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WO2003061601A2 - Atpase d'un transporteur d'aminophospholipides - Google Patents

Atpase d'un transporteur d'aminophospholipides Download PDF

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Publication number
WO2003061601A2
WO2003061601A2 PCT/US2003/002162 US0302162W WO03061601A2 WO 2003061601 A2 WO2003061601 A2 WO 2003061601A2 US 0302162 W US0302162 W US 0302162W WO 03061601 A2 WO03061601 A2 WO 03061601A2
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protein
antibody
cdna
seq
molecules
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PCT/US2003/002162
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WO2003061601A3 (fr
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Amy K. W. Lasek
Kavitha Thangavelu
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Incyte Genomics, Inc.
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Priority to AU2003207674A priority Critical patent/AU2003207674A1/en
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Publication of WO2003061601A3 publication Critical patent/WO2003061601A3/fr

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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • This invention relates to a cDNA which encodes an aminophospholipid transporter ATPase and to the use of the cDNA and the encoded protein in the diagnosis and treatment of cancers, in particular a colon or lung cancer.
  • 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).
  • Colon cancer is causally related to both genes and the environment.
  • Several molecular pathways have been linked to the development and progression of colon cancer, and the expression of key genes in any of these pathways may be lost by inherited or acquired mutation or by hypermethylation.
  • Lung cancer is the leading cause of cancer death in the United States affecting more than
  • the P-type ATPases comprise a diverse family of integral membrane proteins which use the energy derived from ATP hydrolysis to drive uphill transport of ions and other polar substrates across cellular membranes.
  • a key feature of this family is the use an aspartyl phosphate as an intermediate in the enzymatic reaction that drives transport (Moller et al. (1996) Biochem et Biophysica Acta 1286: 1- 51).
  • a subfamily of these proteins has been identified that transport aminophospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC; Halleck et al. (1998) Genome Res 8:354-361; Tang et al. (1996) Science 272: 1495-1497).
  • aminophospholipid transporter (APLT) subfamily As many as 17 different genes in the aminophospholipid transporter (APLT) subfamily are expressed in mammals in a tissue-specific manner and may represent isoforms that transport a variety of amphipathic molecules (Halleck et al. (1999) Amer. Physiol. Soc, 1:139-150).
  • the structural and functional organization of the P-type ATPase transporters is well understood and consists of some 8-10 transmembrane domains with a large cytosolic domain between transmembrane domains 4 and 5 that contains the catalytic aspartate phosphorylation site (DKTGTLT), and 4-6 additional transmembrane domains C-terminal to this region (Moller, supra). Additional conserved sequence motifs are found throughout the protein that are characteristic of all classes of the P-type ATPase transporter family, as well as some which are potentially diagnostic of the APLT subfamily. Members of the APLT subfamily share about 30-35% amino acid seqeuence identity and 50-60% similarity to one another (Halleck (1998) supra).
  • the ATP10C genes encode a putative APLT that maps to a region in chromosome 15 associated with Angelman syndrome, a neurological condition associated with severe mental retardation, ataxia, and epilepsy.
  • ATP10C is adjacent to the UBE3A gene in this region, a gene in which mutation is known to cause the Angelman syndrome phenotype (Herzing et al. (2001) Am J Hum Genet 68: 1501-1505).
  • ATP10C also shows a similar maternal expression to UBE3A in human brain, suggesting that ATP10C may also contribute to the Angelman syndrome phenotype.
  • APLTs Another gene encoding an APLT, IFC1, also maps to a region of chromosome 18 associated with both recurrent and progressive familial intrahepatic cholestasis and is mutated in both forms of the disease (Bull et al. (1998) Nature Genetics 18:219-224).
  • APLTs play a critical role in the recognition of cells undergoing apoptosis.
  • the downregulation of APLT in lymphocytes and neutrophils undergoing apoptosis enhances the exposure of PS on the cell surface triggering apoptosis by macrophages (Verhoven et al. (1995) J Exp Med 182:1597-1601; Pradhan et al. (1997) Mol Biol Cell 8:767-778).
  • expression of APLTs may provide a controling step in apoptosis, an important pathway in preventing growth of abnormal cells such as cancers.
  • compositions which are useful in the diagnosis and treatment of cancer, particularly a colon and lung cancer.
  • the invention is based on the discovery of a cDNA encoding an aminophospholipid transporter ATPase, APTA, which is useful in the diagnosis and treatment of cancer, particularly a colon or lung cancer.
  • the invention provides an isolated cDNA comprising a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1.
  • the invention also provides an isolated cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:2, a fragment of SEQ ID NO:2 selected from SEQ ID NOs:3-14, and a variant of SEQ ID NO:2 selected from SEQ ID NOs: 17-18.
  • the invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding APTA.
  • the invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make Aminophospholipid transporter ATPase.
  • the invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding APTA.
  • the invention additionally provides a fragment, a variant, or the complement of the cDNA selected from the group consisting of SEQ ID NOs:3-14 and 17-18.
  • the invention provides a substrate containing at least one of these fragments or variants or the complements thereof.
  • the invention provides a probe comprising a cDNA or the complement thereof which can be used in methods of detection, screening, and purification, hi a further aspect, the probe is a single-stranded complementary RNA or DNA molecule.
  • the invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample 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 cDNA 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 cDNA is used to diagnose cancer, in particular, a colon or stomach cancer.
  • the cDNA or a fragment or a variant or the complements thereof may comprise an element on an array.
  • the invention additionally provides a method for using a cDNA or a fragment or a variant or the complements thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA , thereby identifying a ligand which specifically binds the cDNA.
  • the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
  • the invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO: 1, a variant having at least 85% identity to the amino acid sequence of SEQ ID NO: 1, an antigenic epitope of SEQ ID NO: 1, and a biologically active portion of SEQ ID NO: 1.
  • the invention also provides a composition comprising the purified protein and a pharmaceutical carrier.
  • the invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein.
  • the molecules or compounds are selected from DNA molecules
  • RNA molecules peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs.
  • the ligand is used to treat a subject with cancer, in particular, a colon or lung cancer.
  • the invention provides a method for using a protein to screen a plurality of antibodies to identify an antibody that specifically binds the protein comprising contacting a plurality of antibodies with the protein under conditions to form an antibody:protein complex, and dissociating the antibody from the antibody-.protein complex, thereby obtaining antibody that specifically binds the protein.
  • the antibodies are selected from intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a bispecific molecule, a multispecific molecule, 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.
  • the invention provides purified antibodies which bind specifically to a protein.
  • the invention also provides methods for using a protein to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein.
  • the method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies.
  • the method for preparing a monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating monoclonal antibodies from culture.
  • the invention further provides purified antibodies which bind specifically to a protein.
  • the invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions for formation of antibody:protein complexes; and detecting complex formation, wherein complex formation indicates expression of the protein in the sample.
  • the amount of complex formation when compared to a standard is diagnostic of a colon or lung cancer.
  • the invention provides a method for immunopurification of a protein comprising attaching an antibody to a substrate, exposing the antibody to a sample containing protein under conditions to allow antibody:protein complexes to form, dissociating the protein from the complex, and collecting purified protein.
  • the invention also provides a composition comprising an antibody that specifically binds the protein and a labeling moiety or pharmaceutical agent; a kit comprising the composition; an array element comprising the antibody; a substrate upon which the antibody is immobilized.
  • the invention further provides a method for using a antibody to assess efficacy of a molecule or compound, the method comprising treating a sample containing protein with a molecule or compound; contacting the protein in the sample with the antibody under conditions for complex formation; determining the amount of complex formation; and comparing the amount of complex formation in the treated sample with the amount of complex formation in an untreated sample, wherein a difference in complex formation indicates efficacy of the molecule or compound.
  • 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, a bispecific molecule that specifically binds the protein, a multispecific molecule 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 cell comprising attaching the pharmaceutical or therapeutic agent to a bispecific molecule that specifically binds the protein and administering the bispecific molecule to a subject in need of therapeutic intervention, wherein the bispecific molecule delivers the pharmaceutical or therapeutic agent to the cell.
  • the invention provides a method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention pharmaceutical agent or a small drug molecule that specifically binds the protein.
  • the invention provides an antisense molecule of 18 to 30 nucleotides in length that specifically binds a portion of a polynucleotide having a nucleic acid sequence of SEQ ID NO:2 or the complement thereof wherein the antisense molecule inhibits expression of the protein encoded by the polynucleotide.
  • the invention also provides an antisense molecule with at least one modified internucleoside linkage or at least one nucleotide analog.
  • the invention further provides that the modified internucleoside linkage is a phosphorothioate linkage and that the modified nucleobase is a 5-methylcytosine.
  • the invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide.
  • the invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:3-14 and 17-18, 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 cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.
  • FIGS. IA through 1O show the aminophospholipid transporter ATPase (APTA;SEQ ID NO: 1) encoded by the cDNA (SEQ ID NO:2). The alignment was produced using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA). Figures 2A through 21 show the amino acid sequence alignments between APTA (SEQ ID NO: 1)
  • aminophospholipid transporter ATPase from mouse (g6457270: SEQ ID NO: 19), and man (gl3878299; SEQ ID NO:20).
  • the alignment was produced using the MEGALIGN program of LASERGENE software (DNASTAR, Madison WI).
  • Figure 3 shows the expression of APTA in various normal adult tissues.
  • the X-axis indicates the tissue type, and the Y-axis the expression of APTA relative to that found in normal colon tissue arbitrarily set at 100%.
  • the analysis was performed by QPCR using the TAQMAN protocol (Applied Biosystems (ABI), Foster City CA)
  • Figure 4 shows the differential expression of APTA in tissues from patients with colon cancer relative to normal colon tissue.
  • the X-axis indicates the patient ID (Donor ID), and the Y-axis the expression of APTA relative to that found in normal colon tissue set at 100%. Tumor samples are displayed in black, and normal tissue in white. The analysis was performed by QPCR using the TAQMAN protocol(ABI).
  • Figure 5 shows the differential expression of APTA in various colon tumor cell lines relative to that found in normal colon tissue set at 100%. The analysis was performed using the TAQMAN protocol (Applied Biosystems).
  • Figure 6 shows the differential expression of APTA in an additional set of samples from colon tumor patients relative to normal colon. The analysis was performed by QPCR using the TAQMAN protocol(ABI).
  • Figure 7 shows the differential expression of APTA in samples from lung tumor patients relative to normal lung tissue from the same donors.
  • the analysis was performed by QPCR using the TAQMAN protocol(ABI).
  • the data was normalized to a pool of normal colon tissue.
  • Table 1 shows the transcript image for APTA at the tissue level produced using the LTFESEQ Gold database (Incyte Genomics, Palo Alto CA).
  • the first column presents the tissue categories; the second column, the number of clones in the tissue category; the third column, the number of libraries in which at least one transcript was found relative to the total number of libraries in that category; the fourth column, the absolute abundance of the transcript (number of transcripts); and the fifth column, percent abundance of the transcript.
  • Table 2 shows the transcript image for APTA in colon tissues of the digestive system category.
  • the first column shows the Library identification, the second column, the library description, the third column the absolute abundance (number of transcripts/library), and the fourth column, the percent abundance of the transcript.
  • Table 3 shows the differential expression of APTA in tissues from patients with colon or lung cancer relative to normal colon or lung tissue, respectively, as determined by microarray analysis.
  • the first column lists the differential expression (DE) between the tumor sample and normal tissue. The results are expressed in terms of the ratio of tumor/normal expression.
  • Column 2 (PI)
  • tissue and patient donor lists the tissue and patient donor (Dn) for microscopically normal samples labeled with fluorescent green dye Cy3.
  • Column 3 lists the tissue and patient donor (Dn) for diseased samples (colon tumor or colon polyps, lung tumor) labeled with fluorescent red dye Cy5.
  • Aminophospholipid transporter ATPase refers to a purified protein obtained from any mammalian species, including bovine, canine, murine, ovine, porcine, rodent, simian, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • 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 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 which specifically binds the protein. Biological activity is not a prerequisite for immunogenicity.
  • Array refers to an ordered arrangement of at least two cDNAs, proteins, or antibodies on a substrate. At least one of the cDNAs, proteins, or antibodies represents a control or standard, and the other cDNA, protein, or antibody of diagnostic or therapeutic interest.
  • the arrangement of at least two and up to about 40,000 cDNAs, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each cDNA 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.
  • the "complement" of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to the cDNA or an mRNA under conditions of high stringency.
  • cDNA refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, represents coding and noncoding 3 ' or 5' sequence, and lacks introns.
  • cDNA encoding a protein refers to a nucleotide sequence that closely aligns with sequences which encode conserved regions, motifs or domains that were identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool) which provides identity within the conserved region (Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410).
  • BLAST Basic Local Alignment Search Tool
  • 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 APTA or fragments of APTA 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.
  • 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; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Derivatization of a cDNA 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 protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group.
  • Derivative molecules retain the biological activities o the naturally occurring molecules but may confer advantages such as 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 the cDNAs and APTA are differentially expressed. Such a disorder includes cancer, including, 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 technologies and mRNAs or cDNAs from a sample.
  • a protein expression profile mirrors the nucleic acid expression profile and uses labeling moieties or antibodies to quantify the 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.
  • Fragments refers to a chain of consecutive nucleotides from about 50 to about 4000 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.
  • a "hybridization complex” is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines ofthe complementary molecule, e.g., 5 -A-G-T-C-3' base pairs with 3'-T-C-A-G-5'.
  • Hybridization conditions, degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
  • Identity 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 et al. (1997) Nucleic Acids Res 25:3389-3402). 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 is an analogous score, but it is calculated with conservative substitutions taken into account; for example, substitution of a valine for a isoleucine or leucine.
  • Labeling moiety refers to any reporter molecule whether a visible or radioactive label, stain or dye than can be attached to or incorporated into a cDNA or protein.
  • Visible labels and dyes include but are not limited to anthocyanins, ⁇ glucuronidase,BIODIPY, Coomassie blue, Cy3 and Cy5, digoxigenin, FTTC, green fluorescent protein (GFP), luciferase, spyro red, silver, 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.
  • 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.
  • 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 amplimer, primer, and oligomer.
  • 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.
  • lipidation 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 a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single-stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.
  • 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 epitope of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison WI).
  • sample refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated.
  • sample is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like.
  • a sample may comprise a bodily fluid; 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 print; a fingerprint, buccal cells, skin, or hair; and the like.
  • Similarity 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) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402).
  • 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 is greater than identity in that conservative substitutions, for example, valine for leucine or isoleucine, are counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
  • Specific binding refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
  • Substrate refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
  • a "transcript image” is a profile of gene transcription activity in a particular tissue at a particular time. Tl 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.
  • “Variant” refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs 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. THE INVENTION
  • the invention is based on the discovery of a cDNA which encodes an aminophospholipid transporter ATPase and on the use of a cDNA, or fragments thereof; a protein, or portions thereof; or an antibody which specifically binds ATPA, or fragments thereof, directly or as compositions in the characterization, diagnosis, prognosis, treatment and evaluation of treatment of a colon or lung cancer.
  • Nucleic acids encoding the aminophospholipid transporter ATPase of the present invention were first identified in Incyte Clone 4028269H1 from a brain library (BRAINOT23) using a computer search for nucleotide and/or amino acid sequence alignments.
  • SEQ ID NO:2 was derived from the following overlapping and/or extended nucleic acid sequences (SEQ ID NO:3-14) and their associated cDNA libraries: Incyte Clones 7362215F6 (BRAJFEE05), 7313608F8 (BRABDIE02), 6772907Jland 6772907H1 (BRAUNOR01), 7032970H1 (BRAXTDR12), 5459667H1, 7582660H1 (BRAIFEC02), 5767060H1 (STOMFET02), 2182261F6 (SIMNTTOl), 72751861V1, 4028269H1 (BRAINOT23), 72794560V1, genomic sequence g7710567_000006_002 (SEQ ID NO: 15) and GenBank sequence gl 1328908 (SEQ ID NO: 16).
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as shown in Figures IA through 1O.
  • APTA is 1461 amino acids in length and has ten N-glycosylation sites at amino acid residues N41, N51, N69, N148, N298, N339, N354, N933, N1191, and N1273.
  • APTA contains three potential cyclic AMP dependent protein kinase phosphorylation sites at T449, S774, and S1451, thirty potential casein kinase H phosphorylation sites at S223, S307, T341, T437, S456, T466, S472, S538, T563, S579, T597, T616, S643, T658, S670, S674, S683, T697, S698, S721, S845, T887, T903, S1052, S1073, T1140, S1198, S1220, T,373, and S1422, twenty-one potential protein kinase C phosphorylation sites at S63, T73, T275, S432, T466, S486, T495, S498, S510, T606, S610, T747, S768, T822, S854, T903, S928, S1348, T1381, S1447, and S1451, and one potential tyrosine kinase phosphorylation sites
  • APTA contains an ATP/GTP binding site motif at G995 to T1002, and the aspartyl phosphorylation site consensus sequence at D 433 KTGTLT.
  • APTA also contains several other sequence motifs identified by Halleck (1998, supra) as both common to all classes to P-type ATPases, and particular to the APLT subfamily (see Fig. 3 at p. 357 of Halleck (1998) supra). These are the sequences E 203 TASLDGETT, S 721 PDEAALV, D 89 ⁇ RLQEGVPDTI, W 912 VLTGDK, and I 1053 GDGANDVSMI, identified by Halleck (1998, supra) as common to all classes of P-type ATPases.
  • APTA also contains a conservative variant of the sequence "CRALNITE" identified by Halleck (1998, supra) as diagnostic ofthe APLT subfamily at C ⁇ RALNIAE in which a conservative amino acid substitution of alanine for threonine is made at amino acid residue 421 of APTA.
  • HMMR analysis indicates the presence of nine transmembrane domains also characteristic of the P-type ATPases as follows: TM-1, amino acid residues 90-107; TM-2, amino acid residues 112-131; TM-3, amino acid residues 317-339; TM-4, amino acid residues 365-387; TM-5, amino acid residues 1114-1133; TM-6, amino acid residues 1143-1165; TM-7, amino acid residues 1195-1217; TM-8, amino acid residues 1250-1272; and TM-9, amino acid residues 1293-1315.
  • the nine TM regions in APTA are designated in Figures 2A through 21.
  • Figures 2C through 2G show that the large cytosolic domain containing the catalytic aspartate phosphorylation site is present in APTA between TM regions 4 and 5.
  • a useful antigenic determinant of SEQ ID NO:l extends from about amino acid residue G340 to about amino acid residue G364 of SEQ ID NO: 1.
  • G340 through G364 represents a unique portion of APTA that is located in the extracellular domain of the molecule between TM-3 and TM-4 (See Fig. 2B and 2C), and would be a useful antibody target for APTA.
  • An antibody which specifically binds APTA is also useful in a diagnostic assay to identify a cancer, in particular, a colon or lung cancer.
  • a fragment of the cDNA of SEQ NO:2 from about nucleotide 1398 to about nucleotide 1476, which encodes the antigenic determinant described above, is also useful in diagnostic assays to detect SEQ ID NO:2.
  • APTA (SEQ ID NO:l) has chemical and structural similarity with both murine and human APLT, g6457270; SEQ ID NO: 19, and gl3878299; SEQ ID NO:20, respectively, hi particular, APTA shares 45% overall identity with either of the two APLTs, which is well within the range of sequence similarity for known members of this family of proteins (see Halleck (1998) supra). In addition, all three proteins share the five sequence motifs found in APTA and identified by Halleck (1998,supra) as common to all classes of P-type ATPases, as well as the sequence "CRALNITE" discussed in the paragraph above.
  • Figure 3 shows the results of various normal adult tissues analyzed for APTA expression by TAQMAN analysis (ABI). The most significant expression of APTA was found in colon and brain indicating that APTA has a relatively restricted normal tissue distribution.
  • Table 1 shows the expression of the APTA across tissue categories, a transcript image of cDNA libraries in the LIFESEQ Gold database (Incyte Genomics). The results are consistent with the analysis in Figure 3, above, in that the highest abundance of transcripts encoding APTA are found in the categories of digestive system (primarily colon) and the nervous system (primarily brain). The differences observed between the results of Table 1 and Figure 3 most likely reflect the high incidence of fetal and diseased tissues in cDNA libraries ofthe LIFESEQ Gold database.
  • Table 2 also shows a transcript image of APTA in colon tissue libraries ofthe digestive system. 14 of the 19 libraries in the digestive system category are from colon tissue, and 9 of these 14 libraries are derived from cancerous colon tissue or colon polyps.
  • Figure 4 shows the expression of APTA in colon cancer tissue samples as compared with normal colon tissue using TAQMAN analysis (ABI). The results show an increased expression of APTA in colon tumors in 5 out of 9 samples examined. The results were considered significant if at least a 1.2-fold difference in expression between cancerous and normal tissue was observed.
  • Figure 5 similarly shows the expression of APTA in various human colon tumor cell lines when compared to normal colon tissue by TAQMAN analysis. APTA is overexpressed nearly 2.5 fold in HT29 cells relative to normal colon tissue.
  • Figure 6 shows the differential expression of APTA in an additional set of samples from colon cancer patients using TAQMAN analysis. The results show increased expression in all 6 of the colon tumor samples relative to normal colon.
  • Figure 7 shows the differential expression of APTA in lung tumor samples relative to normal lung using TAQMAN analysis.
  • the results show increased expression in 5 of 15 tumor samples examined relative to matched normal lung tissue (donor IDs 7173, 9752, 9753, 9754, and 9764).
  • Differential expression was considered significant if at least a 1.5-fold difference in expression between cancerous and normal tissue was observed and a cycle threshold (C ⁇ ) value of no greater than 31 was observed for the tumor sample determination (see QPCR Analysis, Example VUI).
  • Table 3 shows the results of microarray analysis comparing the expression of APTA in colon and lung tumor tissues relative to normal tissue from the same donor. The results confirm the overexpression of APTA in colon and lung tumors observed by TAQMAN analysis ( Figures 4 and 7, respectively). Differential expression (column 1) was considered significant if at least a 1.5-fold difference in expression between cancerous and normal tissue was observed.
  • Mammalian variants ofthe cDNA encoding APTA were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics). These preferred variants have from about 85% to 89% identity as shown in the table below.
  • the first colum represents the SEQ ID NO: for variant cDNAs; the second column, the clone number for the variant cDNAs; the third column, the species; the fourth column, the percent identity to the human cDNA; and the fifth column, the alignment ofthe variant cDNA to the human cDNA.
  • the cDNAs of SEQ DD NOs:3-14 and 17-18 may be used in hybridization, amplification, and screening technologies to identify and distinguish among SEQ ID NO:2 and related molecules in a sample.
  • the mammalian cDNAs, SEQ ID NOs: 17-18 may be used to produce transgenic cell lines or organisms which are model systems for human colon or lung cancer and upon which the toxicity and efficacy of potential therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the cDNAs, proteins, antibodies and molecules and compounds identified using the cDNAs and proteins of the present invention.
  • mRNA 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 aprepared as described in the EXAMPLES.
  • the consensus sequences are chemically and/or electronically assembled from fragments including Incyte cDNAs and extension and/or shotgun sequences using computer programs such as PHRAP (P Green, University of Washington, Seattle WA), and AUTOASSEMBLER application (Applied Biosystems, Foster City CA). After verification of the 5' and 3' sequence, at least one representative cDNA which encodes APTA is designated a reagent. Sequencing
  • 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).
  • Machines commonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNA sequencing systems (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (APB), and the like.
  • the sequences may be analyzed using a variety of algorithms well known in the art and 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 cDNAs 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, or deleted sequences can be removed or restored, respectively, organizing the incomplete assembled sequences into 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 Applied Biosystems
  • nested primers and commercially available cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence.
  • primers may be designed using commercially available 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
  • a sequence to recover regulatory elements it is preferable to use genomic, rather than cDNA libraries.
  • 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 APTA, 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:2-10 and 12-16.
  • 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 cDNA 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 commercially available kits such as those provided by APB.
  • the stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature, hi 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.
  • 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
  • formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis MO) 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 cDNAs 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., Brennan et al. (1995) 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; and Heller et al. (1997) 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 (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial PI constructions, or the cDNAs of libraries made from single chromosomes.
  • Quantitative real-time PCR 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 ABI PRISM 7700 detection system 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 "TAQMAN" probe.
  • ABI PRISM 7700 detection system which consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics 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 ⁇ s, preparing a standard curve, and determining starting copy number is performed by the SEQUENCE DETECTOR 1.7 software (ABI). Expression
  • Any one of a multitude of cDNAs encoding APTA 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, cDNA, 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; plant cell systems transformed with expression vectors containing viral and/or bacterial elements, or animal cell systems (Ausubel supra, unit 16).
  • 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 insect cell systems transformed with baculovirus expression vectors
  • plant cell systems transformed with expression vectors containing viral and/or bacterial elements or animal cell systems (Ausubel supra, unit 16).
  • an adenovirus transcription/translation complex may be utilized in mammalian cells. 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
  • 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 PSPORT1 plasmid (Life Technologies). 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 cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification techniques.
  • 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 available from the ATCC (Manassas VA) 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.
  • Manassas VA Manassas VA
  • 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 commercially available affinity matrices such as immobilized glutathione and metal-chelate resins, respectively.
  • FLAG and MYC are purified using commercially available 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) and are commercially available. Protein Identification
  • Proteins are separated by 2DE employing isoelectric focusing (JEF) in the first dimension followed by SDS-PAGE in the second dimension.
  • JEF isoelectric focusing
  • an immobilzed 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
  • Individual spots of interest 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 mass spectrometer of the MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time of Flight ), ESI (Electrospray lonization), and TOF-TOF (Time of Flight/Time of Flight) machines are used to determine a set of highly accurate peptide masses.
  • 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 usually required 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 (CTD) to produce an ion series.
  • CCD 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 Co put 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 cc-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 main 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 ofthe 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
  • mice 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, St. Louis MO), and dinitrophenol may be used to increase immunological response, humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum are preferable.
  • the antigenic determinant may be an oligopeptide, peptide, or protein.
  • 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)
  • 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 88: 10134-10137).
  • 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 ofthe antibody molecule and Fab fragments generated by reducing the disulfide bridges ofthe 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 preferred for use 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 ofthe protein, preferably in active form, from the antibody (Catty (1988) Antibodies, Volume I: A Practical
  • 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 widely available (Catty (supra): Ausubel (supra) pp. 11.1-11.31).
  • 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 m 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 APTA, on transformation of the cell to a neoplastic state.
  • Immunological Assays can be used, for example, to assess the effect of transfecting a cell with a gene such as APTA, on transformation of the cell to a neoplastic state.
  • 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 enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. 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.
  • DIAGNOSTICS Differential expression of APTA, as detected using APTA, cDNA encoding APTA, or an antibody that specifically binds APTA, and at least one of the assays below can be used to diagnose a colon or lung cancer. Labeling of Molecules for Assay
  • 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 commercially available kits (Promega, Madison WI) 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-metnionine (APB).
  • APB 32 P-dCTP
  • Cy3-dCTP Cy3-dCTP
  • Cy5-dCTP Qiagen Operon, Alameda CA
  • amino acid such as 35 S-metnionine (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, Eugene OR). Nucleic Acid Assays
  • the cDNAs, fragments, oligonucleotides, complementary RNA and DNA molecules, and PNAs may be used to detect and quantify differential gene expression for diagnosis of a disorder.
  • antibodies which specifically bind APTA may be used to quantitate the protein.
  • Disorders associated with differential expression include cancer, in particular, a colon or 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.
  • the cDNA 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.
  • 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 significantly altered (higher or lower) 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 cDNA 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 that disorder.
  • 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.
  • Detection and quantification of a protein using either labeled amino acids or specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include two- dimensional polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). These assays and their quantitation against purifed, labeled standards are well known in the art (Ausubel, supra, unit 10.1- 10.6).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • APTA SEQ ID NO:l
  • APTA differential expression of APTA is highly associated with a colon or lung cancer as shown in Table 3 and Figures 4, 6 and 7.
  • APTA clearly plays a role in cancer, in particular, colon or lung cancer.
  • an inhibitor, antagonist, antibody and the like or a pharmaceutical agent containing one or more of these molecules may be delivered. Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the protein. Neutralizing antibodies which inhibit dimer formation are generally preferred for therapeutic use.
  • the protein, an agonist, an enhancer and the like or a pharmaceutical agent containing one or more of these molecules may be delivered.
  • 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 cDNAs, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, and their ligands 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 APTA. 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. In: Huber and Carr (1994) Molecular and Jmmunologic 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 cDNAs 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 ofthe 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.
  • RNA Interference RNA interference also known as double-stranded RNA (dsRNA)-induced gene silencing, is a method of interfering with the transcription of specific mRNAs through the production of small RNAs (siRNAs) and short hairpin RNAs (shRNAs).
  • RNAs are naturally formed in a multicomponent nuclease complex (RISC) in the presence of an RNAse III family nuclease (Dicer), and they serve as a guide to identify and destroy complementary transcripts.
  • RISC multicomponent nuclease complex
  • Dicer RNAse III family nuclease
  • Transient infection of cells with RNAs capable of interference can bypass the need for Dicer and result in silencing of a gene for the lifespan of the introduced RNAs, usually from about 2 to about 7 days. See Paddison and Hannon (2002) Cancer Cell 2:17-23.
  • RNAi pathway is believed to have evolved in early eukaryotes as a cell-based immunity against viral and genetic parasites. It is considered, however, to have great potential as a method of identifying gene function particularly in diseases such as cancer, as well as providing a highly specific means for nucleic acid-based therapies for cancer and other disorders.
  • cDNA Therapeutics are believed to have evolved in early eukaryotes as a cell-based immunity against viral and genetic parasites. It is considered, however, to have great potential as a method of identifying gene function particularly in diseases such as cancer, as well as providing a highly specific means for nucleic acid-based therapies for cancer and other disorders.
  • 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; Verbs 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). Screening and Purification Assays
  • the cDNA encoding APTA may be used to screen a library or a plurality of molecules or compounds for specific binding affinity.
  • the libraries may be DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, or repressors, 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 single-stranded or double-stranded molecule.
  • the cDNA 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 cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA 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 cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art.
  • the cDNA 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 cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA 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 or a portion thereof to purify a ligand would involve combining the protein or a portion thereof 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.
  • APTA 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 their 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 DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs or any other ligand, which specifically binds the protein.
  • this invention comtemplates 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, diagnostic, 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, antibodies specifically binding the protein, antagonists, inhibitors, or mimetics of the protein.
  • 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.
  • sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol
  • compositions may be administered by any number of routes including oral, intravenous, intramuscular, intra-arterial, intrameduUary, 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. Toxicity and Therapeutic Efficacy
  • 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 and generally available to practitioners. 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, reproductive potential, 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 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.
  • 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.
  • 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.) i 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 (ES) stem cells isolated from rodent embryos retain the potential 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.
  • telomeres 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 In 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.
  • 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.
  • 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 potential 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.
  • the cDNAs 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 cDNAs that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • BRATNOT23 The BRATNOT23 library was constructed using RNA isolated from right temporal lobe tissue removed from a 45-year-old Black male during a brain lobectomy. Pathology for the matched tumor tissue indicated dysembryoplastic neuroepithelial tumor of the right temporal lobe.
  • the frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a POLYTRON homogenizer (Brinkmann Instruments, Westbury NJ).
  • the lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in an L8-70M ultracentrifuge (Beckman Coulter, Fullerton CA) for 18 hours at 25,000 rpm at ambient temperature.
  • the RNA was extracted with acid phenol, pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse- free water, and DNAse treated at 37 °C. Extraction with acid phenol, pH 4.7, and precipitation with sodium acetate and ethanol was repeated.
  • the mRNA was isolated with the OLIGOTEX kit (Qiagen, Chatsworth CA) and used to construct the cDNA library.
  • the mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies) which contains a Notl primer-adaptor designed to prime the first strand cDNA synthesis at the poly(A) tail of mRNAs. Double stranded cDNA was blunted, ligated to EcoRI adaptors and digested with Notl (New England Biolabs, Beverly MA). The cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics). The plasmid pINCY was subsequently transformed into DH5 ⁇ competent cells (Life Technologies).
  • the plasmid was constructed by digesting the pSPORTl plasmid (Life Technologies) with EcoRI restriction enzyme (New England Biolabs, Beverly MA) and filling the overhanging ends using Klenow enzyme (New England Biolabs) and 2'-deoxynucleotide 5'-triphosphates (dNTPs). The plasmid was self-ligated and transformed into the bacterial host, E. coli strain JM109.
  • a kit consists of a 96- well block with reagents for 960 purifications. The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks MD) with carbenicillin at 25 mg/1 and glycerol at 0.4%; 2) after inoculation, the cells were cultured for 19 hours and then lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4C.
  • the cDNAs were prepared for sequencing using the MICROLAB 2200 system (Hamilton) in combination with the DNA ENGINE thermal cyclers (MJ Research).
  • the cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM 377 sequencing system (Applied Biosystems) or the MEGABACE 1000 DNA sequencing system (APB). Most of the isolates were sequenced according to standard ABI protocols and kits (Applied Biosystems) with solution volumes of 0.25x-1.0x concentrations. In the alternative, cDNAs were sequenced using solutions and dyes from APB. IV Extension of cDNA Sequences
  • 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 ofthe known fragment.
  • the initial primers were designed using commercially available 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 more than one extension was necessary, 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 are used to obtain regulatory elements, especially extension into the 5' promoter binding region.
  • Step 1 94C, three min
  • Step 2 94C, 15 sec
  • Step 3 57C, one min
  • Step 4 68C, two min
  • Step 5 Steps 2, 3, and 4 repeated 20 times
  • Step 6 68C, five min
  • Step 7 storage at
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ l 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, 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 ofthe 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: Step 1: 94C, three min; Step 2: 94C, 15 sec; Step 3: 60C, one min; Step 4: 72C, two min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72C, five min; Step 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.
  • the cDNAs ofthe 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 "14 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; http://www.ncbi.nlm.nih.gov/gorf/bl2.html), 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.
  • cDNAs of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics).
  • Component sequences from cDNA, 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 dete ⁇ nined 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; http://pfam.wustl.edu/).
  • the cDNA 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.
  • a transcript image was performed using the LIFESEQ GOLD database (Sep2001 release, Incyte Genomics). This process allowed 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 were categorized by system, organ/tissue and cell type.
  • All sequences and cDNA libraries in the LIFESEQ database have been categorized by system, organ tissue and cell type. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category. For each , library, the number of cDNAs were counted and shown over the total number of cDNAs in that library.
  • all normalized or subtracted libraries which have high copy number sequences 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 guilt-by-association and hybridization analyses. The results of this analysis are presented in Tables 1 and 2.
  • 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 cDNAs presented in the Sequence Listing have been mapped. Any of the fragments of the cDNA encoding APTA 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.
  • 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.
  • Normal colon tissue used as a 100% control in the comparisons in Figures 3, 4, 5, and 6 was a pool of normal colon tissue from 3 donors.
  • the following colorectal adenocarcinoma cell lines were obtained from The American Type Culture Collection (ATCC, Manassas VA) and were cultured according to the suppliers specifications; LS 123, LS 174, HCTl 16, CaCo2, HT29, SW480, Colo205, and SW620.
  • the last two cell lines are metastases of colon carcinoma derived from ascitic fluid and lymph node tissue, respectively. Immobilization of cDNAs on a Substrate
  • the cDNAs are applied to a substrate by one of the following methods.
  • a mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer.
  • the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library.
  • the cDNAs are then arranged on a substrate by one of the following methods.
  • 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 ( STRATALINKER UV-crosslinker (Stratagene).
  • cDNAs 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.
  • 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, Acton MA) 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 STRATALINKER 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.
  • Probes for the QPCR were prepared according to the ABI protocol. Probe Preparation for Membrane Hybridization Hybridization probes derived from the cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs 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 cDNA is then added to a REDffRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged.
  • ABI protocol Probe Preparation for Membrane Hybridization
  • Probe Preparation for Polymer Coated Slide Hybridization Five ⁇ l of [ 32 P]dCTP is added to the tube, and the contents are incubated at 37C for 10 min. The labeling reaction 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. Probe Preparation for Polymer Coated Slide Hybridization
  • Hybridization probes derived from mRNA isolated from samples are employed for screening cDNAs 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 ⁇ 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.
  • Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W.
  • control mRNAs 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.
  • 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 SPDSf+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.
  • Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl, 0.1 M Na 2 HPO 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 lmM Tris (pH 8.0).
  • XOMAT-AR film Eastman Kodak, Rochester NY is exposed to the membrane overnight at -70C, developed, and examined visually.
  • 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 IxSSC, 0.1% SDS, and three times for 10 min each at 45C in 0. IxSSC, and dried.
  • Hybridization reactions are performed in absolute or differential hybridization formats.
  • 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.
  • 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
  • A/D conversion board Analog Devices, Norwood MA
  • 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 BL). 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.
  • cDNA was obtained from Human MTC panels (Clontech).
  • PCR primers and probes (5' 6-FAM-labeled, 3' TAMRA) were designed using ABI Primer Express 1.5 software (ABI) and synthesized by Biosearch Technologies (Novato CA) or ABI.
  • QPCR reactions were performed using an ABI PRISM 7700 sequencing system (ABI) in 25 ul total volume with 5 ul cDNA template, lx TAQMAN UNIVERSAL 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.
  • ABI PRISM 7700 sequencing system (ABI) in 25 ul total volume with 5 ul cDNA template, lx TAQMAN UNIVERSAL PCR master mix (ABI), 100 nM each PCR primer, 200 nM probe, and lx VIC-labeled beta-2-microglobulin endogenous control (ABI).
  • Antisense molecules complementary to the cDNA from about 5 (PNA) to about 5000 bp (complement of a cDNA insert), are used to detect or inhibit gene expression. Detection is described in Example VH.
  • the complementary molecule is designed to bind to the most unique 5' sequence and includes nucleotides of the 5' UTR upstream ofthe 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.
  • the pUB6/V5-His vector system (Invitrogen, Carlsbad CA) is used to express APTA in CHO cells.
  • the vector contains the selectable bsd gene, multiple cloning sites, the promoter/enhancer sequence from the human ubiquitin C gene, a C- terminal V5 epitope for antibody detection with anti-V5 antibodies, and a C-terminal polyhistidine (6xHis) sequence for rapid purification on PROBOND resin (Invitrogen). Transformed cells are selected on media containing blasticidin.
  • Spodoptera frugiperda (Sf9) insect cells are infected with recombinant Autographica californica nuclear polyhedrosis virus (baculovirus).
  • the polyhedrin gene is replaced with the cDNA by homologous recombination and the polyhedrin promoter drives cDNA transcription.
  • the protein is synthesized as a fusion protein with 6xhis which enables purification as described above. Purified protein is used in the following activity and to make 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.
  • the amino acid sequence of the protein is analyzed using readily available commercial software 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 ABI 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.
  • 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 cDNA, 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, Eugene OR), respectively.
  • Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled cDNA 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, Palo Alto CA), is used to screen for peptides that bind the protein of the invention.
  • a cDNA 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 cDNA 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.

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Abstract

L'invention concerne un ADNc codant une ATPase d'un transporteur d'aminophospholipides. Cette invention a également trait à l'utilisation d'ADNc, de fragments, de compléments et de variants de la protéine codée et correspondants, des portions associées et des anticorps associés, dans le cadre du diagnostic et du traitement de troubles, notamment le cancer du côlon et le cancer des poumons. Ladite invention a aussi pour objet des vecteurs d'expression et des cellules hôtes destinés la production de la protéine et d'un système de modèle transgénique.
PCT/US2003/002162 2002-01-22 2003-01-21 Atpase d'un transporteur d'aminophospholipides WO2003061601A2 (fr)

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AU2003207674A AU2003207674A1 (en) 2002-01-22 2003-01-21 Aminophospholipid transporter atpase

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US35125402P 2002-01-22 2002-01-22
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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENEMBL [Online] August 2001 DOE JOINT GENOME INSTITUTE AND STANDORD HUMAN GENOME CENTER Database accession no. AC008456 *
DATABASE GENEMBL [Online] Database accession no. AB018258 *

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