[go: up one dir, main page]

WO2005076012A2 - Nouveaux variants d'epissure et methodes d'utilisation associees - Google Patents

Nouveaux variants d'epissure et methodes d'utilisation associees Download PDF

Info

Publication number
WO2005076012A2
WO2005076012A2 PCT/US2005/002695 US2005002695W WO2005076012A2 WO 2005076012 A2 WO2005076012 A2 WO 2005076012A2 US 2005002695 W US2005002695 W US 2005002695W WO 2005076012 A2 WO2005076012 A2 WO 2005076012A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cell
polypeptide
variant
modulator
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2005/002695
Other languages
English (en)
Other versions
WO2005076012A3 (fr
Inventor
Keting Chu
Amy L. Tsui Collins
Ernestine Lee
Justin G.P. Wong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Five Prime Therapeutics Inc
Original Assignee
Five Prime Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Five Prime Therapeutics Inc filed Critical Five Prime Therapeutics Inc
Publication of WO2005076012A2 publication Critical patent/WO2005076012A2/fr
Publication of WO2005076012A3 publication Critical patent/WO2005076012A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • This invention relates to human polynucleotides and their encoded polypeptides, which are highly expressed in cancer tissues, including adenocarcinomas and squamous cell carcinomas, such as lung cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, colon cancer, kidney cancer, and pancreatic cancer.
  • the invention also relates to modulators of such polynucleotides and polypeptides (for example, antibodies) that specifically bind to or interfere with the activity of these polypeptides, polynucleotides, their fragments, variants, and antagonists.
  • the invention further relates to compositions containing such polypeptides, polynucleotides, or modulators thereof and uses of such compositions in methods of treating immune and proliferative disorders, including cancer and psoriasis, as well as inflammations, infections, and infectious diseases.
  • the invention additionally relates to methods of diagnosing immune disorders and proliferative disorders, such as cancer, by detecting these polynucleotides, polypeptides, or antibodies thereto in patient samples.
  • the invention provides diagnostic tests that identify the polypeptides and polynucleotides herein, which correlate with particular disorders.
  • Detection of cancer cell-specific biomarkers provides an effective screening strategy. Their early detection provides not only early diagnosis, but also the ability to screen for and detect post-operative residual tumor cells, and for occult metastases, an early indicator of tumor recurrence. Early detection could thus improve survival in patients by facilitating early diagnosis, and also patients undergoing treatment and while in remission.
  • Such diseases include proliferative diseases such as cancer, psoriasis, immune-regulated diseases such as autoimmune diseases, and inflammatory disorders such as, for example, multiple myeloma, rheumatoid arthritis, and inflammatory bowel disease.
  • the diagnostic and therapeutic agents ofthe present invention thus include polypeptides, polynucleotides, and modulators of such that are either agonists or antagonists, for example, antibody modulators.
  • the antibody modulators specifically bind to the targets or to ligands or receptors of such targets and may modulate the biological effect ofthe target.
  • the modulators include not only antibodies, but also small molecule drugs, RNAi molecules, ribozymes, anti-sense molecules, aptamers, and soluble receptors or extracellular fragments of receptors or transmembrane proteins.
  • the polypeptides and polynucleotides ofthe present invention are characterized in that they are highly expressed in tumor tissues in comparison with their expression levels in normal tissue.
  • Targets ofthe invention include splice variants. These splice variants may, in some instances, antagonize the activity ofthe wild type. Targets ofthe invention may possess or may lack Pfam domains present in the wild type.
  • the target molecule may antagonize the activity ofthe wild type.
  • the targets ofthe present invention are useful in screening assays for screening for modulators as above that have the desired agonist or antagonist effect.
  • Methods ofthe invention include methods for determining the presence of and the interactions of polynucleotides, polypeptides, and modulators ofthe invention. Methods ofthe invention include using the modulators to modulate biological activity.
  • the polypeptides of the present nvention are transmembrane or secreted proteins, or fragments thereof, that are particularly suitable as targets for the production of modulators.
  • the antibody modulators ofthe invention can bind to transmembrane proteins on cell surfaces, such as a tumor cell surface, to induce an antibody dependent cellular cytotoxicity (ADCC) response or a cell dependent cytotoxicity (CDC) response. They can inhibit functional signaling or target delivery of cytotoxic molecules.
  • the antibody modulators and aptamers herein can also bind secreted proteins or interfere with the binding of secreted proteins to inhibit their activity.
  • the small molecule modulators and the soluble receptors or extracellular fragments of transmembrane proteins can block ligand/receptor or enzyme/substrate interaction and interfere with cell signaling.
  • the RNAi molecules, anti-sense molecules, and ribozymes can block expression ofthe target polypeptides.
  • Certain targets described herein are particularly desirable for production of modulators, such as antibodies, because ofthe low level of expression of such polypeptides in normal tissues, such as in normal lung, heart, kidney, and/or liver.
  • Certain of the polypeptides herein can induce apoptosis, and that therapeutic compositions comprising such polypeptides can be used for treatment in which induction of apoptosis is desirable, such as in cancer.
  • the present invention also provides methods for treatment of the above- mentioned diseases using compositions comprising the compounds ofthe invention, where such treatment includes administering, either systemically or locally, an appropriate composition containing the polypeptides, polynucleotides, or modulators to a subject.
  • the present invention provides methods for diagnosis of diseases using the disclosed polypeptides, polynucleotides, and modulators; and kits useful in the diagnosis.
  • the present invention provides vectors and host cells containing the polypeptides, polynucleotides, and modulators, such as antibodies, as mentioned above.
  • the present invention also provides methods of making and using the polypeptides, polynucleotides, vectors, host cells, antibodies as mentioned above.
  • polynucleotide refers to polymeric forms of nucleotides of any length.
  • the polynucleotides can contain deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives.
  • a "splice site” is the locus of a polynucleotide at which an exon or part of an exon is added or removed. Molecules ofthe invention can differ from related molecules in the public domain by having different splice sites.
  • host cell includes an individual cell, cell line, cell culture, or in vivo cell, which can be, or has been, a recipient of any recombinant vector(s) or isolated polynucleotide(s).
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology, physiology, or in total DNA, RNA, or polypeptide complement) to the original parent cell, due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide ofthe invention.
  • a host cell which comprises a recombinant vector ofthe invention may be called a "recombinant host cell.”
  • polypeptide refers to a sequence of at least three, or at least four, or at least five, or at least six contiguous amino acid residues.
  • polypeptides include full length proteins that include a signal peptide or leader sequence, if present, or a mature protein after cleavage ofthe signal peptide or leader sequence, the signal peptide or leader sequence, or active fragments ofthe full length or mature protein, for example, those containing the Pfam domains or extracellular domains.
  • An active portion or fragment of a polypeptide is one that has activity such as the ability to act as an epitope for generation of antibodies, or one that contains a Pfam or enzymatic domain, or one that is sufficient to participate in a signal transduction pathway, or has the ability to bind an antibody or an antigen, for example.
  • a "signal peptide,” or a “leader sequence,” comprises a sequence of amino acid residues, typically, at the N terminus of a polypeptide, which directs the intracellular trafficking ofthe polypeptide.
  • Polypeptides that contain a signal peptide or leader sequence typically also contain a signal peptide or leader sequence cleavage site. Such polypeptides, after cleavage at the cleavage sites, generate mature polypeptides after extracellular secretion or after being directed to the appropriate intracellular compartment.
  • An “epitope” is a sequence of amino acid residues in a polypeptide that may or may not be contiguous, and constitutes part of an antigen to which an antibody will bind.
  • the term "antibody” refers to protein generated by the immune system that is capable of recognizing and binding to a specific antigen; the term is commonly known in the art.
  • the term includes active fragments, including for example, at least one of an antigen binding fragment of an immunoglobulin, a variable and/or constant region of a heavy chain, a variable and/or constant region of a light chain, a complementarity determining region (cdr), or a framework region.
  • the "antigen binding fragment (Fab fragment)” is a disulfide-linked heterodimer, each chain of which contains one immunoglobulin constant region (C) domain and one variable region (V) domain; the juxtaposition ofthe V domains forms the antigen-binding site.
  • the two Fab fragments of an intact immunoglobulin molecule correspond to its two arms, which typically contain light chain regions paired with the V and CI domains ofthe heavy chains.
  • an “isolated,” “purified,” “substantially isolated,” or “substantially purified” antibody is one that has been manipulated to exist in a higher concentration than in nature.
  • a subject antibody is isolated, purified, substantially isolated, or substantially purified when at least 10%, or 20%, or 40%, or 50%, or 70%, or 90% of non-subject-antibody materials with which it is associated in nature have been removed.
  • an “isolated,” “purified,” “substantially isolated,” or “substantially purified” polypeptide includes recombinant antibodies.
  • a “humanized” antibody is an antibody that contains mostly human immunoglobulin sequences.
  • a humanized antibody may include a human antibody that contains entirely human immunoglobulin sequences, such as those produced by Abgenix's XenoMouse (Fremont, California, USA).
  • ADCC antibody-dependent cell cytotoxicity
  • Cell-dependent cytotoxicity is an adverse effect on a cell that results from an action ofthe cellular immune system.
  • antibody target refers to a polypeptide or a polynucleotide that can be used as an immunogen in the production of antibodies that specifically bind to such polypeptide or polynucleotide.
  • the term "binds specifically,” in the context of antibody binding, refers to high avidity and/or high affinity binding of an antibody to a specific polypeptide or a portion ofthe polypeptide, that is, an epitope of a polypeptide.
  • Antibody binding to a specific epitope can be stronger than binding ofthe same antibody to any other epitopes, particularly other epitopes that can be present in molecules in association with, or in the same sample as the polypeptide of interest. For example, when an antibody binds more strongly to one epitope than to another, adjusting the binding conditions can result in antibody binding almost exclusively to the specific epitope and not to any other epitopes on the same polypeptide, and not to any other polypeptide which does not comprise the epitope.
  • Antibodies that bind specifically to a subject polypeptide may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less ofthe binding shown to the polypeptide of interest).
  • antibodies ofthe invention bind to a specific polypeptide with an association constant of 10 '7 M or greater (e.g., 10 "8 M, 10 "9 M, 10 "10 M, 10 "u M, etc.).
  • the term "receptor” refers to a polypeptide that binds to a specific ligand, which is usually an extracellular molecule, and upon binding, usually initiates a cellular response.
  • ligand refers to any molecule that binds to a specific site on another molecule, usually a receptor.
  • Apoptosis or programmed cell death, is a regulated process leading to cell death via a series of well-defined morphological changes. Programmed cell death provides a balance for cell growth and multiplication, eliminating unnecessary cells.
  • disease refers to any abnormality, deficiency, condition, infection, disorder, or syndrome that requires medical intervention or for which medical intervention is desirable. Such medical intervention includes treatment, diagnosis, and/or prevention.
  • Cancer is herein defined as any abnormal cell or tissue growth, e.g., a tumor; the growth can be malignant or non-malignant. It is characterized by uncontrolled proliferation of cells that may or may not invade the surrounding tissue and, hence, may or may not metastasize to new body sites.
  • carcinomas which are cancers of epithelial cells; carcinomas include squamous cell carcinoma, adenocarcinoma, melanomas, and hepatomas.
  • Cancer also encompasses sarcomas, which are tumors of mesenchymal origin, and includes osteogenic sarcomas, leukemias, and lymphomas. Cancers can involve one or more neoplastic cell type.
  • the term “overexpressed” or “highly expressed” refers to a state wherein there exists any measurable increase in expression over normal or baseline levels.
  • a molecule that is overexpressed in a disease is one that is manifest in a measurably higher level in the presence ofthe disease than in the absence ofthe disease. Such an increase can be at least two-fold, at least three-fold, or more.
  • "Biological sample,” as used herein, includes biological fluids such as blood, serum, plasma, urine, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid, lavage fluid, semen, and other liquid samples or tissues of biological origin.
  • a biological sample can be used in a diagnostic or monitoring assay.
  • subject refers to mammals, including, but not limited to, rodents, simians, humans, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • a "biologically active” or “active” entity is one having structural, regulatory, or biochemical functions of a naturally occurring molecule. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a nucleic acid, or polypeptide, or antibody ofthe present invention.
  • the biological activity ofthe fragments can include an improved desired activity, or a decreased undesirable activity.
  • a biologically active fragment of a polynucleotide includes one that can be detected as unique for the polynucleotide molecule, or that can be used as a primer in PCR; and a biologically active fragment of a polypeptide includes one that can participate in a biological reaction, for example, in ligand/receptor interaction, in eliciting an immune response, such as production of antibodies, or that can participate in signal transduction, such as by binding to receptors, activating enzymes or substrates.
  • agonist refers to a substance that mimics the function of an active molecule.
  • Agonists include, but are not limited to, antibodies, growth factors, cytokines, lymphokines, small molecule drugs, hormones, and neurotransmitters, as well as analogues and fragments thereof.
  • antagonist refers to a molecule that interferes with the activity or binding of an agonist such as by competing for the binding sites of an agonist, but does not induce an active response.
  • modulate encompasses an increase or a decrease, a stimulation, inhibition, interference, or blockage in a measured activity when compared to a suitable control.
  • a “modulator” ofthe polypeptides or polynucleotides or an “agent” herein is an agonist or antagonist that interferes with the binding or activity of such polypeptides or polynucleotides.
  • modulators or agents include, for example, polypeptide variants, whether agonist or antagonist; antibodies, whether agonist or antagonist; soluble receptors, usually antagonists; small molecule drugs, whether agonist or antagonist; RNAi, usually an antagonist; antisense molecules, usually antagonists; and ribozymes, usually antagonists.
  • an agent is a subject polypeptide, where the subject polypeptide itself is administered to an individual.
  • an agent is an antibody specific for a subject "target" polypeptide.
  • an agent is a chemical compound such as a small molecule that may be useful as an orally available drug.
  • modulation includes the recruitment of other molecules that directly effect the modulation.
  • an antibody that modulates the activity of a subject polypeptide that is a receptor on a cell surface may bind to the receptor and fix complement, activating the complement cascade, and resulting in lysis ofthe cell.
  • An agent which modulates a biological activity of a subject polypeptide or polynucleotide increases or decreases the activity or binding at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 80%, or at least about 2-fold, at least about 5-fold, or at least about 10-fold or more when compared to a suitable control.
  • Modulating a level of active subject polypeptide includes increasing or decreasing activity of a subject polypeptide, increasing or decreasing a level of active polypeptide protein, and increasing or decreasing a level of mRNA encoding active subject polypeptide.
  • Treatment covers any treatment of a disease in a mammal, including a human, and includes preventing the disease from occurring or recurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, inhibiting the disease, i.e., arresting its development, or relieving the condition or disease, i.e., causing its regression, or restoring or repairing a lost, missing, or defective function, and/or stimulating an inefficient process.
  • a “composition” of modulators, polypeptides, or polynucleotides herein refers to a composition that usually contains a vehicle, that is conventional in the art.
  • a pharmaceutical composition contains a vehicle such as a pharmaceutically acceptable carrier or excipient and is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes.
  • a vehicle such as a pharmaceutically acceptable carrier or excipient
  • Compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders.
  • a "pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any conventional type.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the carrier for a formulation containing polypeptides does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
  • Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof.
  • the carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness ofthe formulation.
  • Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents can be added as necessary.
  • Percutaneous penetration enhancers such as Azone can also be included.
  • the invention encompasses each intervening value between the upper and lower limits ofthe range to at least a tenth ofthe lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values. Moreover, the invention also encompasses ranges excluding either or both ofthe upper and lower limits ofthe range, unless specifically excluded from the stated range.
  • Beta-l,4-Galactosyltransferase (B4GALT) [054]
  • the UDP-GalbetaGlcNAc beta 1,4,-galactosyltransferase 4 (B4GALT) gene is present on chromosome 1, and is localized to Ip33-p34. It is comprised of 1888 base pairs, and is predicted to encode an amino acid gene product of 373 amino acids (Lo et al, 1998).
  • Beta-l,4-galactosylrransferases are localized in the trans-Golgi compartment of most eukaryotic cells, where they participate in the elongation of oligosaccharide chains on glycoproteins and glycolipids.
  • the inventors have found that this gene is highly expressed in human lung adenocarcinomas, lung squamous cell carcinomas, and colon adenocarcinomas. It is also expressed in normal human lung, heart, kidney and liver. In paired comparisons, this gene is overexpressed in malignant bladder, liver, ovary, stomach, breast, and lung. The inventors have discovered a novel variant of beta-l,4-galactosyltransferase, as shown in the Tables and Sequence Listing.
  • Chrl Orf9 Chromosome 1 Open Reading Frame 9; Membrane Protein CHI (Chrl Orf9)
  • the Chrl Orf9 gene comprises 5556 base pairs, and encodes an open reading frame of 1254 amino acids (Rosok et al, 2000). It is located on human chromosome 1 at region lq24, spans approximately 78.7 kb and is organized into at least 24 exons (Rosok et al, 2000). The sequence of Chrl Orf9 is disclosed through the NCBI as NM_014283.
  • Chrl Orf9 is expressed in normal human adrenals, heart, kidney, liver, lung, and pancreas. This gene is overexpressed in malignant human bladder, liver, ovary, breast, pancreas, and colon adenocarcinomas.
  • the inventors have discovered a novel variant ofthe Chrl Orf9 gene, as shown in the Tables and Sequence Listing.
  • the human claudin-3 gene comprises 1249 base pairs and is located on chromosome 7, at region 7ql 1.23. It has been reported to encode a 23,318 dalton protein comprising 220 amino acids (Peacock et al, 1997). Claudin-3 is an integral membrane protein and a component of tight junctions. It is also a low-affinity receptor for Clostridium perfringens enterotoxin, and shares amino acid sequence similarity with a putative apoptosis-related rat protein (Coyne et al, 2003). The inventors have discovered a novel variant ofthe claudin-3 gene, as shown in the Tables and Sequence Listing.
  • the human comichon-like protein gene also called TGAM77, comprises 14,454 base pairs, and is located on human chromosome 14, at region 14q22.2.
  • Human comichon-like protein is differentially expressed in alloactivated T-cells. Up regulation of human comichon-like protein mRNA has been observed in the early phase of human T-cell activation. Human comichon-like protein mRNA is expressed in a variety of human tissues with various expression levels.
  • Ectonucleoside Triphosphate Diphosphohydrolase 6 [060] The human ectonucleoside triphosphate diphosphohydrolase 6 gene comprises 30,999 base pairs and is located on chromosome 20 at location 20pl 1.21.
  • Human ectonucleoside triphosphate diphosphohydrolase 6 is a member ofthe CD39-like gene family. Members of this family have been shown to have E-type apyrase activity, which hydrolyzes extracellular ATP and ADP, thus modulating homotypic adhesion, platelet aggregation, and platelet removal by activated lymphocytes (Chadwick et al, 1998).
  • the inventors have discovered a novel variant ofthe ectonucleoside triphosphate diphosphohydrolase 6 gene, as shown in the Tables and Sequence Listing. Jagged2
  • the human jagged2 gene comprises 25,075 base pairs and is located on chromosome 14 at location 14q32.33 (Deng et al, 2000). It encodes a ligand for the notch receptor, and has been reported to coordinate differentiation of progenitor cells. Sustained activation ofthe jagged2/notch signal transduction pathway was observed to induce continuous cell cycling, overcoming density-dependent inhibition of cell division, in confluent rabbit-skin fibroblasts; it therefore may contribute to neoplastic transformation (Cereseto et al, 2000). The inventors have discovered a novel variant of the jagged2 gene, as shown in the Tables and Sequence Listing.
  • Phosphatidic Acid Phosphatase Type 2C PAP2C or PPAP2C
  • the human phosphatidic acid phosphatase type 2C (PAP2C) gene is present on human chromosome 19, and localized to 19pl3. It comprises 1327 base pairs, and encodes a gene product of 288 amino acids, with a predicted molecular mass of 32,577 daltons (Roberts et al, 1998). PAP2C encodes integral membrane gene products with six transmembrane regions, a single consensus N-gfycosylation site at amino acid residue 140, and a catalytic site for membrane-associated PAP activity. The catalytic sites are located in the second and third extracellular loops.
  • PAP2A, PAP2B, and PAP2C are widely divergent.
  • Three alternatively spliced transcript variants encoding distinct isoforms have been reported for the PAP2C gene (Roberts et al, 1998).
  • Phosphatidic acid phosphatases convert phosphatidic acid to diacylglycerol in the biosynthetic pathway of stractural membrane lipids, contributing to the de novo synthesis of glycerohpids.
  • Phosphatidic acid and glycerohpids are mediators of lipid signal transduction, in particular, transduction mediated by phospholipase D.
  • PPAP are involved in regulating lipid-mediated signal transduction.
  • the inventors have discovered that PAP2C (sometimes also referred to as PPAP2C) is highly expressed in human tumors such as malignant bladder, liver, ovary, breast, colon, kidney, pancreas, and lung, including adenocarcinomas and squamous cell carcinomas.
  • TM7XN1 [065] The human TM7XN1 gene comprises 44,986 base pairs and is present on chromosome 16 at location 16ql3. It encodes a 77,737 dalton protein of 693 amino acids and belongs to the LN-TM7 subfamily of G-protein coupled receptors.
  • TM7XN1 expression was observed in poorly and intermediately metastasizing cell lines, and was markedly down regulated in highly metastatic cell lines (Zendman et al, 1999).
  • the inventors have discovered a novel variant ofthe TM7XN1 gene, as shown in the Tables and Sequence Listing.
  • Panel [067] These mmor markers can be used in combination, e.g. , in a panel that comprises two or more markers. It is expected that almost all lung cancers will overexpress at least one of these genes, and that combining these markers into a panel will provide a comprehensive screen for certain cancers.
  • the present invention utilized probes and primers that were either purchased directly from Applied Biosystems, Inc. ("ABI") (Foster City, California, USA) Assay- On-Demand or were designed using software PrimerExpress. The exact probe and primer sequences that were purchased from ABI were not released. However, the approximate amplicon sequences could be estimated based on the information provided from ABI.
  • PPAP2C amplicon sequences can be estimated by searching the Assay ID HsOO 186575 from the website: http://myscience.appliedbiosystems. com/cdsEntry/Form/gene_expression_keyword.jsp. Under "Interrogated Sequence,” on the webpage, it is shown that the amplicon covers exon boundaries of exon 3 and exon 4.
  • the "assay location" nucleotide 579 was shown to be within the amplicon sequence when using RefSeq sequence number NM_003712 (available from NCBI).
  • the "context sequence” provided by ABI was shown to be a sequence within the amplicon.
  • the map view link also provided some information about the amplicon. Taken together, this information suggests the amplicon was about 75-150 bp in length and covered the "assay location" nucleotide, the "context sequence,” as well as the exon 3 and 4 boundary.
  • tissue equals 2 Ct(gene_n)-Ct(18S_n) f ⁇ no ⁇ m&l tissue
  • PAP2C Gene Expression of PAP2C
  • PAP2C was found to be highly expressed in at least 8 out of 9 human lung adenocarcinomas, 9 out of 11 human lung squamous cell carcinomas, and 10 out of 10 human colon adenocarcinomas of cancer patients, as compared to an average expression level in normal tissues of normal individuals.
  • the expression ofthe PAP2C gene in normal lung, heart, kidney, and liver tissues was found to be low or very low.
  • This gene was also found to be expressed at a high level in 1 out of 3 normal human lung samples and 3 out of 7 normal human heart samples. This gene was also expressed at a significant level in 1 out of 3 normal human lung samples, 1 out of 7 normal human heart samples, and 2 out of 4 normal human kidney samples. [076] Interrogation ofthe GeneLogic database showed this gene to be expressed in normal adrenals, heart, kidney, liver, lung, and pancreas. This gene was overexpressed, as compared to the corresponding normal patient tissue samples, in malignant bladder, liver, ovarian, breast, and pancreatic tumors.
  • B4GALT Gene Expression of B4GALT [077] This gene was found to be highly expressed in 7 out of 9 human lung adenocarcinomas, 10 out of 11 human lung squamous cell carcinomas, and 9 out of 10 human colon adenocarcinomas from cancer patients. This gene was also found to be expressed at a high or significant level in 2 out of 3 normal human lung samples, 6 out of 7 normal human heart samples, 4 out of 4 normal human kidney samples, and 4 out of 4 normal human liver samples.
  • Genes that are uniquely or differentially expressed in cancerous cells or tissues may potentially serve as cancer cell markers in bodily fluids, e.g., serum.
  • a reliable marker must be specific to cancer, and expressed only when the patient has cancer.
  • polypeptides of the present invention comprise identifiable protein family domains (Pfam).
  • Pfam is an organization of protein sequence classification and analysis, based on conserved protein domains; it can be publicly accessed in a number of ways, for example, at http://pfam.wustl.edu.
  • Protein domains are portions of proteins that have a tertiary structure and sometimes have enzymatic or binding activities; multiple Pfam domains can be connected by flexible polypeptide regions within a protein.
  • Pfam domains can comprise the N-terminus or the C-terminus of a protein, or can be situated at any point in between.
  • the Pfam system identifies protein families based on these domains and provides an annotated, searchable database that classifies proteins into families.
  • Sequences encompassed by the invention include, but are not limited to, the polypeptide and polynucleotide sequences ofthe molecules shown in the Tables, Figures, and Sequence Listing. Sequences ofthe invention can comprise genes or gene segments designated in the application, and their gene products, t.e., RNA and polypeptides.
  • nucleic acid, polypeptide, and modulator compositions ofthe invention find use as therapeutic agents in situations where one wishes to modulate an activity of a subject polypeptide in a host, particularly the activity of the subject polypeptides, or to provide or inhibit the activity at a particular anatomical site.
  • the compositions are useful in treating disorders associated with an activity of a subject polypeptide.
  • active agents ofthe present invention The following provides further details of active agents ofthe present invention.
  • Antisense Oligonucleotides is an agent that modulates, and generally decreases or down regulates, the expression of a gene encoding a target protein in a host, i.e., antisense molecules.
  • Anti-sense reagents include antisense oligonucleotides (ODN), i.e., synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA.
  • ODN antisense oligonucleotides
  • the antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression ofthe targeted gene products.
  • Antisense molecules inhibit gene expression through various mechanisms, e.g., by reducing the amount of mRNA available for translation, through activation of RNase H, or steric hindrance.
  • One or a combination of antisense molecules can be administered, where a combination can comprise multiple different sequences.
  • Antisense molecules can be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule.
  • the antisense molecule is a synthetic oligonucleotide.
  • Antisense oligonucleotides can be chemically synthesized by methods known in the art (Wagner et al, 1993; MiUigan et al, 1993) Oligonucleotides can be chemically modified from the native phosphodiester structure to increase their intracellular stability and binding affinity, for example, as described in detail above.
  • Antisense oligonucleotides will generally be at least about 7, at least about 12, or at least about 20 nucleotides in length, and not more than about 500, not more than about 50, or not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, and specificity, including absence of cross-reactivity, and the like. Short oligonucleotides, of from about 7 to about 8 bases in length, can be strong and selective inhibitors of gene expression (Wagner et al, 1996).
  • a specific region or regions ofthe endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide can use an empirical method, where several candidate sequences are assayed for inhibition of expression ofthe target gene in an in vitro or animal model. A combination of sequences can also be used, where several regions ofthe mRNA sequence are selected for antisense complementation.
  • catalytic nucleic acid compounds e.g., ribozymes, or anti-sense conjugates can be used to inhibit gene expression.
  • Ribozymes can be synthesized in vitro and administered to the patient, or can be encoded in an expression vector, from which the ribozyme is synthesized in the targeted cell (WO95/23225; Beigelman et al, 1995). Examples of oligonucleotides with catalytic activity are described in WO95/06764. Conjugates of anti-sense ODN with a metal complex, e.g., terpyridyl Cu(II), capable of mediating mRNA hydrolysis are described in Bashkin et al, 1995. Interfering RNA [087] In some embodiments, the active agent is an interfering RNA (RNAi), including dsRNAi.
  • RNAi interfering RNA
  • RNA interference provides a method of silencing eukaryotic genes. Double stranded RNA can induce the homology-dependent degradation of its cognate mRNA in C. elegans, fungi, plants, Drosophila, and mammals (Gaudilliere et al. , 2002) .
  • Use of RNAi to reduce a level of a particular mRNA and/or protein is based on the interfering properties of double-stranded RNA derived from the coding regions of a gene. The technique reduces the time between identifying an interesting gene sequence and understanding its function, and thus is an efficient high-throughput method for disrupting gene function (O'Neil, 2001). RNAi can also help identify the biochemical mode of action of a drug and to identify other genes encoding products that can respond or interact with specific compounds.
  • complementary sense and antisense RNAs derived from a substantial portion ofthe subject polynucleotide are synthesized in vitro.
  • the resulting sense and antisense RNAs are annealed in an injection buffer, and the double-stranded RNA injected or otherwise introduced into the subject, i.e., in food or by immersion in buffer containing the RNA (Gaudilliere et al, 2002; O'Neil et al, 2001; WO99/32619).
  • dsRNA derived from a gene of the present invention is generated in vivo by simultaneously expressing both sense and antisense RNA from appropriately positioned promoters operably linked to coding sequences in both sense and antisense orientations.
  • the active agent is a peptide. Suitable peptides include peptides of from about 3 amino acids to about 50, from about 5 to about 30, or from about 10 to about 25 amino acids in length. In some embodiments, a peptide has a sequence of from about 3 amino acids to about 50, from about 5 to about 30, or from about 10 to about 25 amino acids of corresponding naturally-occurring protein.
  • a peptide exhibits one or more of the following activities: inhibits binding of a subject polypeptide to an interacting protein or other molecule; inhibits subject polypeptide binding to a second polypeptide molecule; inhibits a signal transduction activity of a subject polypeptide; inhibits an enzymatic activity of a subject polypeptide; or inhibits a DNA binding activity of a subject polypeptide.
  • Peptides can include naturally-occurring and non-naturally occurring amino acids.
  • Peptides can comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids, etc.) to convey special properties.
  • peptides can be cyclic.
  • Peptides can include non-classical amino acids in order to introduce particular conformational motifs. Any known non-classical amino acid can be used.
  • Non-classical amino acids include, but are not limited to, 1,2,3,4- tetrahydroisoquinoline-3-carboxylate; (2S,3S)-methylphenylalanine, (2S,3R)-methyl- phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine; 2- aminotetrahydronaphthalene-2-carboxylic acid; hydroxy- 1,2,3, 4- tetrahydroisoquinoline-3-carboxylate; ⁇ -carboline (D and L); HIC (histidine isoquinoline carboxylic acid); and HIC (histidine cyclic urea).
  • Amino acid analogs and peptidomimetics can be incorporated into a peptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2- propenidone-6-carboxylic acid), a ⁇ -turn inducing dipeptide analog; ⁇ -sheet inducing analogs; ⁇ -turn inducing analogs; ⁇ -helix inducing analogs; ⁇ -turn inducing analogs; Gly- Ala turn analogs; amide bond isostere; or tretrazol, and the like.
  • a peptide can be a depsipeptide, which can be linear or cyclic (Kuisle et al. , 1999).
  • Linear depsipeptides can comprise rings formed through S-S bridges, or through an hydroxy or a mercapto group of an hydroxy-, or mercapto-amino acid and the carboxyl group of another amino- or hydroxy-acid but do not comprise rings formed only through peptide or ester links derived from hydroxy carboxylic acids.
  • Cyclic depsipeptides contain at least one ring formed only through peptide or ester links, derived from hydroxy carboxylic acids.
  • Peptides can be cyclic or bicyclic.
  • the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement ofthe - OH or the ester (-OR) ofthe carboxyl group or ester respectively with the N- terminal amino group to form a cyclic peptide.
  • the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH 2 C1 2 ), dimethyl formamide (DMF) mixtures.
  • DCC dicyclohexylcarbodiimide
  • the cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization as opposed to polymerization can be enhanced by use of very dilute solutions. Methods for making cyclic peptides are well known in the art.
  • a desamino or descarboxy residue can be incorporated at the terminal ends of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict conformation.
  • C-terminal functional groups include amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.
  • a peptide or peptidomimetic can be modified with or covalently coupled to one or more of a variety of hydrophilic polymers to increase solubility and circulation half-life of the peptide.
  • Suitable nonproteinaceous hydrophilic polymers for coupling to a peptide include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran, and dextran derivatives.
  • hydrophilic polymers have an average molecular weight ranging from about 500 to about 100,000 daltons, from about 2,000 to about 40,000 daltons, or from about 5,000 to about 20,000 daltons.
  • the peptide can be derivatized with or coupled to such polymers using any ofthe methods set forth in Zallipsky (1995); Monfardini et al. (1995); U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337, or WO95/34326.
  • Peptide Aptamers [095]
  • Another suitable agent for modulating an activity of a subject polypeptide is a peptide aptamer.
  • Peptide aptamers are peptides or small polypeptides that act as dominant inhibitors of protein function. Peptide aptamers specifically bind to target proteins, blocking their functional ability (Kolonin and Finley, 1998). Due to the highly selective nature of peptide aptamers, they can be used not only to target a specific protein, but also to target specific functions of a given protein (e.g., a signaling function). Further, peptide aptamers can be expressed in a controlled fashion by use of promoters which regulate expression in a temporal, spatial or inducible manner. Peptide aptamers act dominantly, therefore, they can be used to analyze proteins for which loss-of-function mutants are not available.
  • Peptide aptamers that bind with high affinity and specificity to a target protein can be isolated by a variety of techniques known in the art. Peptide aptamers can be isolated from random peptide libraries by yeast two-hybrid screens (Xu et al, 1997). They can also be isolated from phage libraries (Hoogenboom et al, 1998) or chemically generated peptides/libraries. Soluble Receptors [097] Extracellular fragments of cell surface receptors can be soluble, and can modulate a target protein. These fragments can act as ligands for binding to receptors on cell surfaces in ligand/receptor interactions, and modulate the receptors and cellular activity downstream ofthe receptors.
  • Small molecule modulators such as those commonly used as therapeutic drags can be used as inhibitors, agonists, antagonists, and the like. Small molecule agents include chemical compounds that bind the polypeptide to modulate activity ofthe polypeptide or a cell containing the polypeptide. Small molecule modulators may permeate the cell, and/or may exert their action at the extracellular surface or on con- cellular structures, such as the extracellular matrix.
  • Antibodies include antibodies, and/or may exert their action at the extracellular surface or on con- cellular structures, such as the extracellular matrix.
  • An antibody ofthe present invention may comprise a monoclonal antibody, polyclonal antibody, single chain antibody, intrabody, and active fragments of any of these.
  • the active fragments include variable regions from either heavy chains or light chains.
  • the antibody can be a fusion molecule linked to a cytotoxic molecule, including a radioisotope, a chemocytotoxic agent or a toxin from a plant, bacterium or fungus.
  • the antibody can comprise a T cell receptor (TCR), a cytotoxic T lymphocyte antigen 4 (CTLA4), a fibronectin, or another backbone.
  • TCR T cell receptor
  • CTL4 cytotoxic T lymphocyte antigen 4
  • fibronectin or another backbone.
  • the present invention further features a targeting antibody, a neutralizing antibody, a stabilizing antibody, an enhancing antibody, an antibody agonist, an antibody antagonist, an antibody that promotes cellular endocytosis of a target antigen, a cytotoxic antibody, and an antibody that mediates, complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).
  • the antibody that mediates ADCC can deliver a payload, such as a cytotoxic component, e.g., a radioisotope, a radioactive molecule, a microbial toxin, a plant toxin, a chemotherapeutic agent, or a chemical substance, such as doxorabicin or cisplatin.
  • the payload can be attached with technology from Seattle Genetics (Bothell, WA), which incorporates synthetic stable linkers and drags that can be used to increase the potency of an antibody.
  • the linkers are stable in the bloodstream but release drug payloads under conditions inside target cells.
  • the invention also features an inhibitory antibody, functioning to specifically inhibit the binding of a cognate polypeptide to its ligand or its substrate, or to specifically inhibit the binding of a cognate peptide as the substrate of another molecule.
  • the antibodies ofthe present invention also encompass a human antibody, a non-human primate antibody, e.g., monkey; a non-primate animal antibody, e.g., a rodent such as a rat, mouse, hamster, or guinea pig; a chicken antibody, a cattle antibody, such as a sheep, pig, cow, horse, or goat; a cat; dog; and rabbit. It also features a humanized antibody, a primatized antibody, and a chimeric antibody.
  • the antibodies and antibody fragments ofthe invention can be produced in vitro or in vivo.
  • the present invention features an antibody produced in a cell-free expression system, a prokaryote expression system or a eukaryote expression system, as described herein.
  • antibody fragments can be made in E. coli.
  • the invention further provides a host cell that can produce an antibody ofthe invention or a fragment thereof.
  • the antibody may also be secreted by the cell.
  • the host cell can be a hybridoma, or a prokaryotic or eukaryotic cell.
  • the invention also provides a bacteriophage or other virus particle comprising an anti7body ofthe invention, or a fragment thereof.
  • the bacteriophage or other virus particle may display the antibody or fragment thereof on its surface, and the bacteriophage itself may exist within a bacterial cell.
  • the antibody may also comprise a fusion protein with a viral or bacteriophage protein.
  • the invention further provides transgenic multicellular organisms, e.g., plants or non-human animals, as well as tissues or organs, comprising a polynucleotide sequence encoding a subject antibody or fragment thereof.
  • the organism, tissues, or organs will generally comprise cells producing an antibody ofthe invention, or a fragment thereof.
  • the present invention features a method of making an antibody by immunizing a host animal (Coligan, 2002).
  • a polypeptide or a fragment thereof, a polynucleotide encoding a polypeptide, or a polynucleotide encoding a fragment thereof is introduced into an animal in a sufficient amount to elicit the generation of antibodies specific to the polypeptide or fragment thereof, and the resulting antibodies are recovered from the animal.
  • the polypeptide or polynucleotide sequence can be chosen from the Sequence Listing or the Tables. Initial immunizations can be with either polynucleotide or polypeptide sequences.
  • Subsequent booster immunizations can be with either polynucleotide or polypeptide sequences.
  • Initial immunization with a polynucleotide can be followed with either polynucleotide or polypeptide immunizations, and an initial immunization with a polypeptide can be followed with either polynucleotide or polypeptide immunizations.
  • the invention provides antibodies that specifically recognize a particular polypeptide. Antibodies are obtained by immunizing a host animal with peptides, polynucleotides encoding polypeptides, or cells, each comprising all or a portion of the target protein. The antibodies ofthe invention can be produced in a variety of species.
  • the host animal will generally be a different species than the immunogen, e.g. , a human protein used to immunize mice.
  • the animal can be a non-human primate, (e.g., a monkey) a rodent (e.g., a rat, a mouse, a hamster, a guinea pig), a chicken, cattle (e.g., a sheep, a goat, a horse, a pig, a cow), a rabbit, a cat, or a dog.
  • Suitable host animals include rodents (e.g., mouse, rat, guinea pig, hamster), cattle (e.g., sheep, pig, cow, horse, goat), cat, dog, chicken, primate, monkey, and rabbit.
  • rodents e.g., mouse, rat, guinea pig, hamster
  • cattle e.g., sheep, pig, cow, horse, goat
  • cat dog, chicken, primate, monkey, and rabbit.
  • Methods of antibody production are well known in the art (Coligan, 2002; Howard and Bethell, 2000; Harlow et al, 1998; Harlow and Lane, 1988).
  • the present invention also features a method of making an antibody by isolating a spleen from an animal injected with a polypeptide or a fragment thereof, a polynucleotide encoding a polypeptide, or a polynucleotide encoding a fragment thereof, and recovering antibodies from the spleen cells. Hybridomas can be made from the spleen cells, and hybridomas secreting specific antibodies can be selected. [0109] The present invention further features a method of making a polynucleotide library from spleen cells, and selecting a cDNA clone that produces specific antibodies, or fragments thereof. The cDNA clone or a fragment thereof can be expressed in an expression system that allows production ofthe antibody or a fragment thereof, as provided herein.
  • the immunogen can comprise a nucleic acid, a complete protein, or fragments and derivatives thereof, or proteins expressed on cell surfaces.
  • Pfam domains can be used as immunogens
  • transmembrane domains can be used as immunogens.
  • Immunogens comprise all or a part of one ofthe subject proteins, where these amino acids contain post-translational modifications, such as glycosylation, found on the native target protein.
  • Immunogens comprising protein extracellular domains are produced in a variety of ways known in the art, e.g., expression of cloned genes using conventional recombinant methods, or isolation from tumor cell culture supematants, etc.
  • the immunogen can also be expressed in vivo from a polynucleotide encoding the immunogenic peptide introduced into the host animal.
  • Polyclonal antibodies are prepared by conventional techniques. These include immunizing the host animal in vivo with the target protein (or immunogen) in substantially pure form, for example, comprising less than about 1% contaminant.
  • the immunogen can comprise the complete target protein, fragments, or derivatives thereof.
  • the target protein can be combined with an adjuvant; suitable adjuvants include alum, dextran, sulfate, large polymeric anions, and oil & water emulsions, e.g., Freund's adjuvant (complete or incomplete).
  • the target protein can also be conjugated to synthetic carrier proteins or synthetic antigens.
  • the target protein is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages.
  • blood from the host will be collected, followed by separation ofthe seram from blood cells.
  • the immunoglobulin present in the resultant antiserum can be further fractionated using known methods, such as ammonium salt fractionation, or DEAE chromatography and the like.
  • Cytokines can also be used to help stimulate immune response. Cytokines act as chemical messengers, recruiting immune cells that help the killer T-cells to the site of attack.
  • cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates the proliferation of antigen-presenting cells, thus boosting an organism's response to a cancer vaccine.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • cytokines can be used in conjunction with the antibodies and vaccines disclosed herein. For example, they can be incorporated into the antigen-encoding plasmid or introduced via a separate plasmid, and in some embodiments, a viral vector can be engineered to display cytokines on its surface.
  • the method of producing polyclonal antibodies can be varied in some embodiments ofthe present invention. For example, instead of using a single substantially isolated polypeptide as an immunogen, one may inject a number of different immunogens into one animal for simultaneous production of a variety of antibodies.
  • the immunogens can be nucleic acids (e.g., in the form of plasmids or vectors) that encode the proteins, with facilitating agents, such as liposomes, microspheres, etc, or without such agents, such as "naked" DNA.
  • Antibodies can also be prepared using a library approach. Briefly, mRNA is extracted from the spleens of immunized animals to isolate antibody-encoding sequences. The extracted mRNA may be used to make cDNA libraries. Such a cDNA library may be normalized and subtracted in a manner conventional in the art, for example, to subtract out cDNA hybridizing to mRNA of non-immunized animals. The remaining cDNA may be used to create proteins and for selection of antibody molecules or fragments that specifically bind to the immunogen. The cDNA clones of interest, or fragments thereof, can be introduced into an in vitro expression system to produce the desired antibodies, as described herein.
  • polyclonal antibodies can be prepared using phage display libraries, conventional in the art.
  • phage display libraries conventional in the art.
  • a collection of bacteriophages displaying antibody properties on their surfaces are made to contact subject polypeptides, or fragments thereof.
  • Bacteriophages displaying antibody properties that specifically recognize the subject polypeptides are selected, amplified, for example, in E. coli, and harvested. Such a method typically produces single chain antibodies.
  • Phage display technology can be used to produce Fab antibody fragments, which can be then screened to select those with strong and/or specific binding to the protein targets.
  • the screening can be performed using methods that are known to those of skill in the art, for example, ELISA, immunoblotting, immunohistochemistry, or immunoprecipitation.
  • Fab fragments identified in this manner can be assembled with an Fc portion of an antibody molecule to form a complete immunoglobulin molecule.
  • Monoclonal antibodies are also produced by conventional techniques, such as fusing an antibody-producing plasma cell with an immortal cell to produce hybridomas.
  • Suitable animals will be used, e.g., to raise antibodies against a mouse polypeptide ofthe invention, the host animal will generally be a hamster, guinea pig, goat, chicken, or rabbit, and the like.
  • the spleen and/or lymph nodes of an immunized host animal provide the source of plasma cells, which are immortalized by fusion with myeloma cells to produce hybridoma cells.
  • Culture supematants from individual hybridomas are screened using standard techniques to identify clones producing antibodies with the desired specificity.
  • the antibody can be purified from the hybridoma cell supematants or from ascites fluid present in the host by conventional techniques, e.g., affinity chromatography using antigen, e.g., the subject protein, bound to an insoluble support, t.e., protein A sepharose, etc.
  • the antibody can be produced as a single chain, instead ofthe normal multimeric stracture ofthe immunoglobulin molecule. Single chain antibodies have been previously described (i.e., Jost et al, 1994).
  • DNA sequences encoding parts of the immunoglobulin, for example, the variable region ofthe heavy chain and the variable region ofthe light chain are ligated to a spacer, such as one encoding at least about four small neutral amino acids, i.e., glycine or serine.
  • the protein encoded by this fusion allows the assembly of a functional variable region that retains the specificity and affinity ofthe original antibody.
  • the invention also provides intrabodies that are intracellularly expressed single-chain antibody molecules designed to specifically bind and inactivate target molecules inside cells. Intrabodies have been used in cell assays and in whole organisms (Chen et al, 1994; Hassanzadeh et al, 1998). Inducible expression vectors can be constracted with intrabodies that react specifically with a protein ofthe invention. These vectors can be introduced into host cells and model organisms. [0120] The invention also provides "artificial" antibodies, e.g., antibodies and antibody fragments produced and selected in vitro. In some embodiments, these antibodies are displayed on the surface of a bacteriophage or other viral particle, as described above.
  • artificial antibodies are present as fusion proteins with a viral or bacteriophage stractural protein, including, but not limited to, Ml 3 gene III protein.
  • Methods of producing such artificial antibodies are well known in the art (U.S. Patent Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988; 5,498,538; 5,403,484; 5,571,698; and 5,625,033).
  • the artificial antibodies selected for example, on the basis of phage binding to selected antigens, can be fused to a Fc fragment of an immunoglobulin for use as a therapeutic, as described, for example, in US 5,116,964 or WO99/61630.
  • Antibodies ofthe invention can be used to modulate biological activity of cells, either directly or indirectly.
  • a subject antibody can modulate the activity of a target cell, with which it has primary interaction, or it can modulate the activity of other cells by exerting secondary effects, i.e., when the primary targets interact or communicate with other cells.
  • the antibodies ofthe invention can be administered to mammals, and the present invention includes such administration, particularly for therapeutic and/or diagnostic purposes in humans.
  • Antibodies may be administered by injection systemically, such as by intravenous injection; or by injection or application to the relevant site, such as by direct injection into a tumor, or direct application to the site when the site is exposed in surgery; or by topical application, such as if the disorder is on the skin, for example.
  • the humanized antibody can be the product of an animal having transgenic human immunoglobulin genes, e.g., constant region genes (e.g., Grosveld and Kolias, 1992(not in reference section); Murphy and Carter, 1993 (not in reference section); Pinkert, 1994 (not in reference section); and International Patent Applications WO90/10077 and WO90/04036).
  • transgenic human immunoglobulin genes e.g., constant region genes (e.g., Grosveld and Kolias, 1992(not in reference section); Murphy and Carter, 1993 (not in reference section); Pinkert, 1994 (not in reference section); and International Patent Applications WO90/10077 and WO90/04036).
  • the antibody of interest can be engineered by recombinant DNA techniques to substitute the CHI, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see, e.g., WO92/02190).
  • Humanized antibodies can also be produced by immunizing mice that make human antibodies, such as Abgenix xenomice, Medarex's mice, or Kirin's mice, (Coligan, 2002). Both polyclonal and monoclonal antibodies made in non-human animals may be humanized before administration to human subjects.
  • Chimeric immunoglobulin genes constructed with immunoglobulin cDNA are known in the art (Liu et al. 1987a; Liu et al. 1987b).
  • Messenger RNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
  • the cDNA of interest can be amplified by the polymerase chain reaction using specific primers (U.S. Patent nos. 4,683,195 and 4,683,202).
  • a library is made and screened to isolate the sequence of interest.
  • the DNA sequence encoding the variable region ofthe antibody is then fused to human constant region sequences.
  • the sequences of human constant regions genes are known in the art (Kabat et al, 1991). Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or antibody-dependent cellular cytotoxicity.
  • IgGl, IgG3 and IgG4 isotypes, and either ofthe kappa or lambda human light chain constant regions can be used.
  • the chimeric, humanized antibody is then expressed by conventional methods.
  • Consensus sequences of heavy (“H") and light (“L”) J regions can be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.
  • C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.
  • a convenient expression vector for producing antibodies is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any NH or VL sequence can be easily inserted and expressed, such as plasmids, retroviruses, YACs, or EBV derived episomes, and the like.
  • splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream ofthe coding regions.
  • the resulting chimeric antibody can be joined to any strong promoter, including retroviral LTRs, e.g., SN-40 early promoter, (Okayama, et al. 1983), Rous sarcoma viras LTR (Gorman et al. 1982), and Moloney murine leukemia viras LTR (Grosschedl et al. 1985), or native immunoglobulin promoters.
  • the antibodies can be fully human antibodies.
  • xenogenic antibodies which are produced in animals that are transgenic for human antibody genes, can be employed.
  • xenogenic human antibodies antibodies that are fully human antibodies, with the exception that they are produced in a non-human host that has been genetically engineered to express human antibodies, (e.g., WO98/50433; WO98/24893 and WO99/53049).
  • Antibody fragments such as Fv, F(ab')2 and Fab can be prepared by cleavage ofthe intact protein, e.g., by protease or chemical cleavage. These fragments can include heavy and light chain variable regions.
  • a trancated gene can be designed, e.g., a chimeric gene encoding a portion ofthe F(ab') 2 fragment that includes DNA sequences encoding the CHI domain and hinge region ofthe H chain, followed by a translational stop codon.
  • the antibodies ofthe present invention may be administered alone or in combination with other molecules for use as a therapeutic, for example, by linking the antibody to cytotoxic agent, as discussed above, or to a radioactive molecule. Radioactive antibodies that are specific to a cancer cell, disease cell, or virus-infected cell may be able to deliver a sufficient dose of radioactivity to kill such cancer cell, disease cell, or virus-infected cell.
  • the antibodies ofthe present invention can also be used in assays for detection ofthe subject polypeptides.
  • the assay is a binding assay that detects binding of a polypeptide with an antibody specific for the polypeptide; the subject polypeptide or antibody can be immobilized, while the subject polypeptide and/or antibody can be detectably- labeled.
  • the antibody can be directly labeled or detected with a labeled secondary antibody. That is, suitable, detectable labels for antibodies include direct labels, which label the antibody to the protein of interest, and indirect labels, which label an antibody that recognizes the antibody to the protein of interest.
  • radioisotopes including, but not limited to 64 Cu, 67 Cu, 90 Y, 124 1, 125 1, 13, 1, 137 Cs, 186 Re, 21 I At, 212 Bi, 213 Bi, 223 Ra, 241 Am, and 244 Cm; enzymes having detectable products (e.g., luciferase, ⁇ -galactosidase, and the like); fluorescers and fluorescent labels, e.g., as provided herein; fluorescence emitting metals, e.g., 152 Eu, or others ofthe lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, or acridinium salts; and bioluminescent compounds, e.g., luciferin, or aequorin (green fluorescent protein), specific binding molecules, e.g., magnetic particles, microspheres, nanosphere
  • specific-binding pairs may be used, involving, e.g., a second stage antibody or reagent that is detectably-labeled and that can amplify the signal.
  • a primary antibody can be conjugated to biotin, and horseradish peroxidase-conjugated strepavidin added as a second stage reagent.
  • Digoxin and antidigoxin provide another such pair.
  • the secondary antibody can be conjugated to an enzyme such as peroxidase in combination with a substrate that undergoes a color change in the presence ofthe peroxidase.
  • immunogenic methods ofthe invention can be used alone or in combination with other conventional or unconventional therapies.
  • immunogenic molecules can be combined with other molecules that have a variety of antiproliferative effects, or with additional substances that help stimulate the immune response, i.e., adjuvants or cytokines.
  • Table 1 lists the sequences in the Sequence Listing. Each is identified by an internal Identification (FP ID) number, a SEQ ID NO. corresponding to the nucleotide coding sequence (SEQ ID NO. (NI)), a SEQ ID NO. corresponding to the encoded polypeptide sequence (SEQ ID NO. (PI)), and a SEQ ID NO. corresponding to the entire nucleotide sequence which may include UTRs (SEQ ID NO. (NO)). Finally, Table 1 lists the Clone ID, another internal designation for the sequence. [0132] Table 2 lists the polypeptides ofthe present invention and certain of their characteristics. FP ID represents an internal identification number. Clone ID represents an internal clone identification number.
  • Each sequence is characterized by a predicted protein length in number of amino acid residues (Pred Prot Len) and a Tree Vote representing the probability ofthe sequence being a secreted protein, where a Tree Vote of greater than 0.5 indicates a high probability the protein is secreted.
  • the Mature Protein Coords designate the amino acid residues where the mature protein starts and ends, with the first amino acid residue at the N terminus being amino acid residue number 1.
  • the alternate Mature Protein Coords represent possible variations in the length ofthe mature protein.
  • the positions ofthe amino acid residues that comprise the signal peptides ofthe proteins ofthe invention are also listed (Signal Peptide Coords).
  • Table 2 also specifies the number of transmembrane domains of each claimed sequence (TM), and the position(s), if any, ofthe amino acid residues comprising the transmembrane domains in each claimed sequence (TM Coords).
  • Non-TM Coords represent the amino acid residues ofthe regions that do not comprise TM domains.
  • Table 2 lists the protein family domains present in the sequence (Pfam).
  • Table 2 lists the closest analog ofthe sequence in the human genome, as described by the public National Center for Information Biotechnology (NCBI) database (Top Human Hit Annotation).
  • NCBI National Center for Information Biotechnology
  • MTM indicates a multi-transmembrane polypeptide.
  • STM indicates a single transmembrane polypeptide.
  • Typel_membrane STM indicates that the protein spans the membrane in an orientation with an amino-terminal extracytoplasmic or luminal domain.
  • TypeII_membrane STM indicates that the protein spans the membrane in an orientation with an amino-terminal cytoplasmic domain.
  • Pred SV Prot Len represents the predicted length ofthe splice variant polypeptide in number of amino acid residues.
  • TM for SV represents the coordinates ofthe TM domains ofthe splice variant.
  • TM for Public represents the coordinates ofthe TM domains of a known sequence in the public domain.
  • Table 4 lists the coordinates ofthe Pfam domains ofthe polypeptides ofthe invention. Each is identified by the FP ID and the Clone ID. The Pfam domains are listed (Pfam) and the Pfam coordinates are listed in terms of amino residues beginning with "1" at the N-terminus ofthe polypeptide, beginning with the beginning ofthe open reading frame. Drawings
  • Figure 1 Alignment of HG1014648P1 CLN00497911 (160 aa) with gi
  • Figure 2. Alignment of HG1014652P1 CLN00177675 (106 aa) with gi
  • the invention provides polynucleotides and polypeptides, listed in the Tables and Sequence Listing.
  • polypeptides and polynucleotides have novel functions, and provide methods of diagnosis, treatment, and prophylaxis for immune disorders and cancer, including cancers ofthe lung, bladder, prostate, breast, liver, pancreas, kidney, ovary, cervix, skin, bone, brain, and gastrointestinal tract, such as esophagus, stomach, colon, and rectum, as well as soft tissue sarcomas, multiple myelomas, leukemias, and lymphomas.
  • Some of these polypeptides comprise regions that correspond to Pfamdomains.
  • the regions ofthe polypeptides that correspond to a particular Pfam domain can exhibit variations among polypeptides.
  • fibroblast growth factor receptors ofthe invention comprise epidermal growth factor (EGF) domains, which have variable polypeptide sequences, and are encoded by variable nucleotide sequences.
  • EGF epidermal growth factor
  • the invention also provides a method of making the polypeptides comprising providing a nucleic acid molecule that comprises a polynucleotide sequence that encodes the polypeptide, introducing the nucleic acid molecule into an expression system, and allowing expression ofthe polypeptide.
  • the expression system can be a cell-free system, such as wheat germ extract, a rabbit reticulocyte, or a frog oocyte expression system. It can also be a bacterial expression system, a yeast expression system, an insect cell expression system, or a mammalian cell expression system.
  • the invention provides an antibody that specifically recognizes, binds to, and/or modulates the biological activity of at least one polypeptide encoded by a polynucleotide sequence according to Table 1 and/or the Sequence Listing.
  • This antibody composition can comprise the antibody and a pharmaceutically acceptable carrier.
  • the invention also provides a non-human animal injected with the antibody composition.
  • the antibody can be a monoclonal antibody, a polyclonal antibody, a single chain antibody, an antibody comprising a backbone of a molecule with an Ig domain, a targeting antibody, a neutralizing antibody, a stabilizing antibody, an enhancing antibody, an antibody agonist, an antibody antagonist, an antibody that promotes endocytosis of a target antigen, an antibody that recruits other cells, a cytotoxic antibody, an antibody that mediates ADCC, a human antibody, a non-human primate antibody, a non-primate animal antibody, a rabbit antibody, a mouse antibody, a rat antibody, a sheep antibody, a goat antibody, a horse antibody, a porcine antibody, a cow antibody, a chicken antibody, a humanized antibody, a primatized antibody, or a chimeric antibody.
  • a cytotoxic antibody ofthe invention can possess radioisotopes, microbial toxins, plant toxins, and/or chemical compounds, e.g., doxorabic
  • the antibody can comprise an active fragment of an immunoglobulin.
  • the fragment can be an antigen-binding fragment. It can be chosen from a variable domain, a constant domain, a heavy chain, a light chain, a cdr fragment, and a framework fragment.
  • the antibody can specifically bind to or interfere with the activity of a polypeptide or a ligand ofthe polypeptide. It can be directed to a polypeptide sequence of at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or at least 22 contiguous amino acid residues chosen from the Sequence Listing and/or Table 1. These contiguous residues can correspond to one or more extracellular domain of a polypeptide, or fragment thereof, analogue thereof, and/or antagonist thereto. These residues can correspond to a Pfam domain.
  • the antibody may recognize one or more antigenic epitope. It may specifically recognize one variant ofthe Pfam domain, or more than one variant.
  • the antibody can be produced either in vivo or in vitro, and can be produced by either a prokaryote or a eukaryote, such as a bacterial cell, a fungal cell, a plant cell, an insect cell, and a mammalian cell.
  • suitable cells include yeast cells, Aspergillus cells, SF9 cells, High Five cells, cereal plant cells, tobacco cells, and tomato cells.
  • the antibody can be isolated.
  • the antibody can function to specifically inhibit the binding ofthe polypeptide to a ligand, specifically inhibit the binding ofthe polypeptide to a substrate, specifically inhibit the binding ofthe polypeptide as a ligand, and/or specifically inhibit the binding ofthe polypeptide as a substrate.
  • the invention provides a bacteriophage that displays an antibody, such as the antibodies described above. The antibody may be displayed on the surface ofthe bacteriophage. The invention also provides a bacterial cell comprising the bacteriophage. It further provides a host cell that secretes an antibody ofthe invention.
  • the invention provides a method for making an antibody by introducing a polypeptide, polynucleotide encoding the polypeptide, or a biologically active fragment thereof, into an animal in sufficient amount to elicit generation of antibodies specific to the polypeptide, wherein the polypeptide is described in the sequence listing, or Table 1, and recovering the antibodies.
  • This method may further entail isolating a spleen from the animal injected with the polypeptide or polynucleotide or a fragment thereof, and recovering the antibodies from the spleen cells. It may also further entail making a hybridoma using spleen cells and selecting a hybridoma that secretes the antibodies.
  • the invention provides making a polynucleotide library from the spleen cells, selecting a cDNA clone that produces the antibodies, and expressing the cDNA clone in an expression system to produce antibodies or fragments thereof.
  • the cDNA clone, or a fragment thereof can be introduced into an expression system to produce the antibody.
  • This expression system can be an in vitro system, such as a cell-free system, a bacterial cell expression system, a yeast expression system, or a mammalian sell expression system.
  • the invention provides a method for modulating biological activity by providing an antibody, such as one described above, and contacting this antibody with a first human or non-human host cell, thereby modulating the activity of a first human or non-human animal host cell, or a second host cell.
  • Modulators also take the form of small molecule modulators.
  • the modulation of biological activity can take the form of enhancing cell activity directly, enhancing cell activity indirectly, inhibiting cell activity directly, and/or inhibiting cell activity indirectly. It can also take the form of modulating signal transduction, transcription, and/or translation. Modulation can result in cell growth, inhibition of cell growth and/or cell death.
  • the first host cell can, for example, be a cancer cell.
  • Either the first or second host cell can be a T cell, B cell, NK cell, dendritic cell, macrophage, muscle cell, stem cell, skin cell, fat cell, blood cell, brain cell, bone marrow cell, endothelial cell, retinal cell, bone cell, kidney cell, pancreatic cell, liver cell, spleen cell, prostate cell, cervical cell, ovarian cell, breast cell, lung cell, liver cell, soft tissue cell, colorectal cell, or gastrointestinal tract cell.
  • the invention provides a method for screening for a modulator of polypeptide activity by providing a composition comprising a polypeptide or an active fragment thereof, wherein the polypeptide is chosen from the Sequence Listing or Table 1, allowing at least one modulator to contact the polypeptide, and selecting a modulator that binds to the polypeptide or interferes with the activity ofthe polypeptide.
  • the polypeptide can be expressed on a cell surface. It can be an antibody.
  • a modulator selected in this manner can be present in a composition with a pharmaceutically acceptable carrier.
  • the invention provides a method for diagnosing a proliferative disease such as cancer, psoriasis, and ulcerative colitis, or an immune or inflammatory disease such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis, by providing an antibody, allowing the antibody to contact a patient sample, and detecting specific binding between the antibody and an antigen in the sample to determine whether the subject has proliferative disease such as cancer.
  • a proliferative disease such as cancer, psoriasis, and ulcerative colitis
  • an immune or inflammatory disease such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis
  • the invention also provides a method for diagnosing a proliferative disease, by providing a polypeptide that specifically binds the antibody, allowing the polypeptide to contact a patient sample, and detecting specific binding between the polypeptide and any interacting molecule in the sample to determine whether the subject has a proliferative disease.
  • the invention provides a method for identifying an agent that modulates the biological activity of a polypeptide by providing a polypeptide, an active fragment thereof, an analogue thereof, and/or an antagonist thereto, wherein the polypeptide comprises at least one amino acid sequence found in the Sequence Listing or Table 1; allowing at least one agent to contact the polypeptide; and selecting an agent that binds the polypeptide or affects the biological activity ofthe polypeptide.
  • the polypeptide can be expressed on a cell surface.
  • the invention provides a related modulator composition comprising a modulator and a pharmaceutically acceptable carrier, wherein the modulator is obtainable by the method described above.
  • the invention provides that the modulator ofthe composition can be an antibody.
  • the invention also provides a method of treating a proliferative disease with a composition such as a nucleic acid composition comprising a pharmaceutically acceptable carrier and one or more molecules comprising sequences from Table 1 and/or the Sequence Listing; or an antibody composition, as described above.
  • a composition such as a nucleic acid composition comprising a pharmaceutically acceptable carrier and one or more molecules comprising sequences from Table 1 and/or the Sequence Listing; or an antibody composition, as described above.
  • the composition can administered, for example, orally, parenterally, by implantation, by inhalation, intranasally, intravenously, intra-arterially, intracardiacally, subcutaneously, intraperitoneally, transdermally, intraventricularly, intracranially, and intrathecally.
  • the invention yet also provides a method of treating a proliferative disease by providing an antibody composition that comprises a first antibody or fragment thereof that specifically binds to a first epitope of a first polypeptide or a biologically active fragment thereof, wherein the first polypeptide is encoded by a polynucleotide sequence or polypeptide sequence found in Table 1 and/or the Sequence Listing, and administering the antibody composition to a subject in need of such treatment.
  • the antibody composition can further comprise a second antibody that binds specifically to or interferes with the activity of a second epitope ofthe first polypeptide or to a first epitope of a second polypeptide.
  • the second polypeptide can be chosen from the Tables and Sequence Listing.
  • the invention provides modulators that can induce apoptosis.
  • the default state ofthe cell is to remain alive.
  • a cell enters the apoptotic pathway when an essential factor is removed from the extracellular environment or when an internal signal is activated.
  • Genes and proteins ofthe invention that suppress the growth of tumors by activating cell death provide the basis for treatment strategies for hyperproliferative disorders and conditions.
  • Apoptosis can be assayed using any known method. Assays can be conducted on cell populations or an individual cell, and include morphological assays and biochemical assays. Procedures to detect cell death based on the TUNEL method are available commercially, e.g., from Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus).
  • the invention provides therapeutic agent screening, such as small molecule drag screening.
  • the invention also provides therapeutic applications, such as in the treatment of a variety of diseases and conditions, including, e.g., cancer, proliferative disorders, immune disorders, inflammatory disorders, and other metabolic disorders.
  • the invention further provides a kit comprising an antibody as described above, and instructions for its use.
  • the invention yet further provides method of gene therapy, comprising providing a polynucleotide comprising a nucleic acid molecule encoding the antibody of claim 1, and administering the polynucleotide to a subject in need of such treatment.
  • the invention provides a method for prophylactically or therapeutically treating a subject by providing a vaccine and administering the vaccine to the subject; wherein the vaccine comprises a polynucleotide or a polypeptide found in the Sequence Listing or Table 1, or a fragment thereof, an analogue thereof, or an antagonist thereto.
  • the vaccine can be a cancer vaccine, and the polypeptide can be a cancer antigen.
  • Therapeutic vaccines can be in the form of nucleic acid or polypeptide vaccines, and can be administered alone, such as naked DNA, or can be facilitated, such as via the use of a viral vector, microsomes, or liposomes.
  • the invention also provides a method of inhibiting transcription or translation of a first polynucleotide encoding a first polypeptide by providing a second polynucleotide that hybridizes to the first polynucleotide, wherein the first polynucleotide comprises a polynucleotide sequence chosen from a polynucleotide or a polypeptide found in the Sequence Listing or Table 1, or a fragment thereof, an analogue thereof, or an antagonist thereto, and allowing the first polynucleotide to contact the second polynucleotide.
  • the second polynucleotide can comprise an antisense molecule, a ribozyme, and/or an interfering RNA (iRNA) molecule.
  • the invention yet also provides a method of treating a proliferative disorder by administering a modulator to a subject in need of such treatment, wherein the modulator binds to a cell surface molecule that is overexpressed in the disorder.
  • the modulator can be an antibody, for example, one that is capable of initiating ADCC.
  • Other embodiments ofthe invention will be apparent to those skilled in the art from consideration ofthe specification and practice ofthe invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit ofthe invention being indicated by the following claims. Examples
  • PAP2C can be expressed in vitro in a cell free expression system, using wheat germ lysate or E. coli lysate.
  • PAP2C can be expressed in a baculovirus system (Doerfler, W., Bohm, P., eds. 1987; Luckow, V. and Summers, M. 1988).
  • the expressed protein can be substantially purified (Deutscher, M.P., et al, eds. 1990) and used for injection into mice for production of antibodies.
  • the mice can be normal mice, in which case, the resulting monoclonal antibodies can be made in accordance to conventional techniques, but will have to be humanized for use in treatment of humans.
  • the expressed protein can also be used for injection into XenoMouse or other similar mice owned by Abgenix, Inc. (Fremont, California, USA), Medarex, Inc. (Princeton, NJ, USA) or Kirin (Japan), which are capable of producing human antibodies.
  • the expressed protein can also be used to screen for binding with Fab fragments of antibodies displayed on bacteriophages, using phage display libraries, such as is available from Cambridge Antibody Technology (Cambridge, U.K.), MorphoSys (Martinsried Kunststoff, Germany) or Dyax Corp. (Cambridge, MA, USA).
  • the Fab fragments that bind the PAP2C polypeptide with high affinity can be validated by immunohistochemistry as binding to tumor tissues.
  • the desired Fab fragment can fused to an appropriate Fc fragment to make a synthetic antibody.
  • RNA expression profiles were analyzed to identify genes that are overexpressed in cancer tissues relative to their normal counterparts. These genes represent a class of targets for cancer therapy.
  • cRNA was prepared from various cancer tissues or from normal tissues ofthe same tissue type. Labeled cRNA was hybridized to sequences on an Affymetrix U133 or a FivePrime custom DNA microarray. Probes populating the FivePrime custom array are designated "prbxxxxxx.” A hybridization score calculated from raw data measured the relative expression level of a RNA in a tissue sample. Expression levels of specific genes in cancerous and non-cancerous tissues were compared. [0183] DNA microarrays containing probe sequences which exactly match the gene sequences for a gene of interest and probe sequences with one mismatched nucleotide were tested. cRNAs that hybridized to both the matching sequences and the mismatched sequences were deemed to hybridize non-specifically.
  • RNAs that hybridized to the matched sequences but not to the mismatched sequences were deemed to hybridize specifically. Sequences which hybridize specifically are herein designated "present.”
  • Differential RNA expression profiles were analyzed to identify genes present in a greater number of cancer tissues samples than their normal counterparts. These genes represent a class of targets for cancer therapy.
  • the following indications are supported for the following respective proteins.
  • Proteins in cluster 180153 lung cancer (including adenocarcinoma) (supported by prbl00544 and 201653), ovarian cancer (supported by 201653), and pancreatic cancer (supported by 201653 and 225553).
  • Proteins in cluster 204091 colorectal cancer (supported by prbl06316), liver cancer (supported by 201704), lung cancer (including adenocarcinoma and squamous cell carcinoma) (supported by 201704 and prbl06316), ovarian cancer (supported by 201704), pancreatic cancer (supported by 201704), prostate cancer (supported by 201704 and prbl06316), skin cancer (supported by 201704), and stomach cancer (supported by 201704).
  • Proteins in cluster 183211 bladder cancer (supported by 209529), breast cancer (supported by 209529), colorectal cancer (supported by 209529 and prbl02183), endometrial cancer (supported by 209529), esophageal cancer (supported by 209529), kidney cancer (supported by 209529), liver cancer (supported by 209529), lung cancer (including adenocarcinoma and squamous cell carcinoma) (supported by 209529 and prbl02183), ovarian cancer (supported by 209529), pancreatic cancer (supported by 209529), prostate cancer (supported by 209529 and prbl02183), stomach cancer (supported by 209529), testicular cancer (supported by 209529), and thyroid cancer (supported by 209529).
  • Proteins in cluster 188909 bladder cancer (supported by 203953 and 203954), brain cancer (supported by 203953 and 203954), breast cancer (supported by 203953 and 203954), endometrial cancer (supported by 203953 and 203954), esophageal cancer (supported by 203953 and 203954), kidney cancer (supported by 203953 and 203954), liver cancer (supported by 203953 and 203954), lung cancer (including lung adenocarcinoma) (supported by 203953 and 203954 and prbl03266 and prbl07027), ovarian cancer (supported by 203953 and 203954), prostate cancer (supported by 203953 and 203954 and prbl03266 and prbl07027), and stomach cancer (supported by 203953 and 203954).
  • bladder cancer supported by 203953 and 203954
  • brain cancer supported by 203953 and 203954
  • breast cancer supported by 203953 and 203954
  • endometrial cancer supported by 203953 and 20
  • compositions and methods ofthe invention are useful in the diagnosis, treatment, and/or prevention of proliferative and immune disorders.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des variants d'épissure de polypeptides qui sont considérablement exprimés dans des tissus tumoraux. De tels polypeptides sont utilisés à des fins thérapeutiques, diagnostiques et prophylactiques. Notamment, lesdits polypeptides sont utilisés en tant que cibles pour le développement de modulateurs dans le traitement de maladies.
PCT/US2005/002695 2004-01-30 2005-01-31 Nouveaux variants d'epissure et methodes d'utilisation associees Ceased WO2005076012A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54074504P 2004-01-30 2004-01-30
US60/540,745 2004-01-30

Publications (2)

Publication Number Publication Date
WO2005076012A2 true WO2005076012A2 (fr) 2005-08-18
WO2005076012A3 WO2005076012A3 (fr) 2006-02-16

Family

ID=34837419

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/002695 Ceased WO2005076012A2 (fr) 2004-01-30 2005-01-31 Nouveaux variants d'epissure et methodes d'utilisation associees

Country Status (1)

Country Link
WO (1) WO2005076012A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007034160A3 (fr) * 2005-09-23 2007-07-26 Astrazeneca Ab Methode diagnostique
CN116284224A (zh) * 2023-05-12 2023-06-23 中国农业大学 一种结合Claudin 18.2的环肽及其应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5968744A (en) * 1997-10-14 1999-10-19 Incyte Pharmaceticals, Inc. Human cornichon molecule
JP2002511231A (ja) * 1997-12-30 2002-04-16 カイロン コーポレイション 骨髄分泌タンパク質およびポリヌクレオチド
NZ523206A (en) * 1999-08-31 2004-12-24 Genentech Inc Antibodies that bind to tumor gene sequences
AU2001249642A1 (en) * 2000-04-06 2001-10-23 Genetics Institute, Llc Polynucleotides encoding novel secreted proteins
CA2460959A1 (fr) * 2001-09-28 2003-05-22 Incyte Genomics, Inc. Enzymes
US7193069B2 (en) * 2002-03-22 2007-03-20 Research Association For Biotechnology Full-length cDNA
AU2003289716A1 (en) * 2002-09-12 2004-04-30 Incyte Corporation Molecules for diagnostics and therapeutics

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007034160A3 (fr) * 2005-09-23 2007-07-26 Astrazeneca Ab Methode diagnostique
CN116284224A (zh) * 2023-05-12 2023-06-23 中国农业大学 一种结合Claudin 18.2的环肽及其应用
CN116284224B (zh) * 2023-05-12 2023-07-25 中国农业大学 一种结合Claudin 18.2的环肽及其应用

Also Published As

Publication number Publication date
WO2005076012A3 (fr) 2006-02-16

Similar Documents

Publication Publication Date Title
JP6957546B2 (ja) ヒト非小細胞肺癌における転座及び変異体ｒｏｓキナーゼ
JP6223822B2 (ja) 未分化リンパ腫キナーゼ（ａｌｋ）キナーゼ阻害剤に耐性である癌の診断および治療のための方法および組成物
US20030044409A1 (en) Immunologic compositions and methods for studying and treating cancers expressing frizzled antigens
DK2715365T3 (en) ROSE KINASE IN LUNG CANCER
EA007471B1 (ru) Растворимый рецептор br43x2 и способы его применения
KR20090111307A (ko) 전립선 특이적 전사물 및 전립선암의 치료 및 진단에 사용되는 이의 용도
US20100015047A1 (en) Lung-Expressed Polypeptides
JP2024164067A (ja) 新規のｄｄｒ１抗体およびその使用
WO2005070965A2 (fr) Compositions pharmaceutiques contenant des antagonistes a lrp4, lrp8 ou a la megaline pour le traitement de maladies
US20090214517A1 (en) Compositions and methods of use for modulators of nectin 4, semaphorin 4b, igsf9, and kiaa0152 in treating disease
US8962808B2 (en) EGFR-related polypeptides and methods of use
JP2008522162A (ja) Ｍｅｒの診断用および治療用の作用薬
US8404810B2 (en) Compositions and methods of use for modulators of polypeptides and polynucleotides in treating breast cancer and melanoma
WO2005076012A2 (fr) Nouveaux variants d'epissure et methodes d'utilisation associees
WO2006014903A2 (fr) Compositions et techniques d'utilisation d'antagonistes adam12 dans le traitement de maladie
EP1439223A1 (fr) Nouveau recepteur des cytokines de classe ii
US8003599B2 (en) Inhibitors of L1 and ADAM10 for the treatment of carcinomas
US7141365B2 (en) Methods for diagnosing a mucin production associated disease condition
JP2012143242A (ja) 乳癌において差次的に発現される遺伝子
US20100028867A1 (en) LRRTM1 Compositions and Methods of Their Use for the Diagnosis and Treatment of Cancer
JP2004534525A (ja) ｕＰＡＲ変異体ｄｅｌ４、ｄｅｌ５およびｄｅｌ４＋５に対する単特異性のポリクローナル抗体の製法並びに診断および治療目的のためのその使用
US20070274988A1 (en) Kiaa0779, Splice Variants Thereof, and Methods of Their Use
US7081517B2 (en) Genes differentially expressed in breast cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

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

AL Designated countries for regional patents

Kind code of ref document: A2

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase