; obtaining a set of polynucleotides representing gene expression in the
control cells; and identifying one or more unique polynucleotides, the unique
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polynucleotides being common to the sample cells and the unique
polynucleotides
being absent or expressed at lower levels in the control cells. Finally, by
determining the genes corresponding to the unique polynucleotides, one or more
secreted biological factors are identified.
S The practice of the invention can be applied to the identification of genes)
that are relevant to any property that differs between one cell (a sample
cell) and
another (a control cell). Such properties may include, but are not limited to,
disease state, infection, drug resistance, cytokine secretion, secretory
protein
expression, state of differentiation, growth regulation, consequences of
exposure
to external environmental stimuli, etc. In addition, the practice of the
invention
can be applied to any cell type including, but not limited to, plants, animals
and
microorganisms.
Materials and Methods
Sample cells
The invention provides methods for identifying and obtaining
polynucleotides, genes and fragments thereof, associated with a selected
phenotype in a sample cell. "Sample cells" include, but are not limited to,
neoplastic cells; drug-resistant neoplastic cells; neoplastic cells which
promote
angiogenesis; de-differentiated cells; differentiated cells; apoptotic cells;
hyperproliferative cells; cells infected with a pathogen or drug-resistant
cells
infected with a pathogen.
Cancers from which cells can be obtained for use in the methods of the
present invention include carcinomas, sarcomas, leukemias, and cancers derived
from cells of the nervous system. These include, but are not limited to: brain
tumors, such as astrocytoma, oligodendroglioma, ependymoma,
medulloblastomas, and Primitive Neural Ectodermal Tumor (PNET); pancreatic
tumors, such as pancreatic ductal adenocarcinomas; lung tumors, such as small
and large cell adenocarcinomas, squamous cell carcinoma and
bronchoalveolarcarcinoma; colon tumors, such as epithelial adenocarcinoma and
liver metastases of these tumors; liver tumors, such as hepatoma and
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cholangiocarcinoma; breast tumors, such as ductal and lobular adenocarcinoma;
gynecologic tumors, such as squamous and adenocarcinoma of the uterine cervix,
and uterine and ovarian epithelial adenocarcinoma; prostate tumors, such as
prostatic adenocarcinoma; bladder tumors, such as transitional, squamous cell
carcinoma; tumors of the reticuloendothelial system (RES), such as B and T
cell
lymphoma (nodular and diffuse), plasmacytoma and acute and chronic leukemia;
skin tumors, such as melanoma; and soft tissue tumors, such as soft tissue
sarcoma and leiomyosarcoma.
Tumor cells are typically obtained from a cancer patient by resection,
Z O biopsy, or endoscopic sampling; the cells may be used directly, stored
frozen, or
maintained or expanded in culture. Samples of both the tumor and the patient's
blood or blood fraction should be thoroughly tested to ensure sterility before
co-
culturing of the cells. Standard sterility tests are known to those of skill
in the art
and are not described in detail herein. The tumor cells can be cultured in
vitro to
15 generate a cell line. Conditions for reliably establishing short-term
cultures and
obtaining at least 10g cells from a variety of tumor types is described in
Dillmar et
al. (1993) J. Immunother. 14:65-69. Alternatively, tumor cells can be
dispersed
from, for example, a biopsy sample, by standard mechanical means before use.
Tumor cells can be obtained by any method known in the art. The
20 following is an example of one method employed by skilled artisans. Using
sterile technique, solid tumors (10-30 g) excised from a patient are dissected
into
mm3 pieces which are immersed in RPMI 1640 medium containing 0.01
hyaluronidase type V, 0.002% DNAse type I, 0.1% collagenase type IV, SO IU/ml
penicillin, 50 ~.g/ml streptomycin and SO ~g/ml gentamycin. This mixture is
25 stirred for 6 to 24 hours at room temperature, after which it is filtered
through a
coarse wire grid to exclude undigested tissue fragments. The resultant tumor
cell
suspension is then centrifuged at 400 x g for 10 minutes. The pellet is washed
twice with Hanks balanced salt solution (HBSS) without Ca2+ or Mg2+ or phenol
red, then resuspended in HBSS and passed through Ficoll-Hypaque gradients.
30 The gradient interfaces, containing viable tumor cells, lymphocytes, and
monocytes, are harvested and washed twice more with HBSS. The harvested cells
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may be frozen for storage in a type-compatible human serum containing 10%
(v/v) DMSO.
The terms "neoplastic cell", "tumor cell", or "cancer cell", used either in
the
singular or plural form, refer to cells that have undergone a malignant
transformation
S that makes them pathological to the host organism. Primary cancer cells
(that is,
cells obtained from near the site of malignant transformation) can be readily
distinguished from non-cancerous cells by well-established techniques,
particularly
histological examination. The definition of a cancer cell, as used herein,
includes nat
only a primary cancer cell, but any cell derived from a cancer cell ancestor.
This
includes metastasized cancer cells, and in vitro cultures and cell lines
derived from
cancer cells. When referring to a type of cancer that normally manifests as a
solid
tumor, a "clinically detectable" tumor is one that is detectable on the basis
of
tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging
(MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings
alone may be insufficient to meet this definition.
The emergence of tumor cell resistance to chemotherapeutic agents poses
a major problem in the treatment of malignancies of the blood and solid
tumors.
This resistance causes cancer patients to fail to respond to any antitumor
agent,
since the transformed tumor cells tend to exhibit clinical resistance to many
drugs,
a phenomenon known as mufti-drug resistance (MDR). Several mechanisms can
account for MDR at a molecular and cellular level, including. decreased drug
uptake or increased drug efflux, altered redox potential, enhanced DNA repair,
and increased drug sequestration mechanisms or amplification of the drug-
target
protein. Drugs of proven antitumor chemotherapeutic value to which MDR has
been observed include vinblastine, vincristine, etoposide, teniposide,
doxorubicin
(adriamycin), daunorubicin, pliamycin, and actinomycin D. Jones et al. (1993)
Cancer (Suppl.) 72:3484-3488. Many tumors are intrinsically mufti-drug
resistant
(e.g., adenocarcinomas of the colon and kidney) while other tumors acquire MDR
during the course of therapy (e.g., neuroblastomas and childhood leukemias).
"A
drug-resistant cancer cell", for the purposes of the present invention,
include a cell
which is resistant to a single antitumor chemotherapeutic agent, as well as a
cell
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resistant to two or more antitumor chemotherapeutic agents. Cytotoxic drugs as
antitumor chemotherapeutic agents can be subdivided into several broad
categories, including:. l ) alkylating agents, such as mechlorethamine,
cyclophosphamide, melphalan, uracil mustard, chlorambucil and carmustine; 2)
antimetabolites such as methotrexate, fluorouracil, azarabine, mercaptopurine,
thioguanine and adenine arabinoside; 3) natural product derivatives such as
vinblastine, vin~ristine, doxorubicin, bleomicine, toposide, teniposide and
mitomycin-c; and 4) miscellaneous agents, such as hydroxyurea, procarbezine
and
mititane.
Sample cells further include neoplastic cells which promote angiogenesis.
Tumors promote angiogenesis (or neovascularization) through a combination of
overexpression of angiogenic factors and local inhibition of angiostatic
factors.
This strategy leads to an angiogenic environment that promotes tumor growth
and
metastases. Angiogenic factors include the CXC family of chemokines (Arenberg
et al. (1997) J. Leukocyte Biol. 62:554-562),
Sample cells also include those expressing an antigen, ar those which
specifically recognize an antigen and which induce an immune response such as
a
T-cell. Sample cells also include antigen expressing cells such as "antigen
presenting cells" or "APCs" which includes both intact whole cells as well as
other molecules which are capable of inducing the presentation of one or more
antigens, preferably in association with class I MHC molecules. Examples of
suitable APCs include, but are not limited to, whole cells such as
macrophages,
dendritic cells, B cells; purified MHC class I molecules complexed to (32-
microglobulin; and foster antigen presenting cells. The term "foster antigen
presenting cells" refers to any modified or naturally occurnng cell (wild-type
or
mutant) with antigen presenting capability that is utilized in lieu of antigen
presenting cells ("APC") that normally contact the immune effector cells they
are
to react with. In other words, it is any functional APC that T cells would not
normally encounter in vivo.
Foster antigen presenting cells can be derived as follows. The human cell
line 174xCEM.T2, referred to as T2, contains a mutation in its antigen
processing
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pathway that restricts the association of endogenous peptides with cell
surface
MHC class I molecules (Zweerink et al. (I993) J. Immunol. 150:1763-1771).
This is due to a large homozygous deletion in the MHC class II region
encompassing the genes TAP1, TAP2, LMP1, and LMP2 which are required for
antigen presentation to MHC class I-restricted CD8+ CTLs. In effect, only
"empty" MHC class I molecules are presented on the surface of these cells:
Exogenous peptide added to the culture medium binds to these MHC molecules
provided that the peptide contains the allele-specific binding motif. These T2
cells are what will be referred to as "foster" APCS.
Sample cells include those transduced with a polynucleotide. The term
"polynucleotide" as used herein refers to a polymeric form of nucleotides of
any
length, either ribonucleotides or deoxyribonucleotides. Thus, this term
includes
double- and single-stranded DNA and RNA. As used herein, "DNA" includes not
only bases A, T, C, and G, but also includes any of their analogs or modified
forms of these bases, such as methylated nucleotides, internucleotide
modifications such as uncharged linkages and thioates, use of sugar analogs,
and
modified and/or alternative backbone structures, such as polyarnides. Included
are polynucleotides which encode one or more proteins, or which can be
transcribed to generate antisense RNA or a ribozyme.
Suitable methods for manipulation of polynucleotides include those
described in a variety of references, including, but not limited to, MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd Ed., Vol. 1-3, eds. Sambrook et al. Cold
Spring Harbor Laboratory Press (1989); and CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, eds. Ausubel et al., Greene Publishing and Wiley-
Interscience: New York (1987) and periodic updates.
Any method in the art can be used for the transformation, or insertion, of
an exogenous polynucleotide into a host cell, for example, lipofection,
transduction, infection or electroporation, using either purified DNA, viral
vectors, or DNA or RNA viruses. The exogenous polynucleotide may be
maintained as a non-integrated vector, for example, a plasmid, or
alternatively,
may be integrated into the host cell genome.
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Sample cells include those infected with a pathogen. "Pathogen" includes
any microorganism which is potentially harmful to a cell, including
prokaryotes,
viruses and single-celled eukaryotes. Such pathogens include, but are not
limited
to viruses such as human immunodeficiency virus, Epstein-Barr virus; fungi;
bacteria capable of infecting mammalian cells, such as Chlamydia spp.,
Legionella pneumophila, Mycobacterium spp. (Sinai and Joiner (1997) Ann. Rev.
Microbiol. 51:415-4b2), Salmonella typhosa, Brucella abortus; protozoan
parasites such as Toxoplasma gondii, Leishmania donovani, Trypanosoma cruzi,
malarial plasmodia.
Control cells
The practice of the invention involves comparison of polynucleotides
corresponding to expressed genes between a sample cell and a control cell. The
selection of the appropriate cell or cell type is dependent on the sample cell
initially selected and the phenotype of the sample cell which is under
investigation.
In one aspect of the invention, the sample cell is a neoplastic cell and one
or more counterparts is another neoplastic cell or non-neoplastic precursors
of the
sample cell can be used as control cells. Counterparts would include, for
example, cell lines established from the same or related cells to those found
in the
sample cell population. For example, the control cell can be any of a
counterpart
normal cell type, a counterpart benign cell type, a counterpart non-metastatic
cell
type and a non-neoplastic precursor of the neoplastic cell.
Alternatively, a sample cell can be selected based on the expression of a
gene coding for peptide which participates in recognition of the sample cell
by an
immune effector cell, e.g., an antigen presenting cell, a suitable control
cell is one
which has a compatible MHC complex but does not express the antigen. Such
control cells are compatible for lysis by a cytotoxic T-lymphocyte for
example,
but are not lysed by the cytotoxic T-lymphocyte.
When the sample cell is a cell which secretes a biological factor, a control
cell is one that does not secrete the factor.
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Polynucleotide Fragments or Expression Tags
Practice of the method of this invention involves analysis of
poiynucleotide fragments of or corresponding to expressed genes. The
polynucleotides are obtained from sample and control cells using methods well
known in the art. Many methods are known in the art to identify differentially
expressed polynucleotides and each can be used to provide these
polynucleotides.
As used herein, the term "polynucleotide" includes SAGE tags (defined above)
as
well as any other nucleic acid obtained from methods that yield
quantitative/comparative gene expression data. Such methods include, but are
not
limited to cDNA subtraction, differential display and expressed sequence tag
methods. Techniques based on cDNA subtraction or differential display can be
quite useful for comparing gene expression differences between two cell types
(described in Hedrick et al. (1984) Nature 308:149 and Lian and Pardee (1992)
Science 257:967). The expressed sequence tag (EST) approach is another
valuable tool for gene discovery (desribed in Adams et al. (1991) Science
252:1651), like Northern blotting, RNase protection, and reverse transcriptase-
polymerase chain reaction (RT-PCR) analysis (described in Sambrook et al.
(1989) supra; Alwine et al. (1977) PNAS 74:5350; Zinn et al. (1983) Cell
34:865;
and Veres et al. (1987) Science 237:415). A father method utilizes
differential
display coupled with real time PCT and representational difference analysis
(described in Lisitisyn and Wigler (1995) Meth. Enzymol. 254:291-304). Another
approach is the technology known as Serial Analysis of Gene Expression (SAGE,
described in U.S. Patent No. 5,695,937). Using SAGE, sequence tags (tags being
used synonymously with polynucleotides) corresponding to expressed genes can
be analyzed.
The sequence tags or polynucleotides corresponding to the expressed
genes are prepared essentially as follows. First, a sample containing the
genes of
interest is provided. Suitable sources of samples include cells, tissue,
cellular
extracts or the like. Preferably, the sample is taken from an individual
having a
particular disease state of interest or at a particular stage in its
development.
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Complementary DNA (cDNA) is then isolated from the sample, for example
using methods known to those skilled in the art. In one embodiment, the cDNA
is
synthesized from mRNA using a biotinylated oligo(dT) primer.
Smaller fragments of cDNA are then be created using a restriction
S endonuclease, preferably one that would be expected to cleave most
transcripts at
least once. Preferably, a 4-base pair recognition site enzyme is used. More
than
one restriction endonuclease can also be used, sequentially or in tandem. The
cleaved cDNA can then be isolated by binding to a capture medium for label
attached to the primer described above. For example, streptavidin beads are
used
to isolate the defined 3' nucleotide sequence polynucleotidewhen the oligo dT
primer for cDNA synthesis is biotinylated. Other capture systems (e.g.,
biotin/streptavidin, digoxigenin/anti-digoxigenin) can also be employed.
In one aspect, the isolated defined nucleotide sequence polynucleotides are
separated into two pools of cDNA. Each pool is ligated using the appropriate
linkers. The linkers can be the same or different, although when the linkers
have
the same sequence, it is not necessary to separate the polynucleotides into
pools.
The first oligonucleotide linker comprises a first sequence for hybridization
of a
PCR primer and the second oligonucleotide linker comprises a second sequence
for hybridization of a PCR primer. In addition, the linkers further comprise a
second restriction endonuclease site. The linkers are designed so that
cleavage of
the ligation products with the second restriction enzyme results in release of
the
linker having a defined nucleotide sequence polynucleotide(e.g., 3' of the
restriction endonuclease cleavage site). The defined nucleotide sequence
polynucleotidemay be from about 6 to 30 base pairs. Preferably, the
polynucleotideis about 9 to 11 base pairs. Therefore, a ditag (i. e. the dimer
of two
sequence tags) is from about 12 to 60 base pairs, and preferably from 18 to 22
base pairs:
Typically, the second restriction endonuclease cleaves at a site distant
from or outside of the recognition site. For example, the second restriction
endonuclease can be a type IIS restriction enzyme. Type IIS restriction
endonucleases cleave at a defined distance up to 20 by away from their
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asymmetric recognition sites (Szybalski W. (1985) Gene 40:169). Examples of
type IIS restriction endonucleases include BsmFI and Fokl. Other similar
enzymes will be known to those of skill in the art (see, CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, supra).
The pool of defined tags ligated to linkers having the same sequence, or
the two pools of defined nucleotide sequence tags Iigated to linkers having
different nucleotide sequences, are randomly ligated to each other "tail to
tail".
The portion of the cDNA polynucleotidefurthest from the linker is referred to
as
the "tail". This creates the ditag (ligated tag pair) having a first
restriction
endonuclease site upstream (S') and a first restriction endonuclease site
downstream (3') of the ditag; a second restriction endonuclease cleavage site
upstream and downstream of the ditag, and a linker oligonucleotide containing
both a second restriction enzyme recognition site and an amplification primer
hybridization site upstream and downstream of the ditag. In other words, the
ditag is flanked by the first restriction endonuclease site, the second
restriction
endonuclease cleavage site and the linkers, respectively.
The ditag can be amplified by utilizing primers which specifically
hybridize to one strand of each linker. Preferably, the amplification is
performed
after the ditags have been ligated together using standard polymerase chain
reaction (PCR) methods as described for example in U.S. Patent No. 4,683,195.
Alternatively, the ditags can be amplified by cloning in prokaryotic-
compatible
vectors or by other amplification methods known to those of skill in the art.
Those of skill in the art can prepare similar primers for amplification based
on the
nucleotide sequence of the linkers without undue experimentation.
Cleavage of the amplified PCR product with the f rst restriction
endonuclease allows isolation of ditags which can then be concatenated by
ligation. After ligation, it may be desirable to clone the concatemers,
although it
is not required. Analysis of the ditags or concatemers, whether or not
amplification was performed, can be performed by standard sequencing methods.
Concatemers generally consist of about 2 to 200 ditags and preferably from
about
8 to 20 ditags. While these are preferred concatemers, it will be apparent
that the
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number of ditags which can be concatenated will depend on the length of the
individual tags and can be readily determined by those of skill in the art
without
undue experimentation. After formation of concatemers, multiple tags can be
cloned into a vector for sequence analysis, or alternatively, ditags or
concatemers
can be directly sequenced without cloning by methods known to those of skill
in
the art, either manually or using automated methods.
Among the standard procedures for cloning the defined nucleotide
sequence tags of the invention is insertion of the tags into vectors such as
plasmids or phage. The ditag or concatemers of ditags produced by the method
described herein are cloned into recombinant vectors for further analysis,
e.g.,
sequence analysis, plaque/plasmid hybridization using the tags as probes, by
methods known to those of skill in the art. Vectors in which the ditags are
cloned
can be transferred into a suitable host cell. "Host cells" are cells in which
a vector
can be propagated and its DNA expressed. The term also includes any progeny of
the subject host cell. It is understood that all progeny may not be identical
to the
parental cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is used. Methods
of stable transfer, meaning that the foreign DNA is continuously maintained in
the
host, are known in the art.
Transformation of a host cell with a vector containing ditag(s) may be
carried out by conventional techniques as are well known to those skilled in
the
art. Where the host is prokaryotic, such as E. coli, competent cells which are
capable of DNA uptake can be prepared from cells harvested after exponential
growth phase and subsequently treated by the CaCl2 method using procedures
well known in the art. Alternatively, MgCl2 or RbCI can be used.
Transformation
can also be performed by electroporation or other commonly used methods in the
~.
The individual tags or ditags, can be hybridized with oligonucleotides
immobilized on a solid support (e.g., nitrocellulose filter, glass slide,
silicon chip).
In addition, either the ditags or oligonucleotide probes are labeled with a
detectable label, for example, with a radioisotope, a fluorescent compound, a
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bioluminescent compound, a chemi-luminescent compound, a metal chelator, or
an enzyme. Those of ordinary skill in the art will know of other suitable
labels for
binding to the ditag, or will be able to ascertain such using routine
experimentation. For example, PCR can be performed with labeled (e.g.,
fluorescein tagged) primers.
The ditags are separated into single-stranded molecules which are
preferably serially diluted and added to a solid support (e.g., a silicon chip
as
described by Fodor et al. Science 251:767, 1991) containing oligonucleotides
representing, for example, every possible permutation of a 10-mer (e.g., in
each
grid of a chip). The solid support is then used to determine differential
expression
of the tags contained within that support (e.g., on a grid on a chip) by
hybridization of the oligonucleotides on the solid support with tags produced
from
cells under digerent conditions (e.g., different stage of development growth
of
cells in the absence and presence of a growth factor, normal versus
transformed
cells, comparison of different tissue expression, etc.). In the case of
fluoresceinated end labeled ditags, analysis of fluorescence is indicative of
hybridization to a particular 10-mer. When the immobilized oligonucleotide is
fluoresceinated, for example, a loss of fluorescence due to quenching (by the
proximity of the hybridized ditag to the labeled oligo) is observed and is
analyzed
for the pattern of gene expression.
Computational Analysis
After the polynucleotide information is obtained, it is analyzed to identify
polynucleotides that correspond to genes that are differentially expressed
between
the two or more cell types. It is within the scope of this invention to
perform the
method described above using previously identified and stored sequence
information that define and identify expressed genes. This information can be
obtained from private, publically available and commercially available
sequence
databases.
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For example, after a cell or tissue is selected for having a phenotype which
is dependent on the presence of one gene product within a sample cell samples,
e.g., cells that secrete, a biological factor whose activity can be measured
in an in
vitro assay, cells that stain with an antibody that recognizes a specific
antigen or
S cells that are lysed by cytotoxic T cells that recognize a specific antigen,
the cells
are further selected to identify sample cells that exhibit extremes of the
chosen
phenotype and ideally are matched in all other respects or phenotypic
characteristics. For example, cells that are matched, e.g., from the same
individual, would minimize having to deal with histocompatability differences
Ideally one selects two examples of sample cells (say "A" and "B") that
exhibit the chosen phenotype prominently and two examples of samples cells
(say
"C" and "D") that do not have the phenotype at all. Using the method of this
invention, polynucleotides present in a library form from each cell sample are
isolated and their relative expression noted. The individual libraries are
sequenced and the information regarding sequence and in some embodiments,
relative expression, is stored in any functionally relevant program, e.g., in
Compare Report using the SAGE software (available through Dr. Ken Kinzler at
Johns Hopkins University). The Compare Report provides a tabulation of the
polynucleotide sequences and their abundance for the samples (say A, B,. C and
D
above) normalized to a defined number of polynucleotides per library (say
25,000). This is then imported into MS-ACCESS either directly or via copying
the data into an Excel spreadsheet first and then from there into MS-ACCESS
for
additional manipulations. Other programs such as SYBASE or Oracle that permit
the comparison of polynucleotide numbers could be used as alternatives to MS-
ACCESS. Enhancements to the software can be designed to incorporate these
additional functions. These functions consist in standard Boolean, algebraic,
and
text search operations, applied in various combinations to reduce a large
input set
of polynucleotides to a manageable subset of polynucleotides of specifically
defined interest.
The researcher may create groups containing one or more projects) by
combining the counts of specific polynucleotides within a group (e.g.,
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GroupNormal = Normal l + Normal2, GroupTumor = PrimaryTumorl +
TumorCellLine). Additional characteristic values are also calculated for each
tag
in the group (e.g., average count, minimum count, maximum count). The
researcher may calculate individual tag count ratios between groups, for
example
the ratio of the average GroupNormal count to the average GroupTumor count for
each polynucleotide. The researcher may calculate a statistical measure of the
significance of observed differences in tag counts between groups.
To identify the polynucleotides within MS-ACCESS, a query to sort
polynucleotide tags based on their abundance in the sample cells is run. The
output from the Query report lists specific polynucleotides (by sequence) that
fit
the sorting criteria and their abundance in the various sample cells
The sorting is based on the principle that the gene product of interest (and
hence the corresponding polynucleotide) is more abundant in the samples that
prominently exhibit the chosen phenotype than in samples that do not exhibit
the
phenotype.
For example, one may query to identify polynucleotides that are present at
a level of 10 or more in samples A and B and less than 1 in samples C and D,
the
results of the search might reveal that 5 different polynucleotides fit the
sorting
criteria hence there are 5 candidates genes to be tested to determine whether
they
confer the phenotype when transferred into samples like C and D that do not
have
the phenotype.
The more stringent the sorting criteria, the more efficient the sorting
should be. Thus if one asked for polynucleotides that are at 5 copies or more
in
samples A and B and less than 5 copies in samples C and D, a large number of
candidates would be generated. However, if one can increase the differential
because the samples manifest extremes of the phenotype (say >10 in samples A
and B and <1 in samples C and D) this restricts the number of candidates that
will
be identified.
Prior knowledge of what amount of gene product (hence abundance of
polynucleotides) is required to confer the phenotype is not essential as one
can
arbitrarily select a set of sorting parameters, run the data analysis, and
identify
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and test candidates. If the desired candidate is not found the stringency of
the
sorting criteria can be reduced (i.e. reduce the differential) and the new
candidates
that are found can be tested. Iterative cycles of sorting and testing
candidates
should eventually culminate in the successful recovery of the desired
candidate
Table 1
Cycle Sorting Number of Number of
Criteria Candidates Candidates
to Evaluate
1 -10 in 10 10
samples A and
B
...1 in
samples C and
D
(minimum
differential=10x)
2 5 in 30 20*
samples A and
B
...2 in
samples C and
D
(minimum
differential=2.5x)
3 5 in 80 50#
samples A and
B
...5 in
samples C and
D
(minimum
differential=1
x)
*Of the 30 candidates, 10 will have already been evaluated in cycle 1 so
only 20 new candidates need to be evaluated
#Of the 80 candidates, 30 will have already been evaluated (10 in cycle 1,
in cycle 2) so only 50 need to be evaluated
Knowledge of what amount of gene product (hence abundance of
polynucleotide) is required to confer the phenotype will permit the rationale
use of
15 stringent sorting criteria and greatly accelerate the search process as the
desired
gene may be captured within a handful of candidates
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Establishing what amount of gene product is required to confer a specific
phenotype will be dependent on the specific phenotype in question and the
sensitivity of assays that measure that phenotype
For instance, the inventor has found that a frequency of 1/5000 (5 copies
of a SAGE tag normalized to a library size of 25,000) correlates with
sufficient
expression of a tumor antigen within the sample cell to render it sensitive to
lysis
by an antigen specific T cell while a frequency of 1/25,000 correlates with
the cell
being weakly sensitive to lysis.
Thus, one could use a sorting criteria of z5 in samples cells that are
susceptible to lysis and <_1 in samples that are not susceptible to lysis to
home in
on a candidate tumor antigen.
Accordingly, one enters the individual polynucleotide sequences from the
Query report into the program to determine if there is a match with any known
genes or whether they are potentially novel (no match=NM).
One then retrieves cDNAs corresponding to specific sequences from the
Query Report and test them individually in an appropriate biological assay to
determine if they confer the phenotype. Of the candidates that correspond to
known genes, it is a relatively easy task to obtain complementary DNAs for
these
candidates and test them individually to determine if they confer the specific
phenotype in question when transferred into cells that do not exhibit the
phenotype. If none of the known genes confer the phenotype, retrieve the cDNAs
corresponding to the No Match sequences of the Query Report by PCR cloning
and test the novel cDNAs individually for their ability to confer the
phenotype. If
the assumptions made up to this point are sound (i.e., a single gene product
can
confer the phenotype; the sorting criteria are not too stringent so as to
exclude the
desired candidate) then a cDNA corresponding to one of the candidates of the
Query Report will be found to confer the phenotype and the search is over. If
however none of the candidates are found to confer the phenotype then one may
need to reduce the stringency of the sorting parameters to "cast a wider net"
and
capture more candidates to be tested as above.
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In one embodiment, the polynucleotide or gene sequence care also be
compared to a sequence database, for example, using a computer method to match
a sample sequence with known sequences. Sequence identity can be determined
by a sequence comparison using, i.e., sequence alignment programs that are
known in the art, such as 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. A preferred alignment program is ALIGN Plus (Scientific and Educational
Software; Pennsylvania), preferably using default parameters, which are as
follows: mismatch = 2; open gap = 0; and extend gap = 2. Another preferred
~ program is the BLAST program for alignment of two nucleotide sequences,
using
default parameters as follows: open gap = 50; extension gap - 2 penalties; gap
x
dropoff = 0; expect = 10; word size =11. The BLAST program is available at the
following Internet address: http://www.ncbi.nlm.nih.gov. Alternatively,
hybridization under conditions of high, moderate and low stringency can also
indicate degree of sequence identity.
Phenotypes amenable to study using the methods of the invention
In one aspect of the invention, genes and gene products associated with
cancer and neoplastic cells are determined. Additionally, the methods of the
present invention can be used to establish correlations between the phenotype
and
the SAGE tag genotype of a variety of other types of cell. For example, in
other
aspects, the methods of the invention can be used in the identification of
gene
products associated with genetic disease, inherited disease and/or acquired
diseases. Gene products associated with drug resistance and drug metabolism
can
also be identified. Identification of genes associated with drug metabolism
will
have important applications in the field of pharmacogenomics, wherein an
individual's response to a particular therapeutic is determined, so as to
maximize
therapeutic value and minimize side effects. In additional aspects, the
methods of
the invention are used in the identification of gene products that confer some
measurable biological activity on a mature or differentiated population of
cells,
wherein the activity is not exhibited by immature or undifferentiated
precursors.
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For example, a class of T-lymphocytes known as cytotoxic T-lymphocytes are
able to recognize and lyse a target cell, whereas other types of T-lymphocyte
are
capable of recognition but incapable of lysis. Using the methods of the
invention,
it is possible to identify genes that are responsible for this difference,
i.e., genes
whose expression specifically enable lysis of a target cell by a cytotoxic T-
lymphocyte.
It will be clear to the skilled artisan that the ability to determine the
phenotype of a cell, and hence to establish a correlation between its
phenotype
and its SAGE genotype, will be dependent upon the sensitivity, specificity
and/or
complexity of the assays used to establish the phenotype of the cell. For
example,
a phenotype, such as metastatic potential, which is likely to depend upon
multiple
factors, may be more difficult to establish than a phenotype whose magnitude
is
dependent on the relative abundance of a single specific transcript.
The following example is intended to illustrate, but not limit, the invention
as defined herein.
There are several alternatives to the use of conventional sequencers to
generate sequence information on the polynucleotides:
1 ) Hybridizing tags, or preferably amplified ditags, against
oligonucleotide sequences fixed to a solid matrix such as nitrocellulose
filters,
glass slides or silicon chips ("parallel sequence analysis", or PSA); or
2) Performing limiting dilutions on the ditag (or concatenate)
preparations and then sequencing individual DNAs, either with or without prior
amplification, by techniques that include, for example, mass spectroscopy
(clonal
sequencing, or "CS").
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PSA:
In a preferred embodiment of PSA, the following steps are carried out with
ditags:
1) Ditags are prepared, amplified and cleaved with the anchoring enzyme
as defined by SAGE technology:
OOOOOOOOOOXXXX~OO~XXXCATG
GTACOOOOOOOOOOXO~XX
3) 4-base oligomers containing an identifier (e.g., a fluorescent moiety,
FL) are prepared that are complementary to the overhangs:
FL-CATG
4) The FL-CATG oligomers {in excess) are ligated to the ditags:
FI-CATGOOOOOOOOOOXXX~~~XXCATG
GTACOOOOOOOOOOXXXXXXXXXXGTAC-FL
S) The ditags are purified and melted to yield single-stranded DNAs:
1 S FI-CATGOOOOOOOOOOXXXX3~O~XXCATG
GTACOOOOOOOOOOXXXXXX~~XGTAC-FL
6) The mixture of single-stranded DNAs is serially diluted.
.. 7) Each serial dilution is hybridized out under appropriate stringency
conditions with solid matrices containing gridded single-stranded
oligonucleotides; all of the oligonucleotides contain a half site of the
anchoring
enzyme cleavage sequence. In the example used herein, the oligonucleotide
sequences contain a CATG sequence at the 5' end:
CATGO000000000, CATGXXX~~~, etc.
(or alternatively a GTAC sequence at the 3' end: OOOOOOOOOGTAC)
The matrices are constructed of any material known in the art and the
oligonucleotide-bearing chips are generated by any procedure known in the art,
e.g. silicon chips containing oligonucleotides prepared by the VLSIP
procedure.
See, for example, U.S. Patent No. 5,424,186.
8) 'The oligonucleotide-bearing matrices are evaluated for the presence or
absence of a fluorescent ditag at each position in the grid.
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In a preferred embodiment, there are 41° or 1,048,576,
oligonucleotides on
the grid of the general sequence CATGOOOOOOOOOO, such that every possible
10-base sequence is represented 3' to the CATG. Since there are estimated to
be
no more than 100,000 to 200,000 different expressed genes in the human genome,
there are enough oligonucleotide sequences to identify all of the possible
sequences adjacent to the 3'-most anchoring enzyme site observed in the cDNAs
from the expressed genes in the human genome.
A determination is made of differential expression by comparing the
fluorescence profile on the grids at different dilutions among different
libraries.
For example:
Library A, Ditags Diluted 1:10
A B C D E
1 FL
2 FL
3 FL FL _
4 FL
5 FL
Librarv B. Ditaes Diluted 1:10
A B C D E
1 FL
2 FL FL
3 FL FL
4
5 FL FL
Library A, Ditags Diluted 1:50
A B C D E
1 FL
2
3 FL
4 FL
5 FL
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Library B, Ditags Diluted 1:50
A B C D E
1 FL
2 ~ FL
3 FL FL
4
Library A, Ditags Diluted 1:100
A B C D E
1 FL
2
3 FL
4 FL
5 FL
5 Library B, Ditags Diluted 1:100
A ~ B C D E
1 FL
2 FL
3 FL
4
5
The individual oligonucleotides thus hybridize to ditags with the following
characteristics:
Dilution 1: 10 1:50 1:100
Lib A Lib B Lib Lib B Lib A Lib B
A
lA + + + + + +
2C + + +
2E + +
3g + + + + + +
3C + + +
4D + + +
SA + + + +
SE ~ + ~
From the summary table, it is concluded that tags hybridizing to 1 A and
3B reflect highly abundant mRNAs that are not differentially expressed (since
the
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tags hybridize to both libraries at all dilutions); that 2C is a highly
abundant
mRNA, but only in Library B, and that 4D is highly abundant, but only in
Library
A. 2E reflects a low abundance transcript (since it is only detected at the
lowest
dilution) that is not found to be differentially expressed; 3C reflects a
moderately
abundant transcript (since it is expressed only at the lower two dilutions} in
Library B that is expressed at low abundance in Library A. 4D reflects a
differentially-expressed, high abundance transcript restricted to Library A;
SA
reflects a transcript that is expressed at high abundance in Library A but
only at
low abundance in Library B; and SE reflects a differentially-expressed (in
Library
B), low abundance transcript.
In another PSA embodiment, step 3 above does not involve the use of a
fluorescent or other identifier; instead, at the last round of amplification
of the
ditags, fluoresceinated dNTPs are used so that half of the molecules are
probed on
the chips.
In yet another PSA embodiment, instead of ditags, a particular portion of
the transcript is used, e.g., the sequence between the 3' terminus of the
transcript
and the first anchoring enzyme site. In that particular case, a double-
stranded
cDNA reverse transcript is generated as described in WO 97/10363. The
transcripts are cut with the anchoring enzyme, a linker is added containing a
PCR
primer and amplification is initiated (using the primer at one end and the A
tail at
the other) while the transcripts are still on the strepavidin bead. At the
last round
of amplification, fluoresceinated dNTPs are used so that half of the molecules
can
be probed on the chip. The linker-primer is optionally removed with the
anchoring enzyme at this point in order to reduce the size of the fragments.
The
soluble fragments are then melted and captured on solid matrices containing
CATGO000000000, as in the previous example. Analysis and scoring (only
of the half of the fragments which contain fluoresceinated bases) are as
described
above.
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CS:
Ditags or concatemers are diluted and added to wells or other receptacles
so that on average the wells contain, statistically, less than one DNA
molecule per
well (as is done in limited dilution for cell cloning). Each well then
receives
reagents for PCR or another amplification process and the DNA in each
receptacle
is sequenced, e.g., by mass spectoscopy. The results are either be a single
sequence (there having been a single sequence in that receptacle), a "null"
sequence (no DNA present) or a double sequence (more than one DNA molecule),
which is discarded. Thereafter, assessment of differential expression is the
same
as defined by SAGE technique.
The preceding discussion and examples are intended merely to illustrate
the art. As is apparent to one of skill in the art, various modifications can
be made
to the above without departing from the spirit and scope of this invention.
Example 1
Investigators have sought to elicit antigen specific T cell responses in the
hopes of creating an anti-tumor cell immune response that might lead to the
eradication of tumor cells. To date, 4 classes of tumor antigens have been
identified: differentiation antigens which are self proteins over-expressed by
tumor cells; viral antigens such as HPV 16E6 and E7; the cancer/testes family
of
antigens typified by MAGE; and mutated proteins such as ras or p53. Of the
differentiation antigens, the vast majority are melanoma associated antigens
and
attempts to identify self antigens over-expressed by lung, prostate, breast or
colon
carcinomas that might be good candidates as targets for cytotoxic T cells have
largely been unsuccessful. Thus the vast majority of cancer immunotherapy
trials
conducted to date have been for the treatment of melanoma and little by way of
immunotherapy is available to offer patients suffering with other malignant
diseases.
The present invention calls for the use of genes differentially expressed in
target cells in the design of a vaccine to generate an immune response against
the
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target cells. The inventors have applied a SAGE analysis (described in U.S.
Patent No. 5,695,937), to identify a variety of transcripts that are
differentially
expressed in cancer cells, that have not previously been associated with tumor
cells.
Melanoma cell lines, differentially susceptible to lysis by a gp100 specific
cytotoxic T lymphocyte (CTL) were subjected to SAGE analysis to determine
which SAGE tags were shared amongst the cell lines that were susceptible to
lysis
against those polynucleotides that were absent or less abundant in cell lines
that
were not susceptible to lysis. Ten SAGE polynucleotides matched the sorting
criteria and were found to be represented at a higher level in cell lines
identified
as 624me1 and 1300me1 (that are susceptible to lysis) than in cell lines
identified
as BA1 and A375 (that are not susceptible to lysis). Two different
polynucleotides corresponding to the differentially spliced forms of the gp100
mRNA were identified within the set of differentially expressed genes
indicating
it is possible to rapidly narrow down the candidates, but in addition, 8 other
tag
sequences were found including a tag corresponding to cdc2-related protein
lcinase (Table 2). At the same time other differentially expressed genes were
identified. Thus, by virtue of the fact that the identified genes were
overexpressed, some may be candidates for use in immunotherapy.
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Table 2
COMPARISON OF MELANOMA CELL LINE
SAGE DATA
<5 <5 >10 >10
BA1 A375 624 1300 GENE
0 0 206 92 gp 100 melanocyte lineage-specific
antigen
0 0 65 18 gp 100 melanocyte lineage-specific
antigen
0 0 60 16 calpain-skeletal muscle protein
1 4 18 25 Mitochondrial
1 4 18 11 Biliary glycoprotein
3 3 47 34 microsomal epoxide hydrolase
gene
3 4 26 14 NM
3 4 18 13 NM
4 4 72 27 cdc2-related protein kinase
mRNA
4 4 20 11 ATP synthase subunit c
NM = no match
It was reasoned that if additional melanoma cell lines were included in
the analysis, one might further be able to restrict the number of SAGE
polynucleotides corresponding to expressed transcripts that could encode the
cognate antigen. Two non-HLA-A2 melanoma cell lines (e.g., NM455 and SK28)
were chosen and phenotypes established (susceptibility to lysis by a gp100
specific CTL) of the cells following transduction with an adenoviral vector
encoding HLA-A2. .While SK28 was a good tafet for the gp100 specific CTL,
NM455 was not. The HLA-A2 negative cell lines were subjected to SAGE
analysis and SAGE polynucleotides were sorted to identify polynucleotides
common to lines that are susceptible to lysis that are less abundant in lines
that are
less susceptible to lysis (see Table 3). Of the two polynucleotides that
matched
the sorting criteria, one was the gp100 tag CCTGGTCAAG. Thus, by conducting
the SAGE analysis of 6 different melanoma cell lines that are differentially
susceptible to lysis by an HLA restricted CTL, one is able to focus on just 2
transcripts that were candidates for the cognate antigen, one of which was the
desired target.
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Table 3
Comparison of Melanoma Cell Line SAGE Data
>2 >2 >2 >5 >5 >5 GENE
A375 BA1 NM455 SK28 624 1300
0 0 1 6 200 89 gp100 antigen
0 1 0 8 6 7 tag 9
Example 2
Melanoma and breast cancer cell lines, exhibiting differential
immunoreactivity to an anti-HER-2 antibody as judged by FRCS analysis were
subjected to SAGE analysis to determine which SAGE polynucleotides were
shared amongst the cell lines that showed a high mean fluorescence signal that
were less abundant in cell lines that showed a lower mean fluorescence signal.
Four SAGE polynucleotides matched the sorting criteria and were found to be
represented at a higher level in cell lines 21PT and 21MT (that show a strong
fluorescence signal) than in cell lines MDA-468, SK28, BA1, NM455 and
1300me1 (that show a weaker fluorescence signal) (Table 4). One tag
corresponding to HER-2 was identified but in addition, 3 other tag sequences
were found including a tag corresponding to integrin alpha-3. While HER-2 has
previously been identified as a target for patient derived T cells, it has not
been
reported that integrin alpha-3 can also be a target for patient derived immune
effector cells or antibodies. Thus, the gene encoding integrin alpha-3 or the
corresponding gene product or peptide fragments thereof can be used to provoke
an immune response to target cells that differentially express integrin alpha-
3.
While integrin alpha-3 was used for this example, any differentially expressed
gene or genes (identified by SAGE) and their corresponding proteins or peptide
fragments could be used to provoke an anti-target cell immune response.
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Table 4
Identification of the Antigen Recognized by an Antibody
Cell Line Mean Fluorescence
21PT 35.2
21MT 33.4
MDA-468 3.1
SK28 7.4
BAl ~ 8.9
NM455 11.1
1300 14.7
>10 <5
A B C D E F G Gene
66 11 2 0 0 0 1 NM
21 21 1 1 0 1 3 AL0096
11 25 0 0 I 2 2 HER2
11 1 0 0 4 3 0 integrin alpha-3
S
NM = no match
