WO2004074436A2 - Methods of use of a gpcr in the diagnosis and treatment of colon and lung cancer - Google Patents

Methods of use of a gpcr in the diagnosis and treatment of colon and lung cancer Download PDF

Info

Publication number
WO2004074436A2
WO2004074436A2 PCT/US2004/004060 US2004004060W WO2004074436A2 WO 2004074436 A2 WO2004074436 A2 WO 2004074436A2 US 2004004060 W US2004004060 W US 2004004060W WO 2004074436 A2 WO2004074436 A2 WO 2004074436A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
colon
antibody
expression
cell
Prior art date
Application number
PCT/US2004/004060
Other languages
French (fr)
Other versions
WO2004074436A3 (en
Inventor
Amy W. Lasek
Original Assignee
Incyte 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 Incyte Corporation filed Critical Incyte Corporation
Publication of WO2004074436A2 publication Critical patent/WO2004074436A2/en
Publication of WO2004074436A3 publication Critical patent/WO2004074436A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to the use of a G protein-coupled receptor (GPCR) expressed in colon and lung cancer, its encoding polynucleotide, and an antibody that specifically binds the protein to diagnose, to stage, to treat, or to monitor the progression or treatment of colon or lung cancer.
  • GPCR G protein-coupled receptor
  • Cancers and malignant tumors are characterized by continuous cell proliferation and cell death and are related causally to both genetics and the environment. Genes whose expression are associated with cancer are of potentially great importance as cancer markers in the early diagnosis and prognosis of various cancers, as well as potential targets in cancer treatment.
  • Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year. Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes. (See Potter (1999) J Natl Cancer Institute 91:916-932 for a review of colorectal cancer.) Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et aL (1990) National Institutes of Health, DHHS Publ No. (NIH)90-2789).
  • Lung cancer is the leading cause of cancer death in the United States affecting more than 100,000 men and 50,000 women each year, and nearly 90% of those diagnosed with lung cancer are cigarette smokers.
  • Tobacco smoke contains substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in exposed bronchial epithelium.
  • the lung cancer has metastasized to pleura, brain, bone, pericardium, and liver.
  • Treatment with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to the identification, staging, and treatment of lung cancer could positively affect prognosis.
  • G protein coupled receptors are a superfamily of seven transmembrane domain proteins that mediate the transduction of extracellular signals across the plasma membrane of cells from a large, diverse number of ligands through interaction with heterotrimeric G proteins. See, e.g., Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6.
  • the glycoprotein hormone receptors are a subfamily of GPCRs activated by the gonadotropins; lutropin (LH), thyrotropin (TSH), follitropin (FSH) and human choriogonadotropin (hCG), essential for the growth and differentiation of the gonads and thyroid gland.
  • glycoprotein hormone GPCRs are characterized by a large N-terminal extracellular domain containing some 9 leucine- repeat domains that function in proteimprotein interactions and are likely important for interaction with their respective protein ligands.
  • Two orphan GPCRs (ligand currently unknown) related to this subfamily, designated HG38 and LGR5 have also been identified which share -35% overall identity with members of the glycoprotein hormone receptor subfamily (McDonald et aL (1998) Biochem Biophys Res Commun 247:266-270; Hsu et al (1998) Molecular Endocrinology 12:1830-1845). Both receptors are expressed primarily in skeletal muscle, placenta, spinal cord, and various regions of the brain.
  • both receptors are characterized by a large extracellular domain containing leucine-rich repeats and a cysteine-rich domain near the junction of the extracellular domain and the first transmembrane domain (Hsu et al. supra).
  • Array technologies and quantitative PCR provide the means to explore the expression profiles of a large number of related or unrelated genes. When an expression profile is examined, arrays provide a platform for examining which genes are tissue-specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder. The application of expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of the disease.
  • the invention is based on the discovery that a GPCR known as HG38 (SEQ ID NO: 1) is differentially expressed in colon and lung cancer, and to the use of the protein, its encoding polynucleotide or the complement thereof, and an antibody that specifically binds the protein to diagnose, to stage, to treat, or to monitor the progression or treatment of a colon or lung cancer.
  • the invention provides a method for using a polynucleotide to detect the differential expression of a nucleic acid in a sample of colon or lung tissue comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the polynucleotide in the sample.
  • the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization.
  • the method showing differential expression of the polynucleotide is used to diagnose a colon or lung cancer.
  • the invention provides a purified protein or a portion thereof comprising an amino acid sequence of SEQ ID NO: 1 for use in the diagnosis of a colon or lung cancer.
  • the invention provides a method for diagnosing a colon or lung cancer comprising performing an assay to quantify the amount of the protein expressed in a sample of colon or lung tissue and comparing the amount of protein expressed to a standard, thereby diagnosing a colon or lung cancer.
  • the assay is selected from antibody arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, 2D-PAGE and scintillation counting, protein arrays, radioimmunoassays, and western analysis.
  • the invention also provides a method for using an antibody to detect differential expression of a protein in a sample of colon or lung tissue, the method comprising combining the antibody with a sample under conditions for formation of antibody:protein complexes, detecting complex formation, comparing complex formation with a standard, wherein differential expression of the protein between the sample and the standard is diagnostic of a colon or lung cancer.
  • the invention further provides an antagonist which specifically binds the protein having the amino acid sequence of SEQ ID NO: 1 for use in the treatment of a colon or lung cancer.
  • the invention provides a method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention an antibody that specifically binds the protein or a composition comprising an antibody and a pharmaceutical agent.
  • the invention also provides a method for delivering a pharmaceutical or therapeutic agent to a colon cancer cell comprising attaching the pharmaceutical or therapeutic agent to ,an antibody that specifically binds the protein and administering the anntibody to a subject in need of therapeutic intervention, wherein the antibody delivers the pharmaceutical or therapeutic agent to the cell.
  • the invention also provides a method for using a polynucleotide to produce a mammalian model system, the method comprising constructing a vector containing the polynucleotide of SEQ ID NO:3, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the polynucleotide in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.
  • Figures 1A-1J show the amino acid sequence (SEQ ID NO:l) of HG38 encoded by the nucleic acid sequence of SEQ ID NO:2.
  • the alignment was produced using MACDNASIS PRO software (Hitachi Software Engineering, San Bruno CA).
  • Figure 2 shows the relative expression of HG38 in various normal adult tissues.
  • the X-axis lists tissue type, and the Y-axis, the relative expression of HG38 normalized to that found in normal colon.
  • QPCR analysis was performed using the TAQMAN protocol (Applied Biosystems (ABI), Foster City CA). Tissues were obtained from Clinomics (Pittsfield MA) and Clontech (Palo Alto CA).
  • Figures 3 and 4 show the differential expression of HG38 in donor-matched normal/tumor colon samples as determined using QPCR (ABI).
  • Figure 3 tissues were obtained from the Huntsman Cancer Institute (HCI; Salt Lake City UT);
  • Figure 4 tissues from Asterand Bioresources, Inc. (Detroit MI).
  • Figure 5 shows the relative expression of HG38 in colon tumor cell lines compared to a normal colon cell line (LS 123) using QPCR (ABI).
  • Cell lines were obtained from the ATCC (Manassas VA).
  • Figure 6 shows the differential expression of HG38 in donor-matched normal/tumor lung samples as determined using QPCR (ABI). Tissue samples were obtained from the Roy Castle institute for Lung Cancer Research (RCI; Liverpool UK).
  • the probe sequence for the QPCR analyses depicted in Figures 2-6 used an oligonucleotide extending from about nucleotide 274 to about nucleotide 291 of SEQ ID NO:2.
  • Figure 7 shows the expression of the transcript encoding HG38 in normal colon tissue. Thin sections were stained with DAPI and hybridized in situ using sense or antisense RNA probes made from a fragment of SEQ ID NO: 2 extending from about nucleotide 274 to about nucleotide 2724 of SEQ ID NO:2.
  • Figure 8 shows the expression of the transcript encoding HG38 in a villous adenocarcinoma of the colon. Thin sections were stained with DAPI and hybridized in situ using sense or antisense RNA probes made from a fragment of SEQ ID NO:2 extending from about nucleotide 274 to about nucleotide 2724 of SEQ ID NO:2.
  • Table 1 shows the differential expression of HG38 in cancerous colon tissue relative to normal colon tissue as determined by microarray analysis. Column 1 shows the differential expression of HG38 in terms of the ratio of the signal intensity for the flourescent dye Cy5 in labeled tumor tissue relative to that for flourescent dye Cy3 in labeled normal colon tissue.
  • Column 2 shows the source of the normal colon tissue as the individual donor (Dn), or pooled tissue from more than one donor (pool), column 3 is a description of the colon tumor sample, and column 4, the source of the tumor sample.
  • Tissue samples were obtained from HCI and Asterand Bioresources.
  • Table 2 shows the differential expression of HG38 in cancerous lung tissue relative to normal lung tissue as determined by microarray analysis. Column headings are the same a those described above for Table 1. Tissue samples were obtained from RCI.
  • Antibody refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab') 2 fragment, an Fv fragment, and an antibody-peptide fusion protein.
  • Antigenic determinant refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody that specifically binds the protein. Biological activity is not a prerequisite for immunogenicity.
  • Array refers to an ordered arrangement of at least two polynucleotides, proteins, or antibodies on a substrate. At least one of the polynucleotides, proteins, or antibodies represents a control or standard, and the other polynucleotide, protein, or antibody is of diagnostic or therapeutic interest.
  • the arrangement of at least two and up to about 40,000 polynucleotides, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each polynucleotide and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
  • HG38 refers to a GPCR that is exactly or highly homologous (>85%) to the amino acid sequence of SEQ ID NO: 1 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • the "complement" of a polynucleotide of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to a nucleic acid molecule under conditions of high stringency.
  • polynucleotide encoding a protein refers to a nucleic acid whose sequence closely aligns with sequences that encode conserved regions, motifs or domains identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool; Altschul (1993) J Mol Evol 36:290-300; Altschul et al. (1990) J Mol Biol 215:403-410) and BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402) which provide identity within the conserved region. Brenner et al.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding HG38 or fragments of HG38 may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • “Derivative” refers to a polynucleotide or a protein that has been subjected to a chemical modification. Derivatization of a polynucleotide can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. These substitutions are well known in the art. Derivatization of a polynucleotide or a protein can also involve the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group (for example, 5-methylcytosine). Derivative molecules retain the biological activities of the naturally occurring molecules but may confer longer lifespan or enhanced activity.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • disorder refers to conditions, diseases or syndromes in which HG38 or the rnRNA encoding HG38 are differentially expressed; in particular, a colon or lung cancer.
  • An "expression profile” is a representation of gene expression in a sample. A nucleic acid expression profile is produced using sequencing, hybridization, or amplification (quantitative PCR) technologies and mRNAs or polynucleotides from a sample.
  • a protein expression profile although time delayed, mirrors the nucleic acid expression profile and may use antibody or protein arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, spatial immobilization such as 2D-PAGE, and radioimmunoassays including radiolabeling and quantification using a scintillation counter and western analysis to detect protein expression in a sample.
  • the nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art. Expression profiles may also be evaluated by methods such as electronic northern analysis, guilt-by-association, and transcript imaging. Expression profiles produced using any of the above methods may be contrasted with expression profiles produced using normal or diseased tissues.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
  • BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. "Similarity" uses the same algorithms but takes conservative substitution of residues into account. In proteins, similarity exceeds identity in that substitution, for example, of a valine for a leucine or isoleucine, is counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of HG38 which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of HG38 which is useful in any of the antibody production methods disclosed herein or known in the art.
  • Labeleling moiety refers to any reporter molecule including radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cofactors, inhibitors, or magnetic particles than can be attached to or incorporated into a polynucleotide, protein, or antibody.
  • a wide variety conjugation techniques include both direct synthesis and chemical conjugation, particularly to amines, thiols and other side groups which may be present.
  • Visible labels and dyes include but are not limited' to anthocyanins, ⁇ glucuronidase, biotin, BIODIPY, Coomassie blue, Cy3 and Cy5, 4,6-diamidino- 2-phenylindole (DAPI), digoxigenin, fluorescein, FITC, gold, green fluorescent protein (GFP), lissamine, luciferase, phycoerythrin, rhodamine, spyro red, silver, streptavidin, and the like.
  • Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
  • Ligand refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of HG38. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HG38.
  • a "multispecific molecule” can bind with at least two different binding specificities to at least two different molecules or two different sites on a molecule. Antibodies can perform as multispecific molecules in that they can bind to both a target protein and a pharmaceutical agent.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • Oligomer refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplicon, amplimer, primer, and oligomer.
  • “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • a “pharmaceutical agent” may be an antibody, an antisense molecule, a bispecific molecule, a multispecific molecule, a peptide, a protein, a radionuclide, a small drug molecule, a cytospecific or cytotoxic drug such as abrin, actinomyosin D, cisplatin, crotin, doxorubicin, 5-fluorouracil, methotrexate, ricin, vincristine, vinblastine,, or any combination of these elements.
  • Post-translational modification" of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
  • Probe refers to polynucleotides encoding HG38, their complements, or fragments thereof, which are used to detect identical, allelic or related polynucleotides.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Protein refers to a polypeptide or any portion thereof.
  • a "portion” of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic determinant of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison WI).
  • An "oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a "regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs).
  • Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense as containing nucleic acids, proteins, and antibodies.
  • a sample may comprise a bodily fluid such as ascites, blood, cerebrospinal fluid, lymph, semen, sputum, urine and the like; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue, a tissue biopsy, or a tissue print; buccal cells, skin, hair, a hair follicle; and the like.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any nat ⁇ ral or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any rigid or semi-rigid support to which polynucleotides, proteins, or antibodies are bound and includes magnetic or nonmagnetic beads, capillaries or other tubing, chips, fibers, filters, gels, membranes, plates, polymers, slides, wafers, and microparticles with a variety of surface forms including channels, columns, pins, pores, trenches, and wells.
  • a "transcript image” is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed polynucleotides in the cDNA libraries of an EST database as described in USPN 5,840,484, incorporated herein by reference.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stable transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating phasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro injection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
  • “Variant” refers to molecules that are recognized variations of a protein or the polynucleotides that encode it. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most 5 preferably at least 400. AUelic variants have a high percent identity to the polynucleotides and may differ by about three bases per hundred bases. "Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.
  • SNP single nucleotide polymorphism
  • the invention is based on a GPCR (HG38) and its encoding polynucleotide that are differrentially expressed in colon and lung cancer, and to the use of the polynucleotide, the protein, and to an antibody that specifically binds the protein in the characterization, diagnosis, prognosis, treatment and evaluation of treatment of colon and lung cancer.
  • Figures 1A-1 J shows the amino acid sequence of SEQ ID NO: 1 (HG38) encoded by the nucleic acid sequence of SEQ ID NO:2.
  • Microarray data first showed that SEQ ID NO:2 was preferentially and differentially expressed in colon and lung cancer (Tables 1 and 2, respectively).
  • the polynucleotide encoding HG38 was overexpressed in 9 of 12 colon tumors (Dn3757, Dn3756, Dn3583, Dn3647, Dn3579, Dn3582,
  • Figure 2 shows that HG38 is expressed at the highest levels in brain and skeletal muscle in normal adult tissues, an expression pattern consistent with the known literature.
  • Figure 5 shows the expression of the transcript encoding HG38 in colon tumor cell lines relative to a non-tumorigenic colon cell line, LS123, using QPCR.
  • the cell lines were obtained from the ATCC.
  • the highest expression of HG38 was observed in SW620, a metastasis of colon carcinoma derived from ascitic fluid.
  • Figure 6 shows the expression of the transcript encoding HG38 in donor-matched normal/tumor lung samples obtained from RCI. The gene was overexpressed in 5/15 tumor samples; Dn7173, Dn7178, Dn7191, Dn9751, and Dn9764.
  • Figure 7 shows expression of the transcript encoding HG38 in the epithelial cells of the colon crypt in normal colon tissue.
  • Transcript expression was visualized using in situ hybridization in a thin- sectioned colon sample using sense or antisense RNA probes made from a fragment extending from about nucleotide 274 to about nucleotide 2724 of SEQ ID NO:2. For contrast, the respective sections were counterstained with DAPI.
  • Figure 8 shows the results of a similar in situ hybridization study in a thin-sectioned villous adenocarcinoma colon sample clearly showing expression of the transcript in the tumor epithelium.
  • Northern analysis conducted using the LDFE8EQ GOLD database also shows the differential expression of the transcript encoding HG38 in colon and lung tumors.
  • the two tables shown below describe cDNA libraries from colon and lung tissues in which the gene was expressed.
  • the first column shows the library name; the second column, the total number of cDNAs sequenced in that library; the third column, a description of the library; the fourth column, the absolute abundance of the transcript encoding HG38 in the library; the fourth column, the percent abundance of the transcript in the library.
  • adenoCA adenocarcinoma
  • Normalized and fetal libraries were excluded from this analysis.
  • the data shows that the expression of this transcript in colon tissue libraries is associated exclusively with diseased colon, in particular with colon cancer and colon polyps, a precancerous condition, and with the inflammatory condition, Crohn's disease. Expression was not found in at least 6 normal adult colon tissues unassociated with disease (COLENOR03, COLENOT01, COLENOT02,
  • LUNGTUT17 is particularly significant because it is matched with (rn/) normal lung tissue from the same donor (LUNGNOT28) in which expression was undetectable. Expression was also not found in at least 10 normal lung tissue libraries unassociated with disease (LUNGNOE02, LUNGNOM01, LUNGNOPOl, LUNGNOT01, LUNGNOT02, LUNGNOT04, LUNGNOT27, LUNGNOT34, LUNGNOT37, and LUNGNOT40).
  • HG38 in colon and lung cancer tissue provides a basis for the use of the protein, polynucleotides encoding the protein, and antibodies that specifically bind the protein in the detection of colon and lung cancer, and for the use if the antibody in the treatment of colon or lung cancer either by the delivery of pharmaceutical agents for cancer bound to the antibody, or by the use of the antibody itself as an antagonist of HG38.
  • the use of the receptor itself as a target for antagonists of HG38 in the treatment of colon and lung cancer is also contemplated.
  • Mammalian variants of the polynucleotide encoding HG38 were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics).
  • a highly homologous polynucleotide having about 85% identity to the majority of the coding region of the human polynucleotide is shown in the table below.
  • the first column represents the SEQ ID NO: for homologous polynucleotides (SEQ ID Var ); the second column, the Incyte ID for the homologous polynucleotide (Incyte ID Var ); the third column, the species; the fourth column, the percent identity to the human polynucleotide; and the fifth column, the nucleotide alignment of the homologous polynucleotide to the human polynucleotide.
  • the mammalian polynucleotide of SEQ ID NO:3 may be used in hybridization, amplification, and screening technologies to identify and distinguish among SEQ ED NO:3 and related molecules in a sample.
  • the mammalian polynucleotide, SEQ ID NO:3 may also be used to produce transgenic cell lines or organisms which are model systems for human colon and lung cancer and upon which the toxicity and efficacy of therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the polynucleotides, proteins, antibodies and molecules and compounds identified using the polynucleotides and proteins of the present invention.
  • cDNA libraries rnRNA is isolated from mammalian cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries.
  • the Incyte cDNAs were isolated from mammalian cDNA libraries prepared as described in the EXAMPLES.
  • the consensus sequence is present in a single clone insert ,or chemically assembled, based on the electronic assembly from sequenced fragments including Incyte polynucleotides and extension and/or shotgun sequences.
  • Computer programs, such as PHRAP (P Green, University of Washington, Seattle WA) and the AUTOASSEMBLER application (ABI) are used in sequence assembly and are described in EXAMPLE V. After verification of the 5' and 3' sequence, at least one representative polynucleotide which encodes HG38 is designated a reagent for research and development. Sequencing
  • Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA).
  • enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA).
  • Sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno NV) and the DNA ENGINE thermal cycler (MJ Research, Watertown MA) and sequencing, with the PRISM 3700, 377 or 373 DNA sequencing systems (ABI) or the MEGABACE 1000 DNA sequencing system (APB).
  • machines such as the MICROLAB 2200 system (Hamilton, Reno NV) and the DNA ENGINE thermal cycler (MJ Research, Watertown MA) and sequencing, with the PRISM 3700, 377 or 373 DNA sequencing systems (ABI) or the MEGABACE 1000 DNA sequencing system (APB).
  • nucleic acid sequences of the polynucleotides presented in the Sequence Listing were prepared by such automated methods and may contain occasional sequencing errors and unidentified nucleotides, designated with an N, that reflect state-of-the-art technology at the time the polynucleotide was sequenced.
  • Vector, linker, and polyA sequences were masked using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • Ns and SNPs can be verified either by resequencing the polynucleotide or using algorithms to compare multiple sequences that overlap the area in which the Ns or SNP occur. Both of these techniques are well known to and used by those skilled in the art.
  • sequences may be analyzed using a variety of algorithms described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
  • Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known.
  • Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the polynucleotides of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, can be removed, and deleted sequences can be restored to complete the assembled, finished sequences. Extension of a Nucleic Acid Sequence
  • sequences of the invention may be extended using various PCR-based methods known in the art.
  • the XL-PCR kit (ABI)
  • nested primers and cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence.
  • primers may be designed using software, such as OLIGO primer analysis software (Molecular Biology Insights, Cascade CO) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C.
  • OLIGO primer analysis software Molecular Biology Insights, Cascade CO
  • genomic, rather than cDNA libraries are used.
  • a probe may be designed or derived from unique regions such as the 5' regulatory region or from a nonconserved region (i.e., 5 ' or 3 ' of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding the HG38, allelic variants, or related molecules.
  • the probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to any of the nucleic acid sequences, SEQ ID NOs:2 or 3.
  • Hybridization probes may be produced using oligolabeling, nick-translation, end-labeling, or PCR amplification in the presence of a reporter molecule.
  • a vector containing the polynucleotide or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using kits such as those provided by APB.
  • the stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature.
  • Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45C (medium stringency) or 68C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, from about 35% to about 50% formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed.
  • buffers such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches.
  • buffers such as 0.2xSSC with 0.1% SDS at either 45C (
  • Arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents.
  • Hybridization probes are also useful in mapping the naturally occurring genomic sequence.
  • the probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction.
  • Such constructions include human artificial chromosomes , yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions, or the cDNAs of libraries made from single chromosomes.
  • QPCR is a method for quantifying a nucleic acid molecule based on detection of a fluorescent signal produced during PCR amplification (Gibson et al. (1996) Genome Res 6:995-1001; Heid et al. (1996) Genome Res 6:986-994).
  • Amplification is carried out on machines such as the PRISM 7700 detection system (ABI) which consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics system.
  • BCI PRISM 7700 detection system
  • CCD charge-coupled device
  • the probe which is designed to anneal between the standard forward and reverse PCR primers, is labeled at the 5' end by a flourogenic reporter dye such as 6-carboxyfluorescein (6-FAM) and at the 3' end by a quencher molecule such as 6-carboxy-tetramethyl-rhodamine (TAMRA).
  • a flourogenic reporter dye such as 6-carboxyfluorescein (6-FAM)
  • a quencher molecule such as 6-carboxy-tetramethyl-rhodamine (TAMRA).
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • a cycle threshold (C ⁇ ) value representing the cycle number at which the PCR product crosses a fixed threshold of detection is determined by the instrument software.
  • the C,- is inversely proportional to the copy number of the template and can therefore be used to calculate either the relative or absolute initial concentration of the nucleic acid molecule in the sample.
  • the relative concentration of two different molecules can be calculated by determining their respective C ⁇ values (comparative C ⁇ method).
  • the absolute concentration of the nucleic acid molecule can be calculated by constructing a standard curve using a housekeeping molecule of known concentration.
  • the process of calculating C ⁇ values, preparing a standard curve, and determining starting copy number is performed using SEQUENCE DETECTOR 1.7 software (ABI).
  • Any one of a multitude of polynucleotides encoding HG38 may be cloned into a vector and used to express the protein, or portions thereof, in host cells.
  • the nucleic acid sequence can be engineered by such methods as DNA shuffling (USPN 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like.
  • the expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and polyadenylated 3' sequence) from various sources which have been selected for their efficiency in a particular host.
  • the vector, polynucleotide, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
  • a variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors or plant cell systems transformed with expression vectors containing viral and/or bacterial elements (Ausubel supra, unit 16).
  • an adenovirus transcriptional/ translational complex may be utilized. After sequences are ligated into the El or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells.
  • the Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.
  • Routine cloning, subcloning, and propagation of nucleic acid sequences can be achieved using the multifunctional pBLUESCRIPT vector (Stratagene, La Jolla CA) or pSPORTl plasmid (invitrogen). Introduction of a nucleic acid sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired polynucleotide is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification.
  • the host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a "prepro" form may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein.
  • Recovery of Proteins from Cell Culture Heterologous moieties engineered into a vector for ease of purification include glutathione S- transferase (GST), 6xHis, FLAG, MYC, and the like.
  • GST and 6-His are purified using affinity matrices such as immobilized glutathione and metal-chelat ⁇ resins, respectively.
  • FLAG and MYC are purified using monoclonal and polyclonal antibodies.
  • a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16).
  • MS mass spectrometry
  • Proteins are separated by 2DE employing isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension.
  • IEF isoelectric focusing
  • an immobilized pH gradient strip is useful to increase reproducibility and resolution of the separation.
  • Alternative techniques may be used to improve resolution of very basic, hydrophobic, or high molecular weight proteins.
  • the separated proteins are detected using a stain or dye such as silver stain, Coomassie blue, or spyro red (Molecular Probes, Eugene OR) that is compatible with MS.
  • Gels may be blotted onto a PVDF membrane for western analysis and optically scanned using a STORM scanner (APB) to produce a computer-readable output which is analyzed by pattern recognition software such as MELANTE (GeneBio, Geneva, Switzerland).
  • the software annotates individual spots by assigning a unique identifier and calculating their respective x,y coordinates, molecular masses, isoelectric points, and signal intensity.
  • Individual spots of interest such as those representing differentially expressed proteins, are excised and proteolytically digested with a site-specific protease such as trypsin or chymotrypsin, singly or in combination, to generate a set of small peptides, preferably in the range of 1-2 kDa.
  • a site-specific protease such as trypsin or chymotrypsin, singly or in combination
  • samples Prior to digestion, samples may be treated with reducing and alkylating agents, and following digestion, the peptides are then separated by liquid chromatography or capillary electrophoresis and analyzed using MS.
  • MS converts components of a sample into gaseous ions, separates the ions based on their mass-to-charge ratio, and determines relative abundance.
  • a MALDI-TOF Microx Assisted Laser Desorption Ionization-Time of Flight
  • ESI Electronpray Ionization
  • TOF-TOF Time of Flight/Time of Flight
  • analytical programs such as TURBOSEQUEST software (Finnigan, San Jose CA)
  • the MS data is compared against a database of theoretical MS data derived from known or predicted proteins. A minimum match of three peptide masses is used for reliable protein identification.
  • Tandem-MS may be used to derive information about individual peptides.
  • a first stage of MS is performed to determine individual peptide masses.
  • selected peptide ions are subjected to fragmentation using a technique such as collision induced dissociation (CJJD) to produce an ion series.
  • CJJD collision induced dissociation
  • the resulting fragmentation ions are analyzed in a second round of MS, and their spectral pattern may be used to determine a short stretch of amino acid sequence (Dancik et al. (1999) J Comput Biol 6:327-342).
  • Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art.
  • Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds ⁇ -amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group.
  • a linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin.
  • the amino acid residues are N- ⁇ -protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9- fluorenylmethoxycarbonyl).
  • the carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin.
  • Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively.
  • Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed.
  • the full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide.
  • Antibodies or immunoglobulins (Ig) are components of immune response expressed on the surface of or secreted into the circulation by B cells.
  • the prototypical antibody is a tetramer composed of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds which binds and neutralizes foreign antigens. Based on their H-chain, antibodies are classified as IgA, IgD, IgE, IgG or IgM.
  • the most common class, IgG is tetrameric while other classes are variants or multimers of the basic structure.
  • Antibodies are described in terms of their two functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. The binding of antibody to antigen triggers destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface Fc receptors that specifically bind to the Fc region of the antibody and allow the phagocytic cells to destroy antibody-bound antigen. Fc receptors are single-pass transmembrane glycoproteins containing about 350 amino acids whose extracellular portion typically contains two or three Ig domains (Sears et al. (1990) J Immunol 144:371-378). Preparation and Screening of Antibodies
  • Various hosts including mice, rats, rabbits, goats, llamas, camels, and human cell lines may be immunized by injection with an antigenic determinant.
  • Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH; Sigma-Aldrich), and dinitrophenol may be used to increase immunological response, hi humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum increase response.
  • the antigenic determinant may be an oligopeptide, peptide, or protein.
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120).
  • Chimeric antibodies may be produced by techniques such as splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; and Takeda et al. (1985) Nature 314:452-454).
  • techniques described for antibody production may be adapted, using methods known in the art, to produce specific, single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci
  • Antibody fragments which contain specific binding sites for an antigenic determinant may also be produced.
  • fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 246:1275-1281).
  • Antibodies may also be produced by inducing production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299).
  • a protein may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • K a association constant
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are commonly used in immunoassays in which the protein-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of the protein, preferably in active form, from the antibody (Catty (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell and Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing about 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of protein-antibody complexes. Procedures for making antibodies, evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are discussed in Catty (supra) and Ausubel (supra) pp. 11.1-11.31. Cell Transformation Assays
  • Cell transformation the conversion of a normal cell to a cancerous cell, is a highly complex and genetically diverse process.
  • certain alterations in cell physiology that are associated with this process can be assayed using either in vitro cell-based systems or in vivo animal models.
  • Known alterations include acquired self-sufficiency relative to growth signals, an insensitivity to growth- inhibitory signals, unlimited replicative potential, evasion of apoptosis, sustained angiogenesis, and cellular invasion and metastasis. See Hanahan and Weinberg (2000) Cell 100:57-70.
  • Such assays can be used, for example, to assess the effect of transfecting a cell with a gene such as HG38, on transformation of the cell.
  • HG38 Differential expression of HG38, as detected using HG38, a polynucleotide encoding HG38, or an antibody that specifically binds HG38, and at least one of the assays below can be used to diagnose a colon or lung cancer.
  • Labeling of Molecules for Assay A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays.
  • Synthesis of labeled molecules may be achieved using kits such as those supplied by Promega (Madison WI) or APB for incorporation of a labeled nucleotide such as 32 P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Qiagen-Operon, Alameda CA), or amino acid such as 35 S-methionine (APB).
  • Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes).
  • Nucleic Acid Assays The polynucleotides, fragments, oligonucleotides, complementary RNAs, and peptide nucleic acids (PNA) may be used to detect and quantify differential gene expression for diagnosis of a disorder. Similarly antibodies which specifically bind HG38 may be used to quantitate the protein. Disorders associated with such differential expression include colon and lung cancer.
  • the diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential gene expression. Qualitative or quantitative methods for this comparison are well known in the art. Expression Profiles
  • An expression profile comprises the expression of a plurality of polynucleotides or protein as measured using standard assays with a sample.
  • the polynucleotides, proteins or antibodies of the invention may be used as elements on a array to produce an expression profile.
  • the array is used to diagnose or monitor the progression of disease.
  • the polynucleotide or probe may be labeled by standard methods and added to a biological sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If complex formation in the patient sample is altered in comparison to either a normal or disease standard, then differential expression indicates the presence of a disorder.
  • normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a polynucleotide under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose or stage that disorder.
  • the invention can be used to formulate a prognosis and to design a treatment regimen.
  • the invention can also be used to monitor the efficacy of treatment.
  • the array is employed to improve the treatment regimen.
  • a dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
  • animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disease, or disorder; or treatment of the condition, disease, or disorder.
  • Novel treatment regimens may be tested in these animal models using arrays to establish and then follow expression profiles over time.
  • arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects.
  • the invention provides the means to rapidly determine the molecular mode of action of a drug.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient.
  • diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years.
  • Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include antibody arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, 2D-PAGE and scintillation counting, protein arrays, radioimmunoassays, and western analysis. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. These assays and their quantitation against purifed, labeled standards are well known in the art (Ausubel, supra, unit 10.1-10.6).
  • a two-site, monoclonal-based immunoassay utilizing antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa NJ).
  • Normal or standard values for protein expression are established by combining body fluids or cell extracts taken from a normal mammalian or human subject with specific antibodies to a protein under conditions for complex formation. Standard values for complex formation in normal and diseased tissues are established by various methods, often photometric means. Then complex formation as it is expressed in a subject sample is compared with the standard values. Deviation from the normal standard and toward the diseased standard provides parameters for disease diagnosis or prognosis while deviation away from the diseased and toward the normal standard may be used to evaluate treatment efficacy.
  • antibody arrays have allowed the development of techniques for high-throughput screening of recombinant antibodies. Such methods use robots to pick and grid bacteria containing antibody genes, and a filter-based ELISA to screen and identify clones that express antibody fragments. Because liquid handling is eliminated and the clones are arrayed from master stocks, the same antibodies can be spotted multiple times and screened against multiple antigens simultaneously. Antibody arrays are highly useful in the identification of differentially expressed proteins. (See de Wildt et al. (2000) Nature Biotechnol 18:989-94.) THERAPEUTICS
  • HG38 Differential expression of polynucleotides encoding HG38 is highly associated with colon and lung cancer as shown in data presented in Figures 3-8, Tables 1-2, and Northern analysis. HG38 clearly plays a role in colon and lung cancer.
  • an antibody , antagonist, inhibitor, a pharmaceutical agent or a composition containing one or more of these molecules may be delivered to a subject in need of such treatment.
  • Such delivery may be effected by methods well known in the art and may include delivery by an antibody that specifically binds the protein.
  • monoclonal antibodies are used to block an active site, inhibit dimer formation, trigger apoptosis and the like.
  • the protein, an agonist, an enhancer, a pharmaceutical agent or a composition containing one or more of these molecules may be delivered to a subject in need of such treatment.
  • Such delivery may be effected by methods well known in the art and may include delivery of a pharmaceutical agent by an antibody specifically targeted to the protein.
  • any of the polynucleotides, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, therapeutic antibodies, and ligands binding the polynucleotide or protein may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular disorder at a lower dosage of each agent. Modification of Gene Expression Using Nucleic Acids
  • Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5', 3', or other regulatory regions of the gene encoding HG38. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. hi: Huber and Carr (1994) Molecular and hnmunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA.
  • a complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA.
  • a library or plurality of polynucleotides may be screened to identify those which specifically bind a regulatory, nontranslated sequence.
  • Ribozymes, enzymatic RNA molecules may also be used to catalyze the specific cleavage of
  • RNA Ribonucleic acid
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule or by the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules.
  • cDNAs of the invention can be used in gene therapy.
  • cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein.
  • cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and bacterial plasmids.
  • Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4): 184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics. Humana Press, Totowa NJ; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego CA).
  • Antibodies, and in particular monoclonal antibodies, that specifically bind a particular protein, enzyme, or receptor and block its overexpression are now being used therapeutically.
  • the first widely accepted therapeutic antibodies were HERCEPTIN (Trastuzumab, Genentech, S. San Francisco CA) and GLEEVEC (imatinib mesylate, Norvartis Pharmaceuticals, East Hanover NJ).
  • HERCEPTIN is a humanized antibody approved for the treatment of HER2 positive metastatic breast cancer. It is designed to bind and block the function of overexpressed HER2 protein.
  • GLEEVEC is indicated for the treatment of patients with Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) in blast crisis, accelerated phase, or in chronic phase after failure of interferon-alpha therapy.
  • a second indication for GLEEVEC is treatment of patients with KIT (CDl 17) positive unresectable and/or metastatic malignant gastrointestinal stromal tumors.
  • KIT CDl 17
  • Other monoclonal antibodies are in various stages of clinical trials for indications such as prostate cancer, lymphoma, melanoma, pneumococcal infections, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and the like.
  • the polynucleotide encoding HG38 may be used to screen a library or a plurality of molecules or compounds for specific binding affinity.
  • the libraries may be antisense molecules, artificial chromosome constructions, branched nucleic acid molecules, DNA molecules, peptides, peptide nucleic acid, proteins such as transcription factors, enhancers, or repressors, RNA molecules, ribozymes, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene.
  • the assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the polynucleotide.
  • the polynucleotide of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (USPN 6,010,849) or a reticulocyte lysate transcriptional assay.
  • the polynucleotide may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the polynucleotide and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay.
  • the polynucleotide may be used to purify a molecule or compound using affinity chromatography methods well known in the art.
  • the polynucleotide is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the polynucleotide.
  • the molecule or compound which is bound to the polynucleotide may be released from the polynucleotide by increasing the salt concentration of the flow-through medium and collected.
  • the protein or a portion thereof may be used to purify a ligand from a sample.
  • a method for using a protein to purify a ligand would involve combining the protein with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
  • HG38 may be used to screen a plurality of molecules or compounds in any of a variety of screening assays.
  • the portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly.
  • viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on then cell surface can be used in screening assays.
  • the cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured.
  • the assay may be used to identify agonists, antagonists, antibodies, DNA molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, peptides, peptide nucleic acids, proteins, and RNA molecules or any other ligand, which specifically binds the protein.
  • this invention contemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in USPN 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding.
  • this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a mammalian model system to evaluate their toxicity or therapeutic potential.
  • compositions may be formulated and administered, to a subject in need of such treatment, to attain a therapeutic effect.
  • Such compositions contain the instant protein, agonists, antagonists, bispecific molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules.
  • Compositions may be manufactured by conventional means such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing.
  • compositions may be provided as a salt, formed with acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic, or as a lyophilized powder which may be combined with a sterile buffer such as saline, dextrose, or water.
  • acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic
  • a sterile buffer such as saline, dextrose, or water.
  • auxiliaries or excipients which facilitate processing of the active compounds.
  • Auxiliaries and excipients may include coatings, fillers or binders including sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol; starches from corn, wheat, rice, or potato; proteins such as albumin, gelatin and collagen; cellulose in the form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; lubricants such as magnesium stearate or talc; disintegrating or solubilizing agents such as the, agar, alginic acid, sodium alginate or cross-linked polyvinyl pyrrolidone; stabilizers such as carbopol gel, polyethylene glycol, or titanium dioxide; and dyestuffs or pigments added for identify the product or to characterize the quantity of active compound or dosage.
  • sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol
  • compositions may be administered by any number of routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.
  • parenteral administration may be formulated in aqueous, physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Suspensions for injection may be aqueous, containing viscous additives such as sodium carboxymethyl cellulose or dextran to increase the viscosity, or oily, containing lipophilic solvents such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.
  • Penetrants well known in the art are used for topical or nasal administration.
  • a therapeutically effective dose refers to the amount of active ingredient which ameliorates symptoms or condition.
  • a therapeutically effective dose can be estimated from cell culture assays using normal and neoplastic cells or in animal models.
  • Therapeutic efficacy, toxicity, concentration range, and route of administration may be determined by standard pharmaceutical procedures using experimental animals.
  • the therapeutic index is the dose ratio between therapeutic and toxic effects ⁇ LD50 (the dose lethal to 50% of the population)/ED50 (the dose therapeutically effective in 50% of the population)-and large therapeutic indices are preferred. Dosage is within a range of circulating concentrations, includes an
  • ED50 with little or no toxicity, and varies depending upon the composition, method of delivery, sensitivity of the patient, and route of administration. Exact dosage will be determined by the practitioner in light of factors related to the subject in need of the treatment. Dosage and administration are adjusted to provide active moiety that maintains therapeutic effect. Factors for adjustment include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
  • Normal dosage amounts may vary from 0.1 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration.
  • the dosage of a particular composition may be lower when administered to a patient in combination with other agents, drugs, or hormones.
  • Guidance as to particular dosages and methods of delivery is provided in the pharmaceutical literature. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA). Model Systems
  • Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, gestation period, numbers of progeny, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene. Toxicology
  • Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess consequences on human health following exposure to the agent.
  • Genotoxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations.
  • Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny.
  • Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
  • Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range-finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
  • Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals. Chronic toxicity tests, with a duration of a year or more, are used to test whether long term administration may elicit toxicity, teratogenesis, or carcinogenesis. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment. Transgenic Animal Models
  • Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents.
  • the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
  • Embryonic (ES) stem cells isolated from rodent embryos retain the ability to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains.
  • Mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art.
  • Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene.
  • the vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages.
  • Lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes.
  • Knockout Analysis hi gene knockout analysis, a region of a gene is enzymatically modified to include a non- mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288- 1292).
  • the modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination.
  • the inserted sequence disrupts transcription and translation of the endogenous gene.
  • Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams.
  • Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene, hi one example, the mammalian gene is a human gene.
  • ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases.
  • knockin technology a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome.
  • Transformed cells are injected into blastulae and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases.
  • NHPs are the first choice test animal.
  • NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from "extensive metabolizers" to "poor metabolizers” of these agents.
  • polynucleotides which encode the protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of polynucleotides that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (Qiagen, Chatsworth CA), or an OLIGOTEX mRNA purification kit (Qiagen).
  • RNA isolation kits such as the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
  • Stratagene was provided with RNA and constructed the cDNA libraries.
  • cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with appropriate restriction enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (APB) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid such as pBLUESCRIPT plasmid or pBK- CMV plasmid (both Stratagene), pSPORTl plasmid or PCDNA2.1 plasmid (both Invitrogen), pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof.
  • Recombinant plasmids were transformed into competent E. coU cells including XLl-Blue, XLl-BlueMRF, or SOLR (Stratagene) or DH5 ⁇ , DH10B, or ElectroMAX DH10B (Invitrogen).
  • Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge
  • plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4C.
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a high- throughput format (Rao (1994) Anal Biochem 216: 1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture.
  • Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the CATALYST 800 (ABI) thermal cycler or the DNA ENGINE thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system.
  • cDNA sequencing reactions were prepared using reagents obtained from APB or supplied in sequencing kits such as the PRISM BIGDYE Terminator cycle sequencing ready reaction kit (ABI). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (APB) or PRISM 373 or 377 sequencing systems (ABI) in conjunction with standard protocols and base calling software. Reading frames within the cDNA sequences were identified using standard methods (Ausubel, supra, unit 7.7). Ill Extension of cDNAs
  • the cDNAs were extended using the cDNA clone and oligonucleotide primers.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other, to initiate 3' extension of the known fragment.
  • the initial primers were designed using primer analysis software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68C to about 72C. Any stretch of nucleotides that would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected cDNA libraries were used as templates to extend the sequence. If extension was performed than one time, additional or nested sets of primers were designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5' or upstream regions of genes. Genomic libraries can be used to obtain regulatory elements extending into the 5' promoter binding region.
  • PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research).
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics):
  • the parameters for the cycles are 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4: 68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min; and 7: storage at 4C.
  • the parameters for primer pair T7 and SK+ were as follows: 1: 94C, three min; 2: 94C, 15 sec; 3: 57C, one min; 4: 68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min; and 7: storage at 4C.
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ PICOGREEN quantitation reagent (0.25% reagent in lx TE, v/v; Molecular Probes) and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Life Sciences, Acton MA) and allowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose minigel to determine which reactions were successful in extending the sequence.
  • the extended clones were desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC18 vector (APB).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • APIB pUC18 vector
  • the digested nucleotide sequences were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and the agar was digested with AGARACE enzyme (Promega).
  • Extended clones were religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37C in 384- well plates in LB/2x carbenicillin liquid media. The cells were lysed, and DNA was amplified using primers, Taq DNA polymerase (APB) and
  • Pfu DNA polymerase (Stratagene) with the following parameters: 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4: 72C, two min; 5: 2, 3, and 4 repeated 29 times; 6: 72C, five min; and 7: storage at 4C.
  • DNA was quantified using PICOGREEN quantitation reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the conditions described above. Samples were diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI). IV Homology Searching of cDNA Clones and Their Deduced Proteins
  • the polynucleotides of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST2 to produce alignments and to determine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10 "25 for nucleotides and 10 "M for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the stringency for an exact match was set from a lower limit of about 40 (with 1-2% error due to uncalled bases) to a 100% match of about 70.
  • the BLAST software suite includes various sequence analysis programs including "blastn” that is used to align nucleotide sequences and BLAST2 that is used for direct pairwise comparison of either nucleotide or amino acid sequences.
  • BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11 ; and Filter: on.
  • Identity is measured over the entire length of a sequence.
  • Brenner (supra) analyzed BLAST for its ability to identify structural homologs by sequence identity and found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40%, for alignments of at least 70 residues.
  • polynucleotides of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics).
  • Component sequences from polynucleotide, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3' ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, and bacterial contamination sequences. Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by "Ns" or masked.
  • Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was determined based on the number and orientation of its component sequences.
  • Bins were compared to one another, and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split. Templates were subjected to analysis by STITCHER/EXON MAPPER algorithms that determine the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri.
  • templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in USSN 08/812,290 and USSN 08/811,758, both filed March 6, 1997; in USSN 08/947,845, filed October 9, 1997; and in USSN 09/034,807, filed March 4, 1998. Then templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the HMMER software package (Washington University School of Medicine, St. Louis MO).
  • the polynucleotide was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
  • GenBank rodent mammalian, vertebrate, prokaryote, and eukaryote databases
  • SwissProt BLOCKS
  • PRINTS PRINTS
  • PFAM Prosite.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. The technique is described below and in Ausubel, supra, units 4.1-4.9 and was used to generate the data presented in the Tables at pages 13 and 14, above.
  • Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or the LJFESEQ database (Incyte Genomics). This analysis is faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • a transcript image was performed using the LIFESEQ GOLD database (Incyte Genomics). This process allows assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries and was described in USPN 5,840,484, incorporated herein by reference. All sequences and cDNA libraries in the LIFESEQ database are categorized by system, organ tissue and cell type. The categories include cardiovascular system, connective tissue, digestive system, embryonic structures, endocrine system, exocrine glands, female and male genitalia, germ cells, hemic/immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed, and the urinary tract. Criteria for transcript imaging are selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.
  • the number of libraries in which the sequence was expressed are counted and shown over the total number of libraries in that category.
  • the number of cDNAs are counted and shown over the total number of cDNAs in that library, hi some transcript images, all enriched, normalized or subtracted libraries, which have high copy number sequences can be removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be excluded where clinical relevance is emphasized.
  • fetal tissue can be emphasized wherever elucidation of inherited disorders or differentiation of particular adult or embryonic stem cells into tissues or organs (such as heart, kidney, nerves or pancreas) would be aided by removing clinical samples from the analysis.
  • Transcript imaging can also be used to support data from other methodologies such as hybridization, guilt-by-association and array technologies.
  • Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the polynucleotides presented in the Sequence Listing have been mapped. Any of the fragments of the polynucleotide encoding HG38 that have been mapped result in the assignment of all related regulatory and coding sequences to the same location.
  • the genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm.
  • Tissue Sample Preparation 5 Normal and cancerous colon and lung tissue samples are described by donor identification number in the table below.
  • the first column shows the donor ID; the second, donor age/sex; the third column, a description of the disorder, the fourth column, classification of the tumor; and the fifth column, the source.
  • CA carcinoma
  • U unknown
  • NA not available hi Figure 2
  • the normalized, first-strand synthesis, polynucleotide preparations of normal, human heart, brain (whole), lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, ovary, small intestine, peripheral blood leukocyte, and colon tissues were obtained from Clontech. Additional polynucleotide preparations of human, adult, normal thyroid, pituitary, and adrenal tissues were obtained from Clinomics Bioscience (Pittsfield MA).
  • the colon cell lines shown in Figure 5 were obtained from ATCC and cultured according to the suppliers specifications.
  • the table below describes cancerous and non-cancerous human cell lines analyzed in Figure 5 for HG38 expression.
  • the first column lists the name of the cell line, the second column, the tissue source, the third column, a description of the cell line, and the fourth and fifth columns whether the cell line is tumorigenic and/or metastatic in mice.
  • the polynucleotides are applied to a substrate by one of the following methods.
  • a mixture of polynucleotides is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer.
  • the polynucleotides are individually ligated to a vector and inserted into bacterial host cells to form a library.
  • the polynucleotides are then arranged on a substrate by one of the following methods, hi the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane.
  • the membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37C for 16 hr.
  • the membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH ), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2xSSC for 10 min each.
  • the membrane is then UV irradiated in a STRATALLNKER UV-crosslinker (Stratagene).
  • polynucleotides are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 ⁇ g.
  • Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above.
  • nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in USPN 5,807,522.
  • Polymer-coated slides are prepared by cleaning glass microscope slides (Corning Life Sciences) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR Scientific Products, West Chester PA), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a HOC oven. The slides are washed extensively with distilled water between and after treatments.
  • the nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATALLNKER UV-crosslinker
  • Hybridization probes derived from the polynucleotides of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the polynucleotides to a concentration of 40-50 ng in 45 ⁇ l TE buffer, denaturing by heating to 100C for five min, and briefly centrifuging. The denatured polynucleotide is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five ⁇ l of [ 32 P]dCTP is added to the tube, and the contents are incubated at 37C for 10 min.
  • APB REDIPRIME tube
  • probe Preparation for QPCR is stopped by adding 5 ⁇ l of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below.
  • Probes for the QPCR were prepared according to the ABI protocol. Probe Preparation for Polymer Coated Slide Hybridization
  • Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 ⁇ l TE buffer and adding 5 ⁇ l 5x buffer, 1 ⁇ l 0.1 M DTT, 3 ⁇ l Cy3 or Cy5 labeling mix, 1 ⁇ l RNAse inhibitor, 1 ⁇ l reverse transcriptase, and 5 ⁇ l lx yeast control mRNAs.
  • GEMbright kit Incyte Genomics
  • Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3: 1, 1: 10, 10: 1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37C for two hr.
  • probes are purified using two successive CHROMA SPLN+TE 30 columns (Clontech, Palo Alto CA).
  • Purified probe is ethanol precipitated by diluting probe to 90 ⁇ l in DEPC-treated water, adding 2 ⁇ l lmg/ml glycogen, 60 ⁇ l 5 M sodium acetate, and 300 ⁇ l 100% ethanol.
  • the probe is centrifuged for 20 min at 20,800xg, and the pellet is resuspended in 12 ⁇ l resuspension buffer, heated to 65C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below. hi situ Hybridization
  • Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl, 0.1 M Na 2 HP0 4 , 5 mM EDTA, pH 7) at 55C for two hr.
  • the probe diluted in 15 ml fresh hybridization solution, is then added to the membrane.
  • the membrane is hybridized with the probe at 55C for 16 hr.
  • the membrane is washed for 15 min at 25C in lmM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25C in ImM Tris (pH 8.0).
  • XOMAT-AR film Eastman Kodak, Rochester NY
  • XOMAT-AR film Eastman Kodak, Rochester NY
  • Polymer Coated Slide-based Hybridization The following method was used in the microarray analyses presented in Tables 1 and 2. Probe is heated to 65C for five min, centrifuged five min at 9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, Westbury NY), and then 18 ⁇ l is aliquoted onto the array surface and covered with a coverslip.
  • the arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5xSSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hr at 60C.
  • the arrays are washed for 10 min at 45C in lxSSC, 0.1% SDS, and three times for 10 min each at 45C in O.lxSSC, and dried.
  • Hybridization reactions are performed in absolute or differential hybridization formats.
  • the absolute hybridization format probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample.
  • the differential hybridization format differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
  • Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Melville NY).
  • the slide containing the array is placed on a computer- controled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers.
  • the two fluorophores are sequentially excited by the laser.
  • Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tubes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (AID) conversion board (Analog Devices, Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS program (Incyte Genomics).
  • cDNA was synthesized from 1 ug total RNA in a 25 ul reaction with 100 units M- MLV reverse transcriptase (Ambion, Austin TX), 0.5 mM dNTPs (Epicentre, Madison WI), and 40 ng/ml random hexamers (Fisher Scientific, Chicago JL). Reactions were incubated at 25C for 10 minutes, 42C for 50 minutes, and 70C for 15 minutes, diluted to 500 ul, and stored at -30C. Alternatively, normal tissues were purchased from Clontech (Palo Alto CA) and Clinomics.
  • PCR primers and probes (5' 6- FAM-labeled, 3 'TAMRA) were designed using PRIMER EXPRESS 1.5 software (ABI) and synthesized by Biosearch Technologies (Novato CA) or ABI.
  • QPCR reactions were performed using an PRISM 7700 detection system (ABI) in 25 ul total volume with 5 ul cDNA template, lx TAQMAN UNLVERSAL PCR master mix (ABI), 100 nM each PCR primer, 200 nM probe, and lx VIC-labeled beta-2-microglobulin endogenous control (ABI). Reactions were incubated at 50C for 2 minutes, 95C for 10 minutes, followed by 40 cycles of incubation at 95C for 15 seconds and 60C for 1 minute.
  • Antisense molecules complementary to the cDNA are used to detect or inhibit gene expression. Detection is described in Example V 1.
  • the complementary molecule is' designed to bind to the most unique 5' sequence and includes nucleotides of the 5' UTR upstream of the initiation codon of the open reading frame.
  • Complementary molecules include genomic sequences (such as enhancers or introns) and are used in triple helix base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.
  • Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy.
  • Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.
  • Purification using polyacrylamide gel electrophoresis or similar techniques is used to isolate protein for immunization of hosts or host cells to produce antibodies using standard protocols.
  • amino acid sequence of the protein is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity.
  • a peptide with high immunogenicity is cleaved, recombinantly-produced, or synthesized and used to raise antibodies by means known to those of skill in the art.
  • Methods for selection of appropriate antigenic determinants such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel, supra, Chap. 11).
  • Oligopeptides of about 15 residues in length are synthesized using an 431 A peptide synthesizer (ABI) using FMOC chemistry and coupled to carriers such as BSA, thyroglobulin, or KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase immunogenicity.
  • BSA thyroglobulin
  • KLH Sigma-Aldrich
  • the coupled peptide is then used to immunize the host.
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by binding the peptide to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio- iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinantly produced protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein.
  • An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the purified protein is collected.
  • Samples containing protein are mixed in 2 x loading buffer, heated to 95 C for 3-5 min, and loaded on 4-12% NUPAGE Bis-Tris precast gel (Invitrogen).
  • the gel is electrophoresced in 1 x MES or MOPS running buffer (Invitrogen) at 200 V for approximately 45 min on a (apparatus, supplier) until the RAINBOW marker (APB) has resolved, and dye front approaches the bottom of the gel.
  • the gel and its supports are removed from the apparatus and soaked in 1 x transfer buffer (Invitrogen) with 10% methanol for a few minutes; and the PVDF membrane soaked in 100% methanol for a few seconds to activate it.
  • the membrane, the gel, and supports are placed on the transfer apparatus (machine, supplier) and a constant current of 350 mAmps is applied for 90 min. Conjugation with Antibody and Visualization
  • the membrane After the proteins are transferred to the membrane, it is blocked in 5% (w/v) non-fat dry milk in 1 x phosphate buffered saline (PBS) with 0.1% Tween 20 detergent (blocking buffer) on a rotary shaker (supplier) for at least lhr at room temperature or at 4° overnight. After blocking, the buffer is removed, and 10 ml of primary antibody in blocking buffer is added and incubated on the rotary shaker for 1 hr at room temperature or overnight at 4 C. The membrane is washed 3 x for 10 min each with PBS-Tween (PBST), and secondary antibody, conjugated to horseradish peroxidase, is added at a 1:3000 dilution in 10 ml blocking buffer. The membrane and solution are shaken for 30 min at room temperature and then washed 3 x for 10 min each with PBST.
  • PBS-Tween PBS-Tween
  • the wash solution is carefully removed, and the membrane moistened with ECL+ chemiluminescent detection system (APB) and incubated for approximately 5 min.
  • the membrane is placed, protein side down, on plastic film (product, supplier) and developed for approximately 30 seconds.
  • an antibody array can be used to study protein-protein interactions and phosphorylation.
  • a variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein: antibody complexes are formed.
  • Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest.
  • a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex.
  • DIG digoxigenin
  • the identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane.
  • Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt, supra)
  • the polynucleotide, or fragments thereof, or the protein, or portions thereof, are labeled with 32 P- dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or FITC (Molecular Probes), respectively.
  • Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled polynucleotide or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
  • a yeast two-hybrid system MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories), is used to screen for peptides that bind the protein of the invention.
  • a polynucleotide encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coli.
  • cDNA prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli to construct a cDNA library.
  • the pLexA plasmid and pB42AD-cDNA library constructs are isolated from E.
  • Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30C until the colonies have grown up and are counted.
  • SD synthetic dropout
  • the colonies are pooled in a minimal volume of lx TE (pH 7.5), replated on SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl ⁇ -d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies.
  • Interaction between expressed protein and polynucleotide fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu).
  • Interaction also activates expression of ⁇ -galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.
  • Positive interactions between expressed protein and polynucleotide fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30C.
  • a sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30C until colonies appear.
  • the sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD containing histidine but not on media lacking histidine have lost the pLexA plasmid.
  • Histidine-requiring colonies are grown on SD/Gal/Raf/X-Gal -Trp/-Ura, and white colonies are isolated and propagated.
  • the pB42AD-cDNA plasmid which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized.
  • the GPCR encoded by SEQ ID NO:2 may be expressed in heterologous expression systems and its biological activity tested utilizing the purinergic receptor system (P 2 u) as published by Erb et al. (1993, Proc Natl Acad Sci 90: 10449-53). Because cultured K562 human leukemia cells lack P 2U receptors, they can be transfected with expression vectors containing either normal or chimeric P 2U and loaded with fura- °s fluorescent probe for Ca ++ . Activation of properly assembled and functional extracellular SP- transmembrane/intracellular P 2U receptors with extracellular UTP or ATP mobilizes intracellular Ca ⁇ which reacts with fura- ⁇ * and is measured spectrofluorometrically.
  • P 2 u purinergic receptor system
  • XVI Cell Transformation Assays Colony-formation Assay in Soft Agar The ability of transformed cells to grow in an anchorage-independent manner is measured by the ability of the cells to form colonies in soft agar (0.35%). The assay is conducted in 12-well culture plates where each well is coated with a solid 0.7% Noble agar (Fisher Scientific, Atlanta GA) in cell growth media.
  • a 3.5% agar solution in PBS is prepared, autoclaved, microwaved and kept liquid in a 55 C water bath with shaking.
  • the agar is diluted 1:5 to 0.7% with an appropriate cell growth media, and 0.5 ml of the diluted agar added to each well of the plate.
  • Culture plates are kept at room temperature for about 15 minutes or until the agar solidifies.
  • Trypsinized cells are diluted to 200 to 4000 cells/ml in growth medium and 0.25 ml of diluted cells is mixed with 2 ml warm 0.35% agar. The diluted cells are added to a well of the culture plate; duplicate wells are prepared for each cell concentration. The plates are allowed to cool for about 30 min at room temperature and then transferred to an incubator at 37 C. After a 1-2 week incubation period, colonies are counted under an inverted, phase contrast microscope. Colony forming efficiency is determined as the percentage colonies formed/total number of cells plated. Apoptosis/Survival Assay
  • the ability of transformed cells to evade apoptosis (programmed cell death) and survive may be measured in an assay in which apoptosis or survival of cultured cells is determined by FACS analysis using a double-staining method with Annexin V and propidium iodide (PI).
  • Annexin V serves as a marker for apoptotic cells by binding to phosphatidyl serine, a cell surface marker for apoptosis.
  • Counterstaining with PI allows differentiation between apoptotic cells, which are Annexin V positive and PI negative, and necrotic cells, which are Annexin V and PI positive.
  • Apoptosis is measured between 0-24 hrs of culture, and cell survival is measured between 24-96 hrs of culture.
  • HMVEC human vascular endothelial cells
  • Cell migration and tissue invasion by transformed tumor cells is determined using the BICOAT Angiogenesis system (BD Biosciences, Franklin Lakes NJ) as described by the manufacturer.
  • the assay is carried out in a BD FALCON multiwell insert plate containing an 8 ⁇ m pore size BD FLUOROBLOK polyethylene terephthalate membrane uniformly coated with a reconstituted BD MATRIGEL basement membrane matrix and inserted into a non-treated multiwell receiver plate.
  • the system provides a barrier to passive diffusion of cells through the membrane but allows active migration by invasive tumor cells. After cells in appropriate culture medium are incubated in the upper portion of the chamber for a suitable period of time, any cells appearing on the underside of the membrane are quantitated. Since the membrane blocks the transmission of light from 490 to 700nm, cells traversing the membrane are detected by their fluorescence which is proportionate to cell number.
  • Dn7168 Human, Lung Tumor, Squamous Cell CA, Dn7168 -1.20 Dn7168 Human, Lung Tumor, Squamous Cell CA, Dn7168
  • Dn7164 Human, Lung Tumor, Carcinoid, Dn7164 -1.40 Dn7164 Human, Lung Tumor, Carcinoid, Dn7164

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Hospice & Palliative Care (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides for the use of a protein, HG38, a polynucleotide encoding the protein, and antibodies that specifically bind the protein in various methods to diagnose, stage, treat, or monitor the treatment of colon and lung cancer.

Description

METHODS OF USE OF A GPCR IN THE DIAGNOSIS AND TREATMENT OF
COLON AND LUNG CANCER
TECHNICAL FIELD
This invention relates to the use of a G protein-coupled receptor (GPCR) expressed in colon and lung cancer, its encoding polynucleotide, and an antibody that specifically binds the protein to diagnose, to stage, to treat, or to monitor the progression or treatment of colon or lung cancer.
BACKGROUND OF THE INVENTION Cancers and malignant tumors are characterized by continuous cell proliferation and cell death and are related causally to both genetics and the environment. Genes whose expression are associated with cancer are of potentially great importance as cancer markers in the early diagnosis and prognosis of various cancers, as well as potential targets in cancer treatment.
Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year. Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes. (See Potter (1999) J Natl Cancer Institute 91:916-932 for a review of colorectal cancer.) Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et aL (1990) National Institutes of Health, DHHS Publ No. (NIH)90-2789).
Lung cancer is the leading cause of cancer death in the United States affecting more than 100,000 men and 50,000 women each year, and nearly 90% of those diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in exposed bronchial epithelium. In nearly 80% of those diagnosed, the lung cancer has metastasized to pleura, brain, bone, pericardium, and liver. Treatment with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to the identification, staging, and treatment of lung cancer could positively affect prognosis.
G protein coupled receptors (GPCRs) are a superfamily of seven transmembrane domain proteins that mediate the transduction of extracellular signals across the plasma membrane of cells from a large, diverse number of ligands through interaction with heterotrimeric G proteins. See, e.g., Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6. The glycoprotein hormone receptors are a subfamily of GPCRs activated by the gonadotropins; lutropin (LH), thyrotropin (TSH), follitropin (FSH) and human choriogonadotropin (hCG), essential for the growth and differentiation of the gonads and thyroid gland. The glycoprotein hormone GPCRs are characterized by a large N-terminal extracellular domain containing some 9 leucine- repeat domains that function in proteimprotein interactions and are likely important for interaction with their respective protein ligands. Two orphan GPCRs (ligand currently unknown) related to this subfamily, designated HG38 and LGR5, have also been identified which share -35% overall identity with members of the glycoprotein hormone receptor subfamily (McDonald et aL (1998) Biochem Biophys Res Commun 247:266-270; Hsu et al (1998) Molecular Endocrinology 12:1830-1845). Both receptors are expressed primarily in skeletal muscle, placenta, spinal cord, and various regions of the brain. Like other members of the glycoprotein hormone receptor family, both receptors are characterized by a large extracellular domain containing leucine-rich repeats and a cysteine-rich domain near the junction of the extracellular domain and the first transmembrane domain (Hsu et al. supra). Array technologies and quantitative PCR provide the means to explore the expression profiles of a large number of related or unrelated genes. When an expression profile is examined, arrays provide a platform for examining which genes are tissue-specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder. The application of expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of the disease.
The discovery of a GPCR and its encoding polynucleotide that are differentially expressed in colon and lung cancer, satisfies a need in the art by providing compositions which are useful to diagnose, to stage, to treat, or to monitor the progression or treatment of colon and lung cancer.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a GPCR known as HG38 (SEQ ID NO: 1) is differentially expressed in colon and lung cancer, and to the use of the protein, its encoding polynucleotide or the complement thereof, and an antibody that specifically binds the protein to diagnose, to stage, to treat, or to monitor the progression or treatment of a colon or lung cancer. The invention provides a method for using a polynucleotide to detect the differential expression of a nucleic acid in a sample of colon or lung tissue comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the polynucleotide in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the polynucleotide is used to diagnose a colon or lung cancer.
The invention provides a purified protein or a portion thereof comprising an amino acid sequence of SEQ ID NO: 1 for use in the diagnosis of a colon or lung cancer. The invention provides a method for diagnosing a colon or lung cancer comprising performing an assay to quantify the amount of the protein expressed in a sample of colon or lung tissue and comparing the amount of protein expressed to a standard, thereby diagnosing a colon or lung cancer. In a one aspect, the assay is selected from antibody arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, 2D-PAGE and scintillation counting, protein arrays, radioimmunoassays, and western analysis.
The invention also provides a method for using an antibody to detect differential expression of a protein in a sample of colon or lung tissue, the method comprising combining the antibody with a sample under conditions for formation of antibody:protein complexes, detecting complex formation, comparing complex formation with a standard, wherein differential expression of the protein between the sample and the standard is diagnostic of a colon or lung cancer.
The invention further provides an antagonist which specifically binds the protein having the amino acid sequence of SEQ ID NO: 1 for use in the treatment of a colon or lung cancer.
The invention provides a method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention an antibody that specifically binds the protein or a composition comprising an antibody and a pharmaceutical agent. The invention also provides a method for delivering a pharmaceutical or therapeutic agent to a colon cancer cell comprising attaching the pharmaceutical or therapeutic agent to ,an antibody that specifically binds the protein and administering the anntibody to a subject in need of therapeutic intervention, wherein the antibody delivers the pharmaceutical or therapeutic agent to the cell. The invention also provides a method for using a polynucleotide to produce a mammalian model system, the method comprising constructing a vector containing the polynucleotide of SEQ ID NO:3, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the polynucleotide in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system. BRIEF DESCRIPTION OF THE FIGURES AND TABLES
Figures 1A-1J show the amino acid sequence (SEQ ID NO:l) of HG38 encoded by the nucleic acid sequence of SEQ ID NO:2. The alignment was produced using MACDNASIS PRO software (Hitachi Software Engineering, San Bruno CA). Figure 2 shows the relative expression of HG38 in various normal adult tissues. The X-axis lists tissue type, and the Y-axis, the relative expression of HG38 normalized to that found in normal colon. QPCR analysis was performed using the TAQMAN protocol (Applied Biosystems (ABI), Foster City CA). Tissues were obtained from Clinomics (Pittsfield MA) and Clontech (Palo Alto CA). Figures 3 and 4 show the differential expression of HG38 in donor-matched normal/tumor colon samples as determined using QPCR (ABI). Figure 3 tissues were obtained from the Huntsman Cancer Institute (HCI; Salt Lake City UT); Figure 4 tissues, from Asterand Bioresources, Inc. (Detroit MI).
Figure 5 shows the relative expression of HG38 in colon tumor cell lines compared to a normal colon cell line (LS 123) using QPCR (ABI). Cell lines were obtained from the ATCC (Manassas VA).
Figure 6 shows the differential expression of HG38 in donor-matched normal/tumor lung samples as determined using QPCR (ABI). Tissue samples were obtained from the Roy Castle institute for Lung Cancer Research (RCI; Liverpool UK). The probe sequence for the QPCR analyses depicted in Figures 2-6 used an oligonucleotide extending from about nucleotide 274 to about nucleotide 291 of SEQ ID NO:2.
Figure 7 shows the expression of the transcript encoding HG38 in normal colon tissue. Thin sections were stained with DAPI and hybridized in situ using sense or antisense RNA probes made from a fragment of SEQ ID NO: 2 extending from about nucleotide 274 to about nucleotide 2724 of SEQ ID NO:2.
Figure 8 shows the expression of the transcript encoding HG38 in a villous adenocarcinoma of the colon. Thin sections were stained with DAPI and hybridized in situ using sense or antisense RNA probes made from a fragment of SEQ ID NO:2 extending from about nucleotide 274 to about nucleotide 2724 of SEQ ID NO:2. Table 1 shows the differential expression of HG38 in cancerous colon tissue relative to normal colon tissue as determined by microarray analysis. Column 1 shows the differential expression of HG38 in terms of the ratio of the signal intensity for the flourescent dye Cy5 in labeled tumor tissue relative to that for flourescent dye Cy3 in labeled normal colon tissue. Column 2 shows the source of the normal colon tissue as the individual donor (Dn), or pooled tissue from more than one donor (pool), column 3 is a description of the colon tumor sample, and column 4, the source of the tumor sample. Tissue samples were obtained from HCI and Asterand Bioresources.
Table 2 shows the differential expression of HG38 in cancerous lung tissue relative to normal lung tissue as determined by microarray analysis. Column headings are the same a those described above for Table 1. Tissue samples were obtained from RCI.
DESCRIPTION OF THE INVENTION It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms "a", "an", and "the" may include plural reference unless the context clearly dictates otherwise. For example, a reference to "a host cell" includes a plurality of such host cells known to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Definitions "Antibody" refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab')2 fragment, an Fv fragment, and an antibody-peptide fusion protein.
"Antigenic determinant" refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody that specifically binds the protein. Biological activity is not a prerequisite for immunogenicity.
"Array" refers to an ordered arrangement of at least two polynucleotides, proteins, or antibodies on a substrate. At least one of the polynucleotides, proteins, or antibodies represents a control or standard, and the other polynucleotide, protein, or antibody is of diagnostic or therapeutic interest. The arrangement of at least two and up to about 40,000 polynucleotides, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each polynucleotide and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
"HG38" refers to a GPCR that is exactly or highly homologous (>85%) to the amino acid sequence of SEQ ID NO: 1 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The "complement" of a polynucleotide of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to a nucleic acid molecule under conditions of high stringency.
The phrase "polynucleotide encoding a protein" refers to a nucleic acid whose sequence closely aligns with sequences that encode conserved regions, motifs or domains identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool; Altschul (1993) J Mol Evol 36:290-300; Altschul et al. (1990) J Mol Biol 215:403-410) and BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402) which provide identity within the conserved region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078) who analyzed BLAST for its ability to identify structural homologs by sequence identity found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40% is a reasonable threshold for alignments of at least 70 residues (Brenner, page 6076, column 2). A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding HG38 or fragments of HG38 may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides. "Derivative" refers to a polynucleotide or a protein that has been subjected to a chemical modification. Derivatization of a polynucleotide can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. These substitutions are well known in the art. Derivatization of a polynucleotide or a protein can also involve the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group (for example, 5-methylcytosine). Derivative molecules retain the biological activities of the naturally occurring molecules but may confer longer lifespan or enhanced activity.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
"Disorder" refers to conditions, diseases or syndromes in which HG38 or the rnRNA encoding HG38 are differentially expressed; in particular, a colon or lung cancer. An "expression profile" is a representation of gene expression in a sample. A nucleic acid expression profile is produced using sequencing, hybridization, or amplification (quantitative PCR) technologies and mRNAs or polynucleotides from a sample. A protein expression profile, although time delayed, mirrors the nucleic acid expression profile and may use antibody or protein arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, spatial immobilization such as 2D-PAGE, and radioimmunoassays including radiolabeling and quantification using a scintillation counter and western analysis to detect protein expression in a sample. The nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art. Expression profiles may also be evaluated by methods such as electronic northern analysis, guilt-by-association, and transcript imaging. Expression profiles produced using any of the above methods may be contrasted with expression profiles produced using normal or diseased tissues. Of note is the correspondence between mRNA and protein expression has been discussed by Zweiger (2001, Transducing the Genome. McGraw-Hill, San Francisco, CA) and Glavas et al. (2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others. The term "hybridization complex" refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C0t or R0t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed). "Identity" as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith- Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul (1997, supra). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. "Similarity" uses the same algorithms but takes conservative substitution of residues into account. In proteins, similarity exceeds identity in that substitution, for example, of a valine for a leucine or isoleucine, is counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art. An "immunogenic fragment" is a polypeptide or oligopeptide fragment of HG38 which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of HG38 which is useful in any of the antibody production methods disclosed herein or known in the art. "Labeling moiety" refers to any reporter molecule including radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cofactors, inhibitors, or magnetic particles than can be attached to or incorporated into a polynucleotide, protein, or antibody. A wide variety conjugation techniques are known in the art and include both direct synthesis and chemical conjugation, particularly to amines, thiols and other side groups which may be present. Visible labels and dyes include but are not limited' to anthocyanins, β glucuronidase, biotin, BIODIPY, Coomassie blue, Cy3 and Cy5, 4,6-diamidino- 2-phenylindole (DAPI), digoxigenin, fluorescein, FITC, gold, green fluorescent protein (GFP), lissamine, luciferase, phycoerythrin, rhodamine, spyro red, silver, streptavidin, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
"Ligand" refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate. The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of HG38. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HG38. A "multispecific molecule" can bind with at least two different binding specificities to at least two different molecules or two different sites on a molecule. Antibodies can perform as multispecific molecules in that they can bind to both a target protein and a pharmaceutical agent.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Oligonucleotide" refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplicon, amplimer, primer, and oligomer. "Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
A "pharmaceutical agent" may be an antibody, an antisense molecule, a bispecific molecule, a multispecific molecule, a peptide, a protein, a radionuclide, a small drug molecule, a cytospecific or cytotoxic drug such as abrin, actinomyosin D, cisplatin, crotin, doxorubicin, 5-fluorouracil, methotrexate, ricin, vincristine, vinblastine,, or any combination of these elements. "Post-translational modification" of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
"Probe" refers to polynucleotides encoding HG38, their complements, or fragments thereof, which are used to detect identical, allelic or related polynucleotides. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
"Protein" refers to a polypeptide or any portion thereof. A "portion" of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic determinant of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison WI). An "oligopeptide" is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
A "recombinant nucleic acid" is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra). The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal. A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
"Sample" is used in its broadest sense as containing nucleic acids, proteins, and antibodies. A sample may comprise a bodily fluid such as ascites, blood, cerebrospinal fluid, lymph, semen, sputum, urine and the like; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue, a tissue biopsy, or a tissue print; buccal cells, skin, hair, a hair follicle; and the like.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natμral or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody. The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated. A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any rigid or semi-rigid support to which polynucleotides, proteins, or antibodies are bound and includes magnetic or nonmagnetic beads, capillaries or other tubing, chips, fibers, filters, gels, membranes, plates, polymers, slides, wafers, and microparticles with a variety of surface forms including channels, columns, pins, pores, trenches, and wells.
A "transcript image" (TI) is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed polynucleotides in the cDNA libraries of an EST database as described in USPN 5,840,484, incorporated herein by reference.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed cells" includes stable transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating phasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro injection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
"Variant" refers to molecules that are recognized variations of a protein or the polynucleotides that encode it. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most 5 preferably at least 400. AUelic variants have a high percent identity to the polynucleotides and may differ by about three bases per hundred bases. "Single nucleotide polymorphism" (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.
10 THE INVENTION
The invention is based on a GPCR (HG38) and its encoding polynucleotide that are differrentially expressed in colon and lung cancer, and to the use of the polynucleotide, the protein, and to an antibody that specifically binds the protein in the characterization, diagnosis, prognosis, treatment and evaluation of treatment of colon and lung cancer.
15 Figures 1A-1 J shows the amino acid sequence of SEQ ID NO: 1 (HG38) encoded by the nucleic acid sequence of SEQ ID NO:2.
Microarray data first showed that SEQ ID NO:2 was preferentially and differentially expressed in colon and lung cancer (Tables 1 and 2, respectively). In particular, the polynucleotide encoding HG38 was overexpressed in 9 of 12 colon tumors (Dn3757, Dn3756, Dn3583, Dn3647, Dn3579, Dn3582,
20 Dn3839, Dn9573, and Dn9576), and 6 of 17 lung tumors (Dn7178, Dn7173, Dn7186, Dn7963, Dn5797, and Dn5796) compared to donor-matched normal colon or lung tissue or, in the case of Dn3757, a pool of normal colon tissue. In these experiments, a value of at least 1.7-fold was considered to be significant differential expression. An average value was considered where duplicate experiments were performed. This discovery led to further studies using QPCR and in situ hybridization studies comparing normal and
25 cancerous tissues and tumor cell lines.
Figure 2 shows that HG38 is expressed at the highest levels in brain and skeletal muscle in normal adult tissues, an expression pattern consistent with the known literature.
QPCR analysis of HG38 expression in donor-matched normal/tumor colon samples obtained from the Hunstman Cancer Institute shows that the gene was overexpressed in 8/10 tumor samples; Dn3579,
30 3580, 3581, 3582, 3583, 3647, 3649, and 3479 (Figure 3). A similar study using samples from an alternate source (Asterand Bioresources, Inc) showed overexpression of HG38 in 4/5 colon tumors compared to donor-matched normal colon; Dn9573, Dn9574, Dn9576, and Dn9577 (Figure 4). The highest expression of HG38 was found in one unmatched colon tumor sample, 8401, relative to a pool of normal colon tissue. It is further noteworthy that 6 of the 8 patient samples that exhibited overexpression of HG38 in colon tumor versus normal colon tissue in the microarray study shown above in Table 1 (Dn3583, 3647, 3579, 3582, 9573 and 9576) likewise showed overexpression of HG38 by QPCR analysis of the same samples, hi addition, 2 patient samples that showed marginal overexpression of HG38 in colon tumors in Table 1, Dn9574 and Dn9477, exhibited more significant overexpression by QPCR analysis. Differences in results between the microarray study shown in Table 1 and the QPCR analysis in Figure 3 are likely due, in part, to the greater sensitivity and larger dynamic range for QPCR analysis than for microarray analysis.
Figure 5 shows the expression of the transcript encoding HG38 in colon tumor cell lines relative to a non-tumorigenic colon cell line, LS123, using QPCR. The cell lines were obtained from the ATCC. The highest expression of HG38 was observed in SW620, a metastasis of colon carcinoma derived from ascitic fluid.
Figure 6 shows the expression of the transcript encoding HG38 in donor-matched normal/tumor lung samples obtained from RCI. The gene was overexpressed in 5/15 tumor samples; Dn7173, Dn7178, Dn7191, Dn9751, and Dn9764.
Figure 7 shows expression of the transcript encoding HG38 in the epithelial cells of the colon crypt in normal colon tissue. Transcript expression was visualized using in situ hybridization in a thin- sectioned colon sample using sense or antisense RNA probes made from a fragment extending from about nucleotide 274 to about nucleotide 2724 of SEQ ID NO:2. For contrast, the respective sections were counterstained with DAPI.
Figure 8 shows the results of a similar in situ hybridization study in a thin-sectioned villous adenocarcinoma colon sample clearly showing expression of the transcript in the tumor epithelium.
Northern analysis conducted using the LDFE8EQ GOLD database (Incyte Genomics, Palo Alto, CA) also shows the differential expression of the transcript encoding HG38 in colon and lung tumors. The two tables shown below describe cDNA libraries from colon and lung tissues in which the gene was expressed. The first column shows the library name; the second column, the total number of cDNAs sequenced in that library; the third column, a description of the library; the fourth column, the absolute abundance of the transcript encoding HG38 in the library; the fourth column, the percent abundance of the transcript in the library. Category: Digestive System (Colon)
Library Name* cDNAs Description of Colon Tissue Abundance % Abundance COLCDIT03 3069 colon, cecum polyp, aw/adenoCA1, 67F 3 0.0978
COLNNOT22 3599 colon mw/Crohn's, 56F 1 0.0278
COLNTUP17 7417 colon tumor, adenoCA, 3' CGAP 1 0.0135
1 adenoCA=adenocarcinoma; * Normalized and fetal libraries were excluded from this analysis. The data shows that the expression of this transcript in colon tissue libraries is associated exclusively with diseased colon, in particular with colon cancer and colon polyps, a precancerous condition, and with the inflammatory condition, Crohn's disease. Expression was not found in at least 6 normal adult colon tissues unassociated with disease (COLENOR03, COLENOT01, COLENOT02,
COLNNOPOl, COLNNOP02, and COLNNOP06).
Category: Respiratory System (Lung) Library Name* cDNAs Description of Colon Tissue Abundance % Abundance LUNGTUT17 3950 lung tumor, adenoCA, 53M, 2 0.0506 m/LUNGNOT28 LUNGTUT07 3873 lung tumor, squamous cell CA, 50M 1 0.0258 *No tissues were excluded from this analysis
The above data shows the expression of this transcript exclusively in lung cancer tissue. LUNGTUT17 is particularly significant because it is matched with (rn/) normal lung tissue from the same donor (LUNGNOT28) in which expression was undetectable. Expression was also not found in at least 10 normal lung tissue libraries unassociated with disease (LUNGNOE02, LUNGNOM01, LUNGNOPOl, LUNGNOT01, LUNGNOT02, LUNGNOT04, LUNGNOT27, LUNGNOT34, LUNGNOT37, and LUNGNOT40).
The differential expression of HG38 in colon and lung cancer tissue relative to normal tissue and, in particular, its localization in epithelial cells of colon tissue provides a basis for the use of the protein, polynucleotides encoding the protein, and antibodies that specifically bind the protein in the detection of colon and lung cancer, and for the use if the antibody in the treatment of colon or lung cancer either by the delivery of pharmaceutical agents for cancer bound to the antibody, or by the use of the antibody itself as an antagonist of HG38. The use of the receptor itself as a target for antagonists of HG38 in the treatment of colon and lung cancer is also contemplated.
Mammalian variants of the polynucleotide encoding HG38 were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics). A highly homologous polynucleotide having about 85% identity to the majority of the coding region of the human polynucleotide is shown in the table below. The first column represents the SEQ ID NO: for homologous polynucleotides (SEQ IDVar); the second column, the Incyte ID for the homologous polynucleotide (Incyte IDVar); the third column, the species; the fourth column, the percent identity to the human polynucleotide; and the fifth column, the nucleotide alignment of the homologous polynucleotide to the human polynucleotide.
SEQ IDVar Incyte IDVar Species Identity NtH Alignment
3 050237_Mm.l Mouse 85% 112-2740
The mammalian polynucleotide of SEQ ID NO:3 may be used in hybridization, amplification, and screening technologies to identify and distinguish among SEQ ED NO:3 and related molecules in a sample. The mammalian polynucleotide, SEQ ID NO:3 may also be used to produce transgenic cell lines or organisms which are model systems for human colon and lung cancer and upon which the toxicity and efficacy of therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the polynucleotides, proteins, antibodies and molecules and compounds identified using the polynucleotides and proteins of the present invention. Characterization and Use of the Invention cDNA libraries rnRNA is isolated from mammalian cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries. The Incyte cDNAs were isolated from mammalian cDNA libraries prepared as described in the EXAMPLES. The consensus sequence is present in a single clone insert ,or chemically assembled, based on the electronic assembly from sequenced fragments including Incyte polynucleotides and extension and/or shotgun sequences. Computer programs, such as PHRAP (P Green, University of Washington, Seattle WA) and the AUTOASSEMBLER application (ABI), are used in sequence assembly and are described in EXAMPLE V. After verification of the 5' and 3' sequence, at least one representative polynucleotide which encodes HG38 is designated a reagent for research and development. Sequencing
Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA). Sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno NV) and the DNA ENGINE thermal cycler (MJ Research, Watertown MA) and sequencing, with the PRISM 3700, 377 or 373 DNA sequencing systems (ABI) or the MEGABACE 1000 DNA sequencing system (APB).
The nucleic acid sequences of the polynucleotides presented in the Sequence Listing were prepared by such automated methods and may contain occasional sequencing errors and unidentified nucleotides, designated with an N, that reflect state-of-the-art technology at the time the polynucleotide was sequenced. Vector, linker, and polyA sequences were masked using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. Ns and SNPs can be verified either by resequencing the polynucleotide or using algorithms to compare multiple sequences that overlap the area in which the Ns or SNP occur. Both of these techniques are well known to and used by those skilled in the art. The sequences may be analyzed using a variety of algorithms described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853). Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the polynucleotides of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, can be removed, and deleted sequences can be restored to complete the assembled, finished sequences. Extension of a Nucleic Acid Sequence
The sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (ABI), nested primers, and cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence. For all PCR-based methods, primers may be designed using software, such as OLIGO primer analysis software (Molecular Biology Insights, Cascade CO) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C. When extending a sequence to recover regulatory elements, genomic, rather than cDNA libraries are used. Hybridization The polynucleotide and fragments thereof can be used in hybridization technologies for various purposes. A probe may be designed or derived from unique regions such as the 5' regulatory region or from a nonconserved region (i.e., 5 ' or 3 ' of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding the HG38, allelic variants, or related molecules. The probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to any of the nucleic acid sequences, SEQ ID NOs:2 or 3. Hybridization probes may be produced using oligolabeling, nick-translation, end-labeling, or PCR amplification in the presence of a reporter molecule. A vector containing the polynucleotide or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using kits such as those provided by APB. The stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45C (medium stringency) or 68C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, from about 35% to about 50% formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY. Arrays may be prepared and analyzed using methods well known in the art. Oligonucleotides or polynucleotides may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymorphisms. Arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., USPN 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; USPN 5,605,662.)
Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction. Such constructions include human artificial chromosomes , yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions, or the cDNAs of libraries made from single chromosomes. QPCR
QPCR is a method for quantifying a nucleic acid molecule based on detection of a fluorescent signal produced during PCR amplification (Gibson et al. (1996) Genome Res 6:995-1001; Heid et al. (1996) Genome Res 6:986-994). Amplification is carried out on machines such as the PRISM 7700 detection system (ABI) which consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics system. To perform QPCR, a PCR reaction is carried out in the presence of a doubly labeled probe. The probe, which is designed to anneal between the standard forward and reverse PCR primers, is labeled at the 5' end by a flourogenic reporter dye such as 6-carboxyfluorescein (6-FAM) and at the 3' end by a quencher molecule such as 6-carboxy-tetramethyl-rhodamine (TAMRA). As long as the probe is intact, the 3' quencher extinguishes fluorescence by the 5' reporter. However, during each primer extension cycle, the annealed probe is degraded as a result of the intrinsic 5' to 3' nuclease activity of Taq polymerase (Holland et al. (1991) Proc Natl Acad Sci 88:7276-7280). This degradation separates the reporter from the quencher, and fluorescence is detected every few seconds by the CCD. The higher the starting copy number of the nucleic acid, the sooner an increase in fluorescence is observed. A cycle threshold (Cτ ) value, representing the cycle number at which the PCR product crosses a fixed threshold of detection is determined by the instrument software. The C,- is inversely proportional to the copy number of the template and can therefore be used to calculate either the relative or absolute initial concentration of the nucleic acid molecule in the sample. The relative concentration of two different molecules can be calculated by determining their respective Cτ values (comparative Cτ method). Alternatively, the absolute concentration of the nucleic acid molecule can be calculated by constructing a standard curve using a housekeeping molecule of known concentration. The process of calculating Cτ values, preparing a standard curve, and determining starting copy number is performed using SEQUENCE DETECTOR 1.7 software (ABI). Expression
Any one of a multitude of polynucleotides encoding HG38 may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The nucleic acid sequence can be engineered by such methods as DNA shuffling (USPN 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and polyadenylated 3' sequence) from various sources which have been selected for their efficiency in a particular host. The vector, polynucleotide, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors or plant cell systems transformed with expression vectors containing viral and/or bacterial elements (Ausubel supra, unit 16). In mammalian cell systems, an adenovirus transcriptional/ translational complex may be utilized. After sequences are ligated into the El or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression. Routine cloning, subcloning, and propagation of nucleic acid sequences can be achieved using the multifunctional pBLUESCRIPT vector (Stratagene, La Jolla CA) or pSPORTl plasmid (invitrogen). Introduction of a nucleic acid sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
For long term production of recombinant proteins, the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired polynucleotide is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification.
The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a "prepro" form may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein. Recovery of Proteins from Cell Culture Heterologous moieties engineered into a vector for ease of purification include glutathione S- transferase (GST), 6xHis, FLAG, MYC, and the like. GST and 6-His are purified using affinity matrices such as immobilized glutathione and metal-chelatβ resins, respectively. FLAG and MYC are purified using monoclonal and polyclonal antibodies. For ease of separation following purification, a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16). Protein Identification
Several techniques have been developed which permit rapid identification of proteins using high performance liquid chromatography and mass spectrometry (MS). Beginning with a sample containing proteins, the method is: 1) proteins are separated using two-dimensional gel electrophoresis (2-DE), 2) selected proteins are excised from the gel and digested with a protease to produce a set of peptides; and 3) the peptides are subjected to mass spectral analysis to derive peptide ion mass and spectral pattern information. The MS information is used to identify the protein by comparing it with information in a protein database (Shevenko et al. (1996) Proc Natl Acad Sci 93: 14440-14445). Proteins are separated by 2DE employing isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension. For IEF, an immobilized pH gradient strip is useful to increase reproducibility and resolution of the separation. Alternative techniques may be used to improve resolution of very basic, hydrophobic, or high molecular weight proteins. The separated proteins are detected using a stain or dye such as silver stain, Coomassie blue, or spyro red (Molecular Probes, Eugene OR) that is compatible with MS. Gels may be blotted onto a PVDF membrane for western analysis and optically scanned using a STORM scanner (APB) to produce a computer-readable output which is analyzed by pattern recognition software such as MELANTE (GeneBio, Geneva, Switzerland). The software annotates individual spots by assigning a unique identifier and calculating their respective x,y coordinates, molecular masses, isoelectric points, and signal intensity. Individual spots of interest, such as those representing differentially expressed proteins, are excised and proteolytically digested with a site-specific protease such as trypsin or chymotrypsin, singly or in combination, to generate a set of small peptides, preferably in the range of 1-2 kDa. Prior to digestion, samples may be treated with reducing and alkylating agents, and following digestion, the peptides are then separated by liquid chromatography or capillary electrophoresis and analyzed using MS. MS converts components of a sample into gaseous ions, separates the ions based on their mass-to-charge ratio, and determines relative abundance. For peptide mass fingerprinting analysis, a MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight), ESI (Electrospray Ionization), and TOF-TOF (Time of Flight/Time of Flight) machines are used to determine a set of highly accurate peptide masses. Using analytical programs, such as TURBOSEQUEST software (Finnigan, San Jose CA), the MS data is compared against a database of theoretical MS data derived from known or predicted proteins. A minimum match of three peptide masses is used for reliable protein identification. If additional information is needed for identification, Tandem-MS may be used to derive information about individual peptides. In tandem-MS, a first stage of MS is performed to determine individual peptide masses. Then selected peptide ions are subjected to fragmentation using a technique such as collision induced dissociation (CJJD) to produce an ion series. The resulting fragmentation ions are analyzed in a second round of MS, and their spectral pattern may be used to determine a short stretch of amino acid sequence (Dancik et al. (1999) J Comput Biol 6:327-342). Assuming the protein is represented in the database, a combination of peptide mass and fragmentation data, together with the calculated MW and pi of the protein, will usually yield an unambiguous identification. If no match is found, protein sequence can be obtained using direct chemical sequencing procedures well known in the art (cf. Creighton (1984) Proteins. Structures and Molecular Properties, WH Freeman, New York NY). Chemical Synthesis of Peptides
Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds α-amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9- fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego CA pp. S1-S20). Automated synthesis may also be carried out on machines such as the 431 A peptide synthesizer (ABI). A protein or portion thereof may be purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY). Antibodies
Antibodies, or immunoglobulins (Ig), are components of immune response expressed on the surface of or secreted into the circulation by B cells. The prototypical antibody is a tetramer composed of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds which binds and neutralizes foreign antigens. Based on their H-chain, antibodies are classified as IgA, IgD, IgE, IgG or IgM. The most common class, IgG, is tetrameric while other classes are variants or multimers of the basic structure.
Antibodies are described in terms of their two functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. The binding of antibody to antigen triggers destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface Fc receptors that specifically bind to the Fc region of the antibody and allow the phagocytic cells to destroy antibody-bound antigen. Fc receptors are single-pass transmembrane glycoproteins containing about 350 amino acids whose extracellular portion typically contains two or three Ig domains (Sears et al. (1990) J Immunol 144:371-378). Preparation and Screening of Antibodies
Various hosts including mice, rats, rabbits, goats, llamas, camels, and human cell lines may be immunized by injection with an antigenic determinant. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH; Sigma-Aldrich), and dinitrophenol may be used to increase immunological response, hi humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum increase response. The antigenic determinant may be an oligopeptide, peptide, or protein. When the amount of antigenic determinant allows immunization to be repeated, specific polyclonal antibody with high affinity can be obtained (Klinman and Press (1975) Transplant Rev 24:41-83). Oligopepetides which may contain between about five and about fifteen amino acids identical to a portion of the endogenous protein may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule. Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120).
Chimeric antibodies may be produced by techniques such as splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; and Takeda et al. (1985) Nature 314:452-454). Alternatively, techniques described for antibody production may be adapted, using methods known in the art, to produce specific, single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci
88:10134-10137). Antibody fragments which contain specific binding sites for an antigenic determinant may also be produced. For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 246:1275-1281).
Antibodies may also be produced by inducing production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299). A protein may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Antibody Specificity Various methods such as Scatchard analysis combined with radioimmunoassay techniques may be used to assess the affinity of particular antibodies for a protein. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of protein-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple antigenic determinants, represents the average affinity, or avidity, of the antibodies. The Ka determined for a preparation of monoclonal antibodies, which are specific for a particular antigenic determinant, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are commonly used in immunoassays in which the protein-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 107 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of the protein, preferably in active form, from the antibody (Catty (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell and Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY). , The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing about 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of protein-antibody complexes. Procedures for making antibodies, evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are discussed in Catty (supra) and Ausubel (supra) pp. 11.1-11.31. Cell Transformation Assays
Cell transformation, the conversion of a normal cell to a cancerous cell, is a highly complex and genetically diverse process. However, certain alterations in cell physiology that are associated with this process can be assayed using either in vitro cell-based systems or in vivo animal models. Known alterations include acquired self-sufficiency relative to growth signals, an insensitivity to growth- inhibitory signals, unlimited replicative potential, evasion of apoptosis, sustained angiogenesis, and cellular invasion and metastasis. See Hanahan and Weinberg (2000) Cell 100:57-70. Such assays can be used, for example, to assess the effect of transfecting a cell with a gene such as HG38, on transformation of the cell. DIAGNOSTICS
Differential expression of HG38, as detected using HG38, a polynucleotide encoding HG38, or an antibody that specifically binds HG38, and at least one of the assays below can be used to diagnose a colon or lung cancer. Labeling of Molecules for Assay A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using kits such as those supplied by Promega (Madison WI) or APB for incorporation of a labeled nucleotide such as 32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Qiagen-Operon, Alameda CA), or amino acid such as 35S-methionine (APB). Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes). Nucleic Acid Assays The polynucleotides, fragments, oligonucleotides, complementary RNAs, and peptide nucleic acids (PNA) may be used to detect and quantify differential gene expression for diagnosis of a disorder. Similarly antibodies which specifically bind HG38 may be used to quantitate the protein. Disorders associated with such differential expression include colon and lung cancer. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential gene expression. Qualitative or quantitative methods for this comparison are well known in the art. Expression Profiles
An expression profile comprises the expression of a plurality of polynucleotides or protein as measured using standard assays with a sample. The polynucleotides, proteins or antibodies of the invention may be used as elements on a array to produce an expression profile. In one embodiment, the array is used to diagnose or monitor the progression of disease.
For example, the polynucleotide or probe may be labeled by standard methods and added to a biological sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If complex formation in the patient sample is altered in comparison to either a normal or disease standard, then differential expression indicates the presence of a disorder.
In order to provide standards for establishing differential expression, normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a polynucleotide under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose or stage that disorder.
By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment. For treatments with known side effects, the array is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disease, or disorder; or treatment of the condition, disease, or disorder. Novel treatment regimens may be tested in these animal models using arrays to establish and then follow expression profiles over time. In addition, arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years. Protein Assays
Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include antibody arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, 2D-PAGE and scintillation counting, protein arrays, radioimmunoassays, and western analysis. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. These assays and their quantitation against purifed, labeled standards are well known in the art (Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa NJ).
These methods are also useful for diagnosing diseases that show differential protein expression. Normal or standard values for protein expression are established by combining body fluids or cell extracts taken from a normal mammalian or human subject with specific antibodies to a protein under conditions for complex formation. Standard values for complex formation in normal and diseased tissues are established by various methods, often photometric means. Then complex formation as it is expressed in a subject sample is compared with the standard values. Deviation from the normal standard and toward the diseased standard provides parameters for disease diagnosis or prognosis while deviation away from the diseased and toward the normal standard may be used to evaluate treatment efficacy.
Recently, antibody arrays have allowed the development of techniques for high-throughput screening of recombinant antibodies. Such methods use robots to pick and grid bacteria containing antibody genes, and a filter-based ELISA to screen and identify clones that express antibody fragments. Because liquid handling is eliminated and the clones are arrayed from master stocks, the same antibodies can be spotted multiple times and screened against multiple antigens simultaneously. Antibody arrays are highly useful in the identification of differentially expressed proteins. (See de Wildt et al. (2000) Nature Biotechnol 18:989-94.) THERAPEUTICS
Differential expression of polynucleotides encoding HG38 is highly associated with colon and lung cancer as shown in data presented in Figures 3-8, Tables 1-2, and Northern analysis. HG38 clearly plays a role in colon and lung cancer.
In one embodiment, when decreased expression or activity of the protein is desired, an antibody , antagonist, inhibitor, a pharmaceutical agent or a composition containing one or more of these molecules may be delivered to a subject in need of such treatment. Such delivery may be effected by methods well known in the art and may include delivery by an antibody that specifically binds the protein. For therapeutic use, monoclonal antibodies are used to block an active site, inhibit dimer formation, trigger apoptosis and the like.
In another embodiment, when increased expression or activity of the protein is desired, the protein, an agonist, an enhancer, a pharmaceutical agent or a composition containing one or more of these molecules may be delivered to a subject in need of such treatment. Such delivery may be effected by methods well known in the art and may include delivery of a pharmaceutical agent by an antibody specifically targeted to the protein.
Any of the polynucleotides, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, therapeutic antibodies, and ligands binding the polynucleotide or protein may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular disorder at a lower dosage of each agent. Modification of Gene Expression Using Nucleic Acids
Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5', 3', or other regulatory regions of the gene encoding HG38. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. hi: Huber and Carr (1994) Molecular and hnmunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative, a library or plurality of polynucleotides may be screened to identify those which specifically bind a regulatory, nontranslated sequence. Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary nucleic acids and ribozymes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis, hi addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule or by the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- groups renders the molecule more resistant to endogenous endonucleases. cDNA Therapeutics
The cDNAs of the invention can be used in gene therapy. cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein. Alternatively, cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and bacterial plasmids. Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4): 184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics. Humana Press, Totowa NJ; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego CA).
Monoclonal Antibody Therapeutics
Antibodies, and in particular monoclonal antibodies, that specifically bind a particular protein, enzyme, or receptor and block its overexpression are now being used therapeutically. The first widely accepted therapeutic antibodies were HERCEPTIN (Trastuzumab, Genentech, S. San Francisco CA) and GLEEVEC (imatinib mesylate, Norvartis Pharmaceuticals, East Hanover NJ). HERCEPTIN is a humanized antibody approved for the treatment of HER2 positive metastatic breast cancer. It is designed to bind and block the function of overexpressed HER2 protein. GLEEVEC is indicated for the treatment of patients with Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) in blast crisis, accelerated phase, or in chronic phase after failure of interferon-alpha therapy. A second indication for GLEEVEC is treatment of patients with KIT (CDl 17) positive unresectable and/or metastatic malignant gastrointestinal stromal tumors. Other monoclonal antibodies are in various stages of clinical trials for indications such as prostate cancer, lymphoma, melanoma, pneumococcal infections, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and the like. Screening and Purification Assays The polynucleotide encoding HG38 may be used to screen a library or a plurality of molecules or compounds for specific binding affinity. The libraries may be antisense molecules, artificial chromosome constructions, branched nucleic acid molecules, DNA molecules, peptides, peptide nucleic acid, proteins such as transcription factors, enhancers, or repressors, RNA molecules, ribozymes, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene. The assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the polynucleotide.
The polynucleotide of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (USPN 6,010,849) or a reticulocyte lysate transcriptional assay. The polynucleotide may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the polynucleotide and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay. The polynucleotide may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the polynucleotide is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the polynucleotide. The molecule or compound which is bound to the polynucleotide may be released from the polynucleotide by increasing the salt concentration of the flow-through medium and collected.
The protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein to purify a ligand would involve combining the protein with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
HG38 may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. For example, in one method, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on then cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured. Depending on the particular kind of molecules or compounds being screened, the assay may be used to identify agonists, antagonists, antibodies, DNA molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, peptides, peptide nucleic acids, proteins, and RNA molecules or any other ligand, which specifically binds the protein. hi one aspect, this invention contemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in USPN 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a mammalian model system to evaluate their toxicity or therapeutic potential. Pharmaceutical Compositions
Pharmaceutical compositions may be formulated and administered, to a subject in need of such treatment, to attain a therapeutic effect. Such compositions contain the instant protein, agonists, antagonists, bispecific molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules. Compositions may be manufactured by conventional means such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing. The composition may be provided as a salt, formed with acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic, or as a lyophilized powder which may be combined with a sterile buffer such as saline, dextrose, or water. These compositions may include auxiliaries or excipients which facilitate processing of the active compounds.
Auxiliaries and excipients may include coatings, fillers or binders including sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol; starches from corn, wheat, rice, or potato; proteins such as albumin, gelatin and collagen; cellulose in the form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; lubricants such as magnesium stearate or talc; disintegrating or solubilizing agents such as the, agar, alginic acid, sodium alginate or cross-linked polyvinyl pyrrolidone; stabilizers such as carbopol gel, polyethylene glycol, or titanium dioxide; and dyestuffs or pigments added for identify the product or to characterize the quantity of active compound or dosage.
These compositions may be administered by any number of routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.
The route of administration and dosage will determine formulation; for example, oral administration may be accomplished using tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, or suspensions; parenteral administration may be formulated in aqueous, physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Suspensions for injection may be aqueous, containing viscous additives such as sodium carboxymethyl cellulose or dextran to increase the viscosity, or oily, containing lipophilic solvents such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Penetrants well known in the art are used for topical or nasal administration.
Toxicity and Therapeutic Efficacy
A therapeutically effective dose refers to the amount of active ingredient which ameliorates symptoms or condition. For any compound, a therapeutically effective dose can be estimated from cell culture assays using normal and neoplastic cells or in animal models. Therapeutic efficacy, toxicity, concentration range, and route of administration may be determined by standard pharmaceutical procedures using experimental animals.
The therapeutic index is the dose ratio between therapeutic and toxic effects~LD50 (the dose lethal to 50% of the population)/ED50 (the dose therapeutically effective in 50% of the population)-and large therapeutic indices are preferred. Dosage is within a range of circulating concentrations, includes an
ED50 with little or no toxicity, and varies depending upon the composition, method of delivery, sensitivity of the patient, and route of administration. Exact dosage will be determined by the practitioner in light of factors related to the subject in need of the treatment. Dosage and administration are adjusted to provide active moiety that maintains therapeutic effect. Factors for adjustment include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
Normal dosage amounts may vary from 0.1 μg, up to a total dose of about 1 g, depending upon the route of administration. The dosage of a particular composition may be lower when administered to a patient in combination with other agents, drugs, or hormones. Guidance as to particular dosages and methods of delivery is provided in the pharmaceutical literature. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA). Model Systems
Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, gestation period, numbers of progeny, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene. Toxicology
Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess consequences on human health following exposure to the agent.
Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range-finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals. Chronic toxicity tests, with a duration of a year or more, are used to test whether long term administration may elicit toxicity, teratogenesis, or carcinogenesis. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment. Transgenic Animal Models
Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., USPN 5,175,383 and USPN
5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
Embryonic Stem Cells
Embryonic (ES) stem cells isolated from rodent embryos retain the ability to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art.
Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes. Knockout Analysis hi gene knockout analysis, a region of a gene is enzymatically modified to include a non- mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288- 1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene, hi one example, the mammalian gene is a human gene. Knockin Analysis
ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases. Non-Human Primate Model
The field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drug evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus and Rhesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and Common Marmosets (Callithrix jacchus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs, early research and toxicological studies are usually carried out in rodent models, hi studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from "extensive metabolizers" to "poor metabolizers" of these agents.
In additional embodiments, the polynucleotides which encode the protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of polynucleotides that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
EXAMPLES
I cDNA Library Construction Cells or tissues were homogenized and lysed in guanidinium isothiocyanate, in phenol or in a suitable mixture of denaturants such as TRIZOL reagent (invitrogen) and guanidine isothiocyanate. The lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNAse. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (Qiagen, Chatsworth CA), or an OLIGOTEX mRNA purification kit (Qiagen). Alternatively, RNA was isolated directly from tissue lysates using RNA isolation kits such as the POLY(A)PURE mRNA purification kit (Ambion, Austin TX). In some cases, Stratagene was provided with RNA and constructed the cDNA libraries. cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with appropriate restriction enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (APB) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid such as pBLUESCRIPT plasmid or pBK- CMV plasmid (both Stratagene), pSPORTl plasmid or PCDNA2.1 plasmid (both Invitrogen), pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant plasmids were transformed into competent E. coU cells including XLl-Blue, XLl-BlueMRF, or SOLR (Stratagene) or DH5α, DH10B, or ElectroMAX DH10B (Invitrogen).
II Isolation, Preparation, and Sequencing of cDNAs
Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or REAL PREP 96 plasmid purification kit from Qiagen. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4C. Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high- throughput format (Rao (1994) Anal Biochem 216: 1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN U fluorescence scanner (Labsystems Oy, Helsinki, Finland).
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the CATALYST 800 (ABI) thermal cycler or the DNA ENGINE thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents obtained from APB or supplied in sequencing kits such as the PRISM BIGDYE Terminator cycle sequencing ready reaction kit (ABI). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (APB) or PRISM 373 or 377 sequencing systems (ABI) in conjunction with standard protocols and base calling software. Reading frames within the cDNA sequences were identified using standard methods (Ausubel, supra, unit 7.7). Ill Extension of cDNAs
The cDNAs were extended using the cDNA clone and oligonucleotide primers. One primer was synthesized to initiate 5' extension of the known fragment, and the other, to initiate 3' extension of the known fragment. The initial primers were designed using primer analysis software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68C to about 72C. Any stretch of nucleotides that would result in hairpin structures and primer-primer dimerizations was avoided.
Selected cDNA libraries were used as templates to extend the sequence. If extension was performed than one time, additional or nested sets of primers were designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5' or upstream regions of genes. Genomic libraries can be used to obtain regulatory elements extending into the 5' promoter binding region.
High fidelity amplification was obtained by PCR using methods such as that taught in USPN 5,932,451. PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)2S04, and β-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): The parameters for the cycles are 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4: 68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min; and 7: storage at 4C. hi the alternative, the parameters for primer pair T7 and SK+ (Stratagene) were as follows: 1: 94C, three min; 2: 94C, 15 sec; 3: 57C, one min; 4: 68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min; and 7: storage at 4C.
The concentration of DNA in each well was determined by dispensing 100 μ\ PICOGREEN quantitation reagent (0.25% reagent in lx TE, v/v; Molecular Probes) and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Life Sciences, Acton MA) and allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose minigel to determine which reactions were successful in extending the sequence.
The extended clones were desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC18 vector (APB). For shotgun sequences, the digested nucleotide sequences were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and the agar was digested with AGARACE enzyme (Promega). Extended clones were religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37C in 384- well plates in LB/2x carbenicillin liquid media. The cells were lysed, and DNA was amplified using primers, Taq DNA polymerase (APB) and
Pfu DNA polymerase (Stratagene) with the following parameters: 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4: 72C, two min; 5: 2, 3, and 4 repeated 29 times; 6: 72C, five min; and 7: storage at 4C. DNA was quantified using PICOGREEN quantitation reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the conditions described above. Samples were diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI). IV Homology Searching of cDNA Clones and Their Deduced Proteins
The polynucleotides of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST2 to produce alignments and to determine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
As detailed in Karlin and Altschul (1993; Proc Natl Acad Sci 90:5873-5877), BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10"25 for nucleotides and 10"M for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the stringency for an exact match was set from a lower limit of about 40 (with 1-2% error due to uncalled bases) to a 100% match of about 70.
The BLAST software suite (NCBI, Bethesda MD), includes various sequence analysis programs including "blastn" that is used to align nucleotide sequences and BLAST2 that is used for direct pairwise comparison of either nucleotide or amino acid sequences. BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11 ; and Filter: on. Identity is measured over the entire length of a sequence. Brenner (supra) analyzed BLAST for its ability to identify structural homologs by sequence identity and found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40%, for alignments of at least 70 residues.
The polynucleotides of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics). Component sequences from polynucleotide, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3' ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, and bacterial contamination sequences. Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by "Ns" or masked.
Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was determined based on the number and orientation of its component sequences.
Bins were compared to one another, and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split. Templates were subjected to analysis by STITCHER/EXON MAPPER algorithms that determine the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri. An exact match was defined as having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs and a homology match as having an E-value (or probability score) of <1 x 10"8. The templates were also subjected to frameshift FASTx against GENPEPT, and homology match was defined as having an E-value of <1 x 10"3. Template analysis and assembly was described in USSN 09/276,534, filed March 25, 1999.
Following assembly, templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in USSN 08/812,290 and USSN 08/811,758, both filed March 6, 1997; in USSN 08/947,845, filed October 9, 1997; and in USSN 09/034,807, filed March 4, 1998. Then templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the HMMER software package (Washington University School of Medicine, St. Louis MO). The polynucleotide was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite. V Northern Analysis, Transcript Imaging Northern analysis
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. The technique is described below and in Ausubel, supra, units 4.1-4.9 and was used to generate the data presented in the Tables at pages 13 and 14, above. Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or the LJFESEQ database (Incyte Genomics). This analysis is faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous. The basis of the search is the product score which was described above in EXAMPLE TV. The results of northern analysis are reported as a list of libraries in which the transcript encoding HG38 occurs. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library. Transcript Imaging
A transcript image was performed using the LIFESEQ GOLD database (Incyte Genomics). This process allows assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries and was described in USPN 5,840,484, incorporated herein by reference. All sequences and cDNA libraries in the LIFESEQ database are categorized by system, organ tissue and cell type. The categories include cardiovascular system, connective tissue, digestive system, embryonic structures, endocrine system, exocrine glands, female and male genitalia, germ cells, hemic/immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed, and the urinary tract. Criteria for transcript imaging are selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.
For each category, the number of libraries in which the sequence was expressed are counted and shown over the total number of libraries in that category. For each library, the number of cDNAs are counted and shown over the total number of cDNAs in that library, hi some transcript images, all enriched, normalized or subtracted libraries, which have high copy number sequences can be removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be excluded where clinical relevance is emphasized. Conversely, fetal tissue can be emphasized wherever elucidation of inherited disorders or differentiation of particular adult or embryonic stem cells into tissues or organs (such as heart, kidney, nerves or pancreas) would be aided by removing clinical samples from the analysis. Transcript imaging can also be used to support data from other methodologies such as hybridization, guilt-by-association and array technologies. VI Chromosome Mapping
Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the polynucleotides presented in the Sequence Listing have been mapped. Any of the fragments of the polynucleotide encoding HG38 that have been mapped result in the assignment of all related regulatory and coding sequences to the same location. The genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm. VII Hybridization and Amplication Technologies and Analyses Tissue Sample Preparation 5 Normal and cancerous colon and lung tissue samples are described by donor identification number in the table below. The first column shows the donor ID; the second, donor age/sex; the third column, a description of the disorder, the fourth column, classification of the tumor; and the fifth column, the source.
Donor Age/Sex* Tissue and Description Stage Source
3579 55/M colon; well differentiated adenoCA Dukes' C; TMN T2N1 HCI 10 3580 38/M colon; poorly differentiated, metastatic adenoCA T3N1MX HCI 3581 U/M rectal; tumor NA HCI 3582 78/M colon; moderately differentiated adenoCA TMN T4N2MX HCI 3583 58/M colon; tubulovillous adenoma (hyperplastic polyp) NA HCI 3647 83/U colon; invasive moderately differentiated adenoCA TMN T3N1MX HCI 15 3649 86/U colon; invasive well-differentiated adenoCA NA HCI 3479 68/M colon; adenoCA NA HCI 3839 59/M colon tumor NA HCI 3757 75/F colon tumor NA HCI 3756 78/U colon tumor NA HCI 20 4614 67/U colon; moderately differentiated adenoCA Dukes' B; TMN T3N0 HCI 9573 60/F colon; moderately differentiated adenoCA Dukes C; T2N2M0 Asterand 9574 34/F colon; well differentiated metastatic adenoCA Dukes C; T2N1M0 Asterand 9575 60/M colon; moderately differentiated metastatic Dukes C; TXN1-2M0 Asterand adenoCA
9576 65/M colon; well differentiated adenoCA Dukes C; T3N2M0 Asterand
25 9577 46/F colon; well differentiated adenoCA Dukes C; TXN1-2M0 Asterand 8401 57/M colon; well-moderately differentiated adenoCA Grade II Asterand 8403 54/F colon; adenoCA Grade π Asterand 7162 73/M lung poorly differentiated,large cell endocrine HB RCI 7164 79/M lung pulmonary carcinoid stage LA RCI
30 7168 75/M lung poorly differentiated adenoCA LB RCI 7173 70/M lung moderately differentiated squamous cell CA ΠB RCI 7175 67/M lung moderately differentiated adenoCA ΓB RCI 7176 72/M lung; poorly differentiated, adenosquamous IB RCI 7178 68/F moderately differentiated squamous cell carcinoma LTJA RCI
35 7186 61/M atypical carcinoid stage IA RCI 7188 54/M poorly differentiated adenoCA LTJA RCI 7189 78/M poorly differentiated adenoCA m RCI 7190 50/F moderately differentiated squamous cell CA RCI 7191 43/M poorly differentiated, squamous cell CA LLB RCI
40 7963 71/M poorly differentiated adenoCA ILLA RCI 9751 70F poorly differentiated, squamous cell CA NA RCI 9752 56/M squamous cell carcinoma NA RCI 9753 66/M moderately differentiated squamous cell CA NA RCI 9754 72/M moderately differentiated squamous cell CA NA RCI
45 9757 58/F lung, adenoCA NA RCI 9758 48/M lung, adenoCA NA RCI 9764 73/M lung, adenoCA NA RCI
5793 73/M lung; moderately differentiated squamous cell CA LLA RCI
5795 71/F lung; moderately differentiated adenoCA IA RCI
5796 66/M lung; moderately differentiated squamous cell CA IB RCI
5797 73/M lung; moderately differentiated squamous cell CA LfB RCI
5798 66/F lung; adenoCA NA RCI
5799 66/F lung; moderately differentiated adenoCA flB RCI
5800 75/F lung; moderately differentiated squamous cell CA IB RCI
* Abbreviations: CA=carcinoma, U=unknown, NA=not available hi Figure 2, the normalized, first-strand synthesis, polynucleotide preparations of normal, human heart, brain (whole), lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, ovary, small intestine, peripheral blood leukocyte, and colon tissues were obtained from Clontech. Additional polynucleotide preparations of human, adult, normal thyroid, pituitary, and adrenal tissues were obtained from Clinomics Bioscience (Pittsfield MA).
The colon cell lines shown in Figure 5 were obtained from ATCC and cultured according to the suppliers specifications. The table below describes cancerous and non-cancerous human cell lines analyzed in Figure 5 for HG38 expression. The first column lists the name of the cell line, the second column, the tissue source, the third column, a description of the cell line, and the fourth and fifth columns whether the cell line is tumorigenic and/or metastatic in mice.
Sample Tissue Description Tumorigenic Metastatic
Figure imgf000043_0001
Immobilization of polynucleotides on a Substrate
The polynucleotides are applied to a substrate by one of the following methods. A mixture of polynucleotides is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the polynucleotides are individually ligated to a vector and inserted into bacterial host cells to form a library. The polynucleotides are then arranged on a substrate by one of the following methods, hi the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37C for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH ), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2xSSC for 10 min each. The membrane is then UV irradiated in a STRATALLNKER UV-crosslinker (Stratagene).
In the second method, polynucleotides are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above. Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in USPN 5,807,522. Polymer-coated slides are prepared by cleaning glass microscope slides (Corning Life Sciences) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR Scientific Products, West Chester PA), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a HOC oven. The slides are washed extensively with distilled water between and after treatments. The nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATALLNKER UV-crosslinker
(Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford MA) for 30 min at 60C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before. Probe Preparation for Membrane Hybridization
Hybridization probes derived from the polynucleotides of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the polynucleotides to a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heating to 100C for five min, and briefly centrifuging. The denatured polynucleotide is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five μl of [32P]dCTP is added to the tube, and the contents are incubated at 37C for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below. Probe Preparation for QPCR
Probes for the QPCR were prepared according to the ABI protocol. Probe Preparation for Polymer Coated Slide Hybridization
The following method was used for the preparation of probes for the microarray analyses presented in Tables 1 and 2 Hybridization probes derived from mRNA isolated from samples are employed for screening polynucleotides of the Sequence Listing in array-based hybridizations. Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 μl TE buffer and adding 5 μl 5x buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5 labeling mix, 1 μl RNAse inhibitor, 1 μl reverse transcriptase, and 5 μl lx yeast control mRNAs. Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3: 1, 1: 10, 10: 1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37C for two hr. The reaction mixture is then incubated for 20 min at 85C, and probes are purified using two successive CHROMA SPLN+TE 30 columns (Clontech, Palo Alto CA). Purified probe is ethanol precipitated by diluting probe to 90 μl in DEPC-treated water, adding 2 μl lmg/ml glycogen, 60 μl 5 M sodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20 min at 20,800xg, and the pellet is resuspended in 12 μl resuspension buffer, heated to 65C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below. hi situ Hybridization
The following method was used in the analyses performed in Figures 7 and 8. Ln situ hybridization was used to determine the expression of HG38 in sectioned tissue. With the digoxygenin protocol, fresh cryosections, 10 microns thick, were removed from the freezer, immediately immersed in 4% paraformaldehyde for 10 min, rinsed in PBS, and acetylated in 0.1 M TEA, pH 8.0, containing 0.25% (v/v) acetic anhydride. After the tissue equilibrated in 5 x SSC, it was prehybridized in hybridization buffer (50% formamide, 5 x SSC, 1 x Denhardt's solution, 10% dextran sulfate, and 1 mg/ml herring sperm DNA). Digoxygenin-labeled HG38-specific RNA probes, sense and antisense nucleotides selected from the polynucleotide of SEQ ID NO: 1 were produced using PCR. Approximately 500 ng/ml of probe was used in overnight hybridizations at 65C in hybridization buffer. Following hybridization, the sections were rinsed for 30 min in 2 x SSC at room temperature, 1 hr in 2 x SSC at 65C, and 1 hr in 0.1 x SSC at 65C. The sections were equilibrated in PBS, blocked for 30 min in 10% DIG kit blocker (Roche
Molecular Biochemicals, Indianapolis IN) in PBS, then incubated overnight at 4C in 1:500 anti-DIG-AP. The following day, the sections were rinsed in PBS, equilibrated in detection buffer (0.1 M Tris, 0.1 M NaCl, 50 mM MgCl2, pH 9.5), and then incubated in detection buffer containing 0.175 mg/ml NBT and 0.35 mg/ml BCJP. The reaction was terminated in TE, pH 8. Tissue sections were counterstained with 1 μg ml DAPI and mounted in VECTASHLELD (Vector Laboratory, Burlingame CA). Membrane-based Hybridization
Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl, 0.1 M Na2HP04, 5 mM EDTA, pH 7) at 55C for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55C for 16 hr. Following hybridization, the membrane is washed for 15 min at 25C in lmM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25C in ImM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the membrane overnight at -70C, developed, and examined visually. Polymer Coated Slide-based Hybridization The following method was used in the microarray analyses presented in Tables 1 and 2. Probe is heated to 65C for five min, centrifuged five min at 9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, Westbury NY), and then 18 μl is aliquoted onto the array surface and covered with a coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5xSSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hr at 60C. The arrays are washed for 10 min at 45C in lxSSC, 0.1% SDS, and three times for 10 min each at 45C in O.lxSSC, and dried.
Hybridization reactions are performed in absolute or differential hybridization formats. In the absolute hybridization format, probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample. Ln the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Melville NY). The slide containing the array is placed on a computer- controled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers. In the differential hybridization format, the two fluorophores are sequentially excited by the laser. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tubes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (AID) conversion board (Analog Devices, Norwood MA) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore. A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS program (Incyte Genomics). QPCR Analysis
For QPCR, cDNA was synthesized from 1 ug total RNA in a 25 ul reaction with 100 units M- MLV reverse transcriptase (Ambion, Austin TX), 0.5 mM dNTPs (Epicentre, Madison WI), and 40 ng/ml random hexamers (Fisher Scientific, Chicago JL). Reactions were incubated at 25C for 10 minutes, 42C for 50 minutes, and 70C for 15 minutes, diluted to 500 ul, and stored at -30C. Alternatively, normal tissues were purchased from Clontech (Palo Alto CA) and Clinomics. PCR primers and probes (5' 6- FAM-labeled, 3 'TAMRA) were designed using PRIMER EXPRESS 1.5 software (ABI) and synthesized by Biosearch Technologies (Novato CA) or ABI. QPCR reactions were performed using an PRISM 7700 detection system (ABI) in 25 ul total volume with 5 ul cDNA template, lx TAQMAN UNLVERSAL PCR master mix (ABI), 100 nM each PCR primer, 200 nM probe, and lx VIC-labeled beta-2-microglobulin endogenous control (ABI). Reactions were incubated at 50C for 2 minutes, 95C for 10 minutes, followed by 40 cycles of incubation at 95C for 15 seconds and 60C for 1 minute. Emissions were measured once every cycle, and results were analyzed using SEQUENCE DETECTOR 1.7 software (ABI) and fold differences, relative concentration of mRNA as compared to standards, were calculated using the comparative Cτ method (ABI User Bulletin #2). QPCR was used to produce the data for Figures 2-6.
VIII Complementary Molecules Antisense molecules complementary to the cDNA, from about 5 bp to about 5000 bp in length, are used to detect or inhibit gene expression. Detection is described in Example V 1. To inhibit transcription by preventing promoter binding, the complementary molecule is' designed to bind to the most unique 5' sequence and includes nucleotides of the 5' UTR upstream of the initiation codon of the open reading frame. Complementary molecules include genomic sequences (such as enhancers or introns) and are used in triple helix base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. To inhibit translation, a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.
Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy. Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.
Stable transformation of dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (USPN 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules to compromise or entirely eliminate activity of the polynucleotide encoding the protein.
IX Production of Specific Antibodies
Purification using polyacrylamide gel electrophoresis or similar techniques is used to isolate protein for immunization of hosts or host cells to produce antibodies using standard protocols.
Alternatively, the amino acid sequence of the protein is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity. A peptide with high immunogenicity is cleaved, recombinantly-produced, or synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate antigenic determinants such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel, supra, Chap. 11). Oligopeptides of about 15 residues in length are synthesized using an 431 A peptide synthesizer (ABI) using FMOC chemistry and coupled to carriers such as BSA, thyroglobulin, or KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase immunogenicity. The coupled peptide is then used to immunize the host. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by binding the peptide to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio- iodinated goat anti-rabbit IgG.
X Immunopurification Using Antibodies Naturally occurring or recombinantly produced protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the purified protein is collected.
XI Western Analysis Electrophoresis and Blotting
Samples containing protein are mixed in 2 x loading buffer, heated to 95 C for 3-5 min, and loaded on 4-12% NUPAGE Bis-Tris precast gel (Invitrogen). The gel is electrophoresced in 1 x MES or MOPS running buffer (Invitrogen) at 200 V for approximately 45 min on a (apparatus, supplier) until the RAINBOW marker (APB) has resolved, and dye front approaches the bottom of the gel. The gel and its supports are removed from the apparatus and soaked in 1 x transfer buffer (Invitrogen) with 10% methanol for a few minutes; and the PVDF membrane soaked in 100% methanol for a few seconds to activate it. The membrane, the gel, and supports are placed on the transfer apparatus (machine, supplier) and a constant current of 350 mAmps is applied for 90 min. Conjugation with Antibody and Visualization
After the proteins are transferred to the membrane, it is blocked in 5% (w/v) non-fat dry milk in 1 x phosphate buffered saline (PBS) with 0.1% Tween 20 detergent (blocking buffer) on a rotary shaker (supplier) for at least lhr at room temperature or at 4° overnight. After blocking, the buffer is removed, and 10 ml of primary antibody in blocking buffer is added and incubated on the rotary shaker for 1 hr at room temperature or overnight at 4 C. The membrane is washed 3 x for 10 min each with PBS-Tween (PBST), and secondary antibody, conjugated to horseradish peroxidase, is added at a 1:3000 dilution in 10 ml blocking buffer. The membrane and solution are shaken for 30 min at room temperature and then washed 3 x for 10 min each with PBST.
The wash solution is carefully removed, and the membrane moistened with ECL+ chemiluminescent detection system (APB) and incubated for approximately 5 min. The membrane is placed, protein side down, on plastic film (product, supplier) and developed for approximately 30 seconds.
XII Antibody Arrays Proteimprotein interactions
Ln an alternative to yeast two hybrid system analysis of proteins, an antibody array can be used to study protein-protein interactions and phosphorylation. A variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein: antibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest. In the alternative, a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex. The identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane. Proteomic Profiles
Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt, supra)
XIII Screening Molecules for Specific Binding with the polynucleotide or Protein
The polynucleotide, or fragments thereof, or the protein, or portions thereof, are labeled with 32P- dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or FITC (Molecular Probes), respectively. Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled polynucleotide or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule. XIV Two-Hybrid Screen
A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories), is used to screen for peptides that bind the protein of the invention. A polynucleotide encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coli. cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli to construct a cDNA library. The pLexA plasmid and pB42AD-cDNA library constructs are isolated from E. coli and used in a 2: 1 ratio to co-transform competent yeast EGY48[p8op- lacZ] cells using a polyethylene glycol lithium acetate protocol. Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30C until the colonies have grown up and are counted. The colonies are pooled in a minimal volume of lx TE (pH 7.5), replated on SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and polynucleotide fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu). Interaction also activates expression of β-galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.
Positive interactions between expressed protein and polynucleotide fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30C. A sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30C until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD containing histidine but not on media lacking histidine have lost the pLexA plasmid. Histidine-requiring colonies are grown on SD/Gal/Raf/X-Gal -Trp/-Ura, and white colonies are isolated and propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized. XV GPCR Activity Assay
The GPCR encoded by SEQ ID NO:2 may be expressed in heterologous expression systems and its biological activity tested utilizing the purinergic receptor system (P2u) as published by Erb et al. (1993, Proc Natl Acad Sci 90: 10449-53). Because cultured K562 human leukemia cells lack P2U receptors, they can be transfected with expression vectors containing either normal or chimeric P 2U and loaded with fura- °s fluorescent probe for Ca++. Activation of properly assembled and functional extracellular SP- transmembrane/intracellular P2U receptors with extracellular UTP or ATP mobilizes intracellular Ca^ which reacts with fura-<* and is measured spectrofluorometrically. Bathing the transfected K562 cells in microwells containing appropriate ligands will trigger binding and fluorescent activity defining effectors t of SP. Once ligand and function are established, the P2U system is useful for defining antagonists or inhibitors which block binding and prevent such fluorescent reactions. XVI Cell Transformation Assays Colony-formation Assay in Soft Agar The ability of transformed cells to grow in an anchorage-independent manner is measured by the ability of the cells to form colonies in soft agar (0.35%). The assay is conducted in 12-well culture plates where each well is coated with a solid 0.7% Noble agar (Fisher Scientific, Atlanta GA) in cell growth media. A 3.5% agar solution in PBS is prepared, autoclaved, microwaved and kept liquid in a 55 C water bath with shaking. The agar is diluted 1:5 to 0.7% with an appropriate cell growth media, and 0.5 ml of the diluted agar added to each well of the plate. Culture plates are kept at room temperature for about 15 minutes or until the agar solidifies.
Trypsinized cells are diluted to 200 to 4000 cells/ml in growth medium and 0.25 ml of diluted cells is mixed with 2 ml warm 0.35% agar. The diluted cells are added to a well of the culture plate; duplicate wells are prepared for each cell concentration. The plates are allowed to cool for about 30 min at room temperature and then transferred to an incubator at 37 C. After a 1-2 week incubation period, colonies are counted under an inverted, phase contrast microscope. Colony forming efficiency is determined as the percentage colonies formed/total number of cells plated. Apoptosis/Survival Assay
The ability of transformed cells to evade apoptosis (programmed cell death) and survive may be measured in an assay in which apoptosis or survival of cultured cells is determined by FACS analysis using a double-staining method with Annexin V and propidium iodide (PI). Annexin V serves as a marker for apoptotic cells by binding to phosphatidyl serine, a cell surface marker for apoptosis. Counterstaining with PI allows differentiation between apoptotic cells, which are Annexin V positive and PI negative, and necrotic cells, which are Annexin V and PI positive. Apoptosis is measured between 0-24 hrs of culture, and cell survival is measured between 24-96 hrs of culture.
Alternatively, the direct effect of a secreted protein, such as HG3, on apoptosis/cell survival may be measured in cultured human vascular endothelial cells (HMVEC) following treatment of HMVEC cells with HG38, or infection of the cells with a recombinant adenovirus containing the cDNA encoding HG38. Apoptosis/survival of the HMVEC cells is measured as described above. Tissue Invasion and Metastasis Assay
Cell migration and tissue invasion by transformed tumor cells is determined using the BICOAT Angiogenesis system (BD Biosciences, Franklin Lakes NJ) as described by the manufacturer. The assay is carried out in a BD FALCON multiwell insert plate containing an 8 μm pore size BD FLUOROBLOK polyethylene terephthalate membrane uniformly coated with a reconstituted BD MATRIGEL basement membrane matrix and inserted into a non-treated multiwell receiver plate. The system provides a barrier to passive diffusion of cells through the membrane but allows active migration by invasive tumor cells. After cells in appropriate culture medium are incubated in the upper portion of the chamber for a suitable period of time, any cells appearing on the underside of the membrane are quantitated. Since the membrane blocks the transmission of light from 490 to 700nm, cells traversing the membrane are detected by their fluorescence which is proportionate to cell number.
All patents and publications mentioned in the specification are incorporated by reference herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.
Figure imgf000054_0001
TABLE 2
Cy5/C Normal Y3 Dn ID Tumor Description umor Dn ID
-1.40 Dn7168 Human, Lung Tumor, Squamous Cell CA, Dn7168 -1.20 Dn7168 Human, Lung Tumor, Squamous Cell CA, Dn7168
-1.20 Dn7164 Human, Lung Tumor, Carcinoid, Dn7164 -1.40 Dn7164 Human, Lung Tumor, Carcinoid, Dn7164
-1.50 Dn7162 Huma , Lung Tumor, Large Cell Endocrine C Dn7162 -2.00 Dn7162 Human, Lung Tumor, Large Cell Endocrine C Dn7162
1.80 Dn7178 Human, Lung Tumor, Left Upper Lobe, Squam Dn7178
-1.60 Dn7176 Human, Lung Tumor, Right Middle Lower Lob Dn7176
2.20 Dn7173 Human, Lung Tumor, Right, Squamous Cell C Dn7173 3.80 Dn7173 Human, Lung Tumor, Right, Squamous Cell C Dn7173
1.30 Dn7190 Huma , Lung Tumor, Left, Squamous Cell CA Dn7190
1.80 Pool Human, Lung Tumor, Right Middle Lobe, Aty Dn7186
2.80 Dn7963 Human, Lung Tumor, Non-Small Cell Lung CA Dn7963
-1.40 Dn7191 Human, Lung Tumor, Left, Squamous Cell CA Dn7191
1.60 Dn5800 Human, Lung Tumor, Squamous Cell CA, Dn5800 1.60 Dn5800 Human, Lung Tumor, Squamous Cell CA, Dn5800
1.10 Dn5799 Human, Lung Tumor, AdenoCA, Dn5799 1.00 Dn5799 Human, Lung Tumor, AdenoCA, Dn5799
-1.20 Dn5798 Human, Lung Tumor, AdenoCA, Dn5798 -1.10 Dn5798 Human, Lung Tumor, AdenoCA, Dn5798
2.10 Dn5797 Human, Lung Tumor, Squamous Cell CA, Dn5797 2.40 Dn5797 Human, Lung Tumor, Squamous Cell CA, Dn5797
1.80 Dn5796 Human, Lung Tumor, Squamous Cell CA, Dn5796
-1.20 Dn5795 Human, Lung Tumor, AdenoCA, Dn5795
1.30 Dn5793 Human, Lung Tumor, Squamous Cell CA, Dn5793 1.20 Dn5793 Human, Lung Tumor, Squamous Cell CA, Dn5793
Figure imgf000055_0001

Claims

What is claimed is:
1. A method for using a polynucleotide to detect a colon or lung cancer comprising: a) hybridizing a composition comprising the polynucleotide of SEQ ID NO:2, or the complement thereof, and a labeling moiety, to nucleic acids of a sample of colon or lung tissue under conditions to form at least one hybridization complex; b) detecting hybridization complex formation; and c) comparing complex formation to a standard, wherein the comparison reflects differential expression of the polynucleotide in the sample relative to the standard and is diagnostic of a colon or lung cancer. 2. The method of claim 1 further comprising amplifying the nucleic acids of the sample prior to hybridization.
3. The method of claim 1 wherein the composition is attached to a substrate.
4. A method for detecting a colon or lung cancer, the method comprising: a) performing an assay to determine the amount of the protein of SEQ ID NO: 1 in a sample of colon or lung tissue; and b) comparing the amount of protein to a standard, thereby detecting expression of the protein in the sample, wherein differential expression of the protein in the sample when compared with the standard is diagnostic of a colon or lung cancer.
5. The method of claim 4 wherein the assay is selected from antibody arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, two dimensional-polyacrylamide gel electrophoresis and scintillation counting, radioimmunoassays, and western analysis.
6. The method of claim 4 comprising: a) combining an antibody specific for SEQ ID NO:l with a sample under conditions which allow the formation of antibody:protein complexes; b) detecting complex formation wherein complex formation indicates expression of the protein in the sample; and c) comparing complex formation with a standard, wherein differential expression of the protein between the sample and the standard is dignostic of a colon or lung cancer.
7. A method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention the antibody of claim 6.
9. A method for delivering a therapeutic agent to a colon cancer cell comprising: a) attaching the therapeutic agent to the antibody of claim 6; and b) administering the antibody to a subject in need of therapeutic intervention, wherein the antibody specifically binds the protein having the amino acid sequence of SEQ ID NO:l thereby delivering the therapeutic agent to the cell.
10. A method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention an antagonist of the protein of claim 4.
PCT/US2004/004060 2003-02-19 2004-02-11 Methods of use of a gpcr in the diagnosis and treatment of colon and lung cancer WO2004074436A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US44895903P 2003-02-19 2003-02-19
US60/448,959 2003-02-19

Publications (2)

Publication Number Publication Date
WO2004074436A2 true WO2004074436A2 (en) 2004-09-02
WO2004074436A3 WO2004074436A3 (en) 2007-02-22

Family

ID=32908678

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/004060 WO2004074436A2 (en) 2003-02-19 2004-02-11 Methods of use of a gpcr in the diagnosis and treatment of colon and lung cancer

Country Status (1)

Country Link
WO (1) WO2004074436A2 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1602930A2 (en) * 2003-08-22 2005-12-07 Hinzmann, Bernd, Dr. Use of compounds that bind to GPR49 for diagnosis and treatment of cancer
ES2277785A1 (en) * 2005-12-21 2007-07-16 Oryzon Genomics, S.A. Method for the analysis of differential expression in colorectal cancer
WO2009005809A3 (en) * 2007-07-02 2009-04-02 Oncomed Pharm Inc Compositions and methods for treating and diagnosing cancer
WO2010016766A2 (en) * 2008-08-08 2010-02-11 Koninklijke Nederlandse Akademie Van Wetenschappen Antibodies recognizing endogenous human lgr5 and/or lgr6
US8680243B2 (en) 2007-11-14 2014-03-25 Chugai Seiyaku Kabushiki Kaisha Diagnosis and treatment of cancer using anti-GPR49 antibody
US8802097B2 (en) 2011-07-15 2014-08-12 Oncomed Pharmaceuticals, Inc. Anti-RSPO1 antibodies
US9181333B2 (en) 2012-07-13 2015-11-10 Oncomed Pharmaceuticals, Inc. RSPO3 binding agents and uses thereof
US10064937B2 (en) 2014-09-16 2018-09-04 Oncomed Pharmaceuticals, Inc. Treatment of dermal fibrosis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002061087A2 (en) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Antigenic peptides, such as for g protein-coupled receptors (gpcrs), antibodies thereto, and systems for identifying such antigenic peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002061087A2 (en) * 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Antigenic peptides, such as for g protein-coupled receptors (gpcrs), antibodies thereto, and systems for identifying such antigenic peptides

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1602930A2 (en) * 2003-08-22 2005-12-07 Hinzmann, Bernd, Dr. Use of compounds that bind to GPR49 for diagnosis and treatment of cancer
EP1602930A3 (en) * 2003-08-22 2006-10-04 Hinzmann, Bernd, Dr. Use of compounds that bind to GPR49 for diagnosis and treatment of cancer
ES2277785A1 (en) * 2005-12-21 2007-07-16 Oryzon Genomics, S.A. Method for the analysis of differential expression in colorectal cancer
WO2007074193A3 (en) * 2005-12-21 2008-10-30 Oryzon Genomics Sa Method for the analysis of differential expression in colorectal cancer
EP3424529A1 (en) * 2007-07-02 2019-01-09 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US9040044B2 (en) 2007-07-02 2015-05-26 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US9610348B2 (en) 2007-07-02 2017-04-04 Oncomed Pharmaceuticals, Inc Compositions and methods for treating and diagnosing cancer
US8158757B2 (en) 2007-07-02 2012-04-17 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US8158758B2 (en) 2007-07-02 2012-04-17 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US8540989B2 (en) 2007-07-02 2013-09-24 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US8628774B2 (en) 2007-07-02 2014-01-14 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
EP2173379B1 (en) 2007-07-02 2015-09-02 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
WO2009005809A3 (en) * 2007-07-02 2009-04-02 Oncomed Pharm Inc Compositions and methods for treating and diagnosing cancer
US8883736B2 (en) 2007-07-02 2014-11-11 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US9717794B2 (en) 2007-07-02 2017-08-01 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
EP3009148A1 (en) * 2007-07-02 2016-04-20 Oncomed Pharmaceuticals, Inc. Compositions and methods for treating and diagnosing cancer
US8680243B2 (en) 2007-11-14 2014-03-25 Chugai Seiyaku Kabushiki Kaisha Diagnosis and treatment of cancer using anti-GPR49 antibody
US9296823B2 (en) 2007-11-14 2016-03-29 Chugai Seiyaku Kabushiki Kaisha Diagnosis and treatment of cancer using anti-GPR49 antibody
WO2010016766A2 (en) * 2008-08-08 2010-02-11 Koninklijke Nederlandse Akademie Van Wetenschappen Antibodies recognizing endogenous human lgr5 and/or lgr6
WO2010016766A3 (en) * 2008-08-08 2010-05-06 Koninklijke Nederlandse Akademie Van Wetenschappen Antibodies recognizing endogenous human lgr5 and/or lgr6
US8802097B2 (en) 2011-07-15 2014-08-12 Oncomed Pharmaceuticals, Inc. Anti-RSPO1 antibodies
US9644034B2 (en) 2011-07-15 2017-05-09 Oncomed Pharmaceuticals, Inc. Anti-RSPO2 antibodies and uses thereof
US9109025B2 (en) 2011-07-15 2015-08-18 Oncomed Pharmaceuticals, Inc. Anti-RSPO2 antibodies
US9109024B2 (en) 2011-07-15 2015-08-18 Oncomed Pharmaceuticals, Inc. Anti-RSPO1 antibodies and uses thereof
US9598497B2 (en) 2012-07-13 2017-03-21 Oncomed Pharmaceuticals, Inc. RSPO3 binding agents and uses thereof
US9181333B2 (en) 2012-07-13 2015-11-10 Oncomed Pharmaceuticals, Inc. RSPO3 binding agents and uses thereof
US10064937B2 (en) 2014-09-16 2018-09-04 Oncomed Pharmaceuticals, Inc. Treatment of dermal fibrosis

Also Published As

Publication number Publication date
WO2004074436A3 (en) 2007-02-22

Similar Documents

Publication Publication Date Title
US20030064397A1 (en) Transmembrane protein differentially expressed in prostate and lung tumors
US20020187472A1 (en) Steap-related protein
US20020102569A1 (en) Diagnostic marker for cancers
US20160282350A1 (en) Methods of diagnosing cancer
US20060275314A1 (en) Transmembrane protein differentially expressed in cancer
US20030186333A1 (en) Down syndrome critical region 1-like protein
US20040018188A9 (en) Sparc-related proteins
US20030103981A1 (en) Methods of use of a prostate-associated protease in the diagnosis and treatment of prostate cancer
WO2004074436A2 (en) Methods of use of a gpcr in the diagnosis and treatment of colon and lung cancer
US20030124543A1 (en) Breast cancer marker
US20030175754A1 (en) RVP-1 variant differentially expressed in crohns disease
US20040214990A1 (en) Transmembrane protein differentially expressed in cancer
US20030211515A1 (en) Novel compounds
US6632617B1 (en) Tumor-associated antigen
US20030165989A1 (en) GPCR diagnostic for brain cancer
US20030082653A1 (en) GPCR differentially expressed in squamous cell carcinoma
US20030129655A1 (en) Nucleic acids encoding GTPase activating proteins
US20030099995A1 (en) Ras association domain containing protein
US20030113317A1 (en) Molecules associated with apoptosis
WO2004033643A2 (en) Methods of use of a dna helicase in the diagnosis and treatment of colon and lung cancer
US20030036102A1 (en) Cathepsin differentially expressed in lung cancer
US20030054385A1 (en) Human ubiquitin-conjugating enzymes
WO2003061601A2 (en) Aminophospholipid transporter atpase
US20030104472A1 (en) Antibody specifically binding human pinch protein homolog
WO2004034974A2 (en) Anti-reg iϝ antibody for treatment of colon cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase