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WO1999037816A1 - Procedes d'identification de cibles therapeutiques - Google Patents

Procedes d'identification de cibles therapeutiques Download PDF

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Publication number
WO1999037816A1
WO1999037816A1 PCT/US1999/001463 US9901463W WO9937816A1 WO 1999037816 A1 WO1999037816 A1 WO 1999037816A1 US 9901463 W US9901463 W US 9901463W WO 9937816 A1 WO9937816 A1 WO 9937816A1
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WIPO (PCT)
Prior art keywords
cell
cells
sample
neoplastic
gene
Prior art date
Application number
PCT/US1999/001463
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English (en)
Inventor
Bruce L. Roberts
Srinivas Shankara
Original Assignee
Genzyme Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genzyme Corporation filed Critical Genzyme Corporation
Priority to JP2000528722A priority Critical patent/JP2002500896A/ja
Priority to EP99903346A priority patent/EP1053349A4/fr
Priority to CA002319148A priority patent/CA2319148A1/fr
Priority to AU23391/99A priority patent/AU756357B2/en
Publication of WO1999037816A1 publication Critical patent/WO1999037816A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • 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.
  • This invention provides methods for identifying therapeutically-relevant genes which are expressed differentially in one cell with respect to another.
  • 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.
  • 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.
  • 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 provide the skilled artisan with knowledge of the sequence and structure of the protein product(s) 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.
  • PCR 2 A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)) and ANIMAL CELL CULTURE (RJ. Freshney, ed. (1987)).
  • a cell includes a plurality of cells, including mixtures thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • T-lymphocytes 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, CD8, or both.
  • 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 “na ⁇ ve” cell is a cell that has never been exposed to an antigen.
  • 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, murines, 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., murine, rat, simian or human.
  • An “antibody” is an immunoglobulin molecule capable of binding an antigen.
  • 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.
  • 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.
  • 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.
  • a "concentrated”, “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 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 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.
  • 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%.
  • 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.
  • 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.
  • stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 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.
  • 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.
  • 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 gene(s) encoding the factor(s) 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 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.
  • the practice of the invention can be applied to the identification of gene(s) 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.
  • the practice of the invention can be applied to any cell type including, but not limited to, plants, animals and microorganisms.
  • 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 cholangiocarcinoma; breast tumors, such as ductal and lobular adenocarcinoma; gynecologic tumors, such as
  • Tumor cells are typically obtained from a cancer patient by resection, 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 generate a cell line. Conditions for reliably establishing short-term cultures and obtaining at least 10 8 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 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 5 mm pieces which are immersed in RPMI 1640 medium containing 0.01%> hyaluronidase type V, 0.002% DNAse type I, 0.1 % collagenase type IV, 50 IU/ml penicillin, 50 ⁇ g/ml streptomycin and 50 ⁇ g/ml gentamycin. This mixture is 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 Ca or Mg or phenol red, then resuspended in HBSS and passed through Ficoll-Hypaque gradients.
  • HBSS Hanks balanced salt solution
  • the gradient interfaces, containing viable tumor cells, lymphocytes, and monocytes, are harvested and washed twice more with HBSS.
  • 10 may be frozen for storage in a type-compatible human serum containing 10% (v/v) DMSO.
  • neoplastic cell refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not 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.
  • 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.
  • MDR multi-drug resistance
  • 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
  • Cytotoxic drugs as antitumor chemotherapeutic agents can be subdivided into several broad categories, including: 1) 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, vincristine, 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.
  • 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, or 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.
  • antigen presenting 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.
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells; purified MHC class I molecules complexed to ⁇ 2- microglobulin; and foster antigen presenting cells.
  • Faster antigen presenting cells refers to any modified or naturally occurring 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
  • T2 cells are what will be referred to as "foster" APCS.
  • Sample cells include those transduced with a polynucleotide. The term
  • polynucleotide 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.
  • 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 polyamides.
  • 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.
  • 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.
  • 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. 51:415-462), Salmonella typhosa, Brucella abortus; protozoan parasites such as Toxoplasma gondii, Leishmania donovani, Trypanosoma cruzi, malarial plasmodia.
  • 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.
  • 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.
  • 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.
  • 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.
  • a suitable control cell is one which has a compatible MHC complex but does not express the antigen.
  • control cells are compatible for lysis by a cytotoxic T-lymphocyte for example, but are not lysed by the cytotoxic T-lymphocyte.
  • a control cell is one that does not secrete the factor.
  • 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.
  • a futher 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.
  • 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.
  • cDNA Complementary DNA
  • cDNA is then isolated from the sample, for example using methods known to those skilled in the art.
  • the cDNA is synthesized from mRNA using a biotinylated oligo(dT) primer.
  • Smaller fragments of cDNA are then be created using a restriction endonuclease, preferably one that would be expected to cleave most transcripts at least once.
  • a restriction endonuclease preferably one that would be expected to cleave most transcripts at least once.
  • 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.
  • streptavidin beads are used to isolate the defined 3' nucleotide sequence polynucleotide when the oligo dT primer for cDNA synthesis is biotinylated.
  • Other capture systems e.g., biotin/streptavidin, digoxigenin anti-digoxigenin
  • biotin/streptavidin digoxigenin anti-digoxigenin
  • 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.
  • 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 polynucleotidefe.g., 3' of the restriction endonuclease cleavage site).
  • the defined nucleotide sequence polynucleotide may be from about 6 to 30 base pairs.
  • the polynucleotide is about 9 to 11 base pairs.
  • a ditag i.e. the dimer of two sequence tags
  • the second restriction endonuclease cleaves at a site distant from or outside of the recognition site.
  • the second restriction endonuclease can be a type IIS restriction enzyme.
  • Type IIS restriction endonucleases cleave at a defined distance up to 20 bp away from their
  • the ditag (ligated tag pair) having a first restriction endonuclease site upstream (5') 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • 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 first 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
  • 17 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.
  • 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.
  • 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.
  • 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 CaCl method using procedures well known in the art. Alternatively, MgCl 2 or RbCl can be used. Transformation can also be performed by electroporation or other commonly used methods in the art.
  • the individual tags or ditags can be hybridized with oligonucleotides immobilized on a solid support (e.g., nitrocellulose filter, glass slide, silicon chip).
  • a solid support e.g., nitrocellulose filter, glass slide, silicon chip.
  • either the ditags or oligonucleotide probes are labeled with a detectable label, for example, with a radioisotope, a fluorescent compound, a
  • bioluminescent compound a chemi-luminescent compound, a metal chelator, or an enzyme.
  • a chemi-luminescent compound a chemi-luminescent compound
  • metal chelator a metal chelator
  • an enzyme e.g., a carboxylate, a carboxylate, or a carboxylate.
  • PCR can be performed with labeled (e.g. , fiuorescein 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).
  • a solid support e.g., a silicon chip as described by Fodor et al. Science 251:767, 1991
  • 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 different 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.).
  • fluoresceinated end labeled ditags analysis of fluorescence is indicative of hybridization to a particular 10-mer.
  • 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.
  • polynucleotide information 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.
  • 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 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.
  • test 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.
  • 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
  • GroupNormal Normal 1 + Normal2
  • GroupTumor Primary Tumor 1 + 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.
  • 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.
  • 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.
  • Query Report and test them individually in an appropriate biological assay to determine if they confer the phenotype.
  • 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.
  • the polynucleotide or gene sequence can 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.
  • the BLAST program is available at the following Internet address: http://www.ncbi.nlm.nih.gov.
  • hybridization under conditions of high, moderate and low stringency can also indicate degree of sequence identity.
  • 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.
  • 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.
  • genes that are responsible for this difference i.e., genes whose expression specifically enable lysis of a target cell by a cytotoxic T- lymphocyte.
  • 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.
  • 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
  • Ditags are prepared, amplified and cleaved with the anchoring enzyme as defined by SAGE technology:
  • oligonucleotide sequences contain a CATG sequence at the 5' end:
  • 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.
  • the oligonucleotide-bearing matrices are evaluated for the presence or absence of a fluorescent ditag at each position in the grid.
  • 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.
  • Library B that is expressed at low abundance in Library A.
  • 4D reflects a differentially-expressed, high abundance transcript restricted to Library A;
  • 5 A reflects a transcript that is expressed at high abundance in Library A but only at low abundance in Library B;
  • 5E reflects a differentially-expressed (in Library B), low abundance transcript.
  • 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.
  • 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.
  • 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
  • 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.
  • tumor antigens which are self proteins over-expressed by tumor cells
  • viral antigens such as HPV16E6 and E7
  • cancer/testes family of antigens typified by MAGE
  • mutated proteins such as ras or p53.
  • 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.
  • 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
  • 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.
  • SAGE analysis described in U.S. Patent No. 5,695,937
  • CTL gplOO specific cytotoxic T lymphocyte
  • 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 gplOO 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.
  • Example 2 Melanoma and breast cancer cell lines, exhibiting differential immunoreactivity to an anti-HER-2 antibody as judged by FACS 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.
  • 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 1300mel (that show a weaker fluorescence signal) (Table 4).
  • 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.
  • 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.
  • 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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Abstract

La présente invention concerne de manière générale un procédé de corrélation du phénotype d'une cellule avec le génotype fonctionnel de celle-ci, à savoir la constellation de séquences exprimés dans cette cellule. En outre, l'invention concerne des moyens d'identification de gènes et produits géniques thérapeutiquement appropriés.
PCT/US1999/001463 1998-01-26 1999-01-25 Procedes d'identification de cibles therapeutiques WO1999037816A1 (fr)

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CA002319148A CA2319148A1 (fr) 1998-01-26 1999-01-25 Procedes d'identification de cibles therapeutiques
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EP1364066A2 (fr) * 2001-02-02 2003-11-26 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procede d'identification d'acides nucleiques fonctionnels

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JP2004287619A (ja) * 2003-03-19 2004-10-14 Ntt Data Corp 疫学情報管理装置、疫学情報管理方法、および、プログラム

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