US20050202496A1 - Secreted and transmembrane polypeptides and nucleic acids encoding the same - Google Patents

Secreted and transmembrane polypeptides and nucleic acids encoding the same Download PDF

Info

Publication number
US20050202496A1
US20050202496A1 US11/110,520 US11052005A US2005202496A1 US 20050202496 A1 US20050202496 A1 US 20050202496A1 US 11052005 A US11052005 A US 11052005A US 2005202496 A1 US2005202496 A1 US 2005202496A1
Authority
US
United States
Prior art keywords
pro
acid sequence
nucleic acid
alternatively
antibody
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.)
Abandoned
Application number
US11/110,520
Inventor
Audrey Goddard
Paul Godowski
Austin Gurney
Victoria Smith
William Wood
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.)
Genentech Inc
Original Assignee
Genentech 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
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=21841414&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20050202496(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from PCT/US2000/032678 external-priority patent/WO2001040466A2/en
Priority claimed from US10/028,072 external-priority patent/US20030004311A1/en
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to US11/110,520 priority Critical patent/US20050202496A1/en
Publication of US20050202496A1 publication Critical patent/US20050202496A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/0005Vertebrate antigens
    • A61K39/0011Cancer antigens
    • A61K39/001102Receptors, cell surface antigens or cell surface determinants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702Regulators; Modulating activity
    • C07K14/4703Inhibitors; Suppressors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546Integrin superfamily
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82Translation products from oncogenes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K17/00Carrier-bound or immobilised peptides; Preparation thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/001Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1048Glycosyltransferases (2.4)
    • C12N9/1051Hexosyltransferases (2.4.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90Isomerases (5.)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/02Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/06Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • G01N33/5011Chemical 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • G01N33/5014Chemical 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 toxicity
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • G01N33/502Chemical 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 non-proliferative effects
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • G01N33/5044Chemical 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 involving specific cell types
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • G01N33/5044Chemical 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 involving specific cell types
    • G01N33/5047Cells of the immune system
    • G01N33/505Cells of the immune system involving T-cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical 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
    • G01N33/5044Chemical 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 involving specific cell types
    • G01N33/5061Muscle cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845Methods of identifying protein-protein interactions in protein mixtures
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00Uses of viruses
    • C12N2799/02Uses of viruses as vector
    • C12N2799/021Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/136Screening for pharmacological compounds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/04Endocrine or metabolic disorders
    • G01N2800/042Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827Proteins from mammals or birds
    • Y10S530/828Cancer
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/866Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain

Definitions

  • the present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides.
  • Extracellular proteins play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms.
  • secreted polypeptides for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones
  • secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.
  • Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors.
  • Most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins.
  • Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents.
  • Efforts are being undertaken by both industry and proficient to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].
  • Membrane-bound proteins and receptors can play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms.
  • membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
  • Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents.
  • Receptor immunoadhesins for instance, can be employed as therapeutic agents to block receptor-ligand interactions.
  • the membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alteratively at least about 96% nucleic acid sequence identity,
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 9
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PRO polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes.
  • nucleic acid fragments are usually at least about 10 nucleotides in length, alternatively at least about 15 nucleotides in length, alternatively at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleo
  • novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
  • the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99%
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described.
  • Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • Another aspect the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated.
  • Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein.
  • the agonist or antagonist is an anti-PRO antibody or a small molecule.
  • the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide.
  • the PRO polypeptide is a native PRO polypeptide.
  • the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody.
  • the invention provides vectors comprising DNA encoding any of the herein described polypeptides.
  • Host cell comprising any such vector are also provided.
  • the host cells may be CHO cells, E. coli, or yeast.
  • a process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
  • the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence.
  • Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
  • the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides.
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
  • the invention provides oligonucleotide probes which may be useful for isolating genomic and cDNA nucleotide sequences, measuring or detecting expression of an associated gene or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences. Preferred probe lengths are described above.
  • the present invention is directed to methods of using the PRO polypeptides of the present invention for a variety of uses based upon the functional biological assay data presented in the Examples below.
  • FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequence PRO5997 cDNA, wherein SEQ ID NO:1 is a clone designated herein as “DNA97005-2687”.
  • FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO:1 shown in FIG. 1 .
  • PRO polypeptide and “PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers, to specific polypeptide sequences as described herein.
  • the terms “PRO/number polypeptide” and “PRO/number” wherein the term “number” is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein).
  • the PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • PRO polypeptide refers to each individual PRO/number polypeptide disclosed herein.
  • PRO polypeptide refers to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually.
  • the term “PRO polypeptide” also includes variants of the PRO/number polypeptides disclosed herein.
  • a “native sequence PRO polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term “native sequence PRO polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.
  • the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons are shown in bold font and underlined in the figures. However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides.
  • the PRO polypeptide “extracellular domain”. or “ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein.
  • an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are comtemplated by the present invention.
  • the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).
  • cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species.
  • These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
  • PRO polypeptide variant means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein.
  • Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence.
  • a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length
  • PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more.
  • Percent (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital, UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B.
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest.
  • amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
  • Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • NCBI-BLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • PRO variant polynucleotide or “PRO variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein.
  • a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 9
  • PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
  • Percent (%) nucleic acid sequence identity with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D.
  • nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to, C.
  • Tables 4 and 5 demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated “Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”, wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acid sequence of interest, “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the “PRO-DNA” nucleic acid molecule of interest is being compared, and “N”, “L” and “V” each represent different hypothetical nucleotides.
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest.
  • nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
  • Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • NCBI-BLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md.
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
  • PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length PRO polypeptide as disclosed herein.
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide.
  • Isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • An “isolated” PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid.
  • An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells.
  • an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • antibody is used in the broadest sense and specifically covers, for example, single anti-PRO monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below).
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5 ⁇ SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 ⁇ Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran
  • Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
  • An example of moderately stringent conditions is overnight incubation at 37° C.
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused.
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • immunoadhesin designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains.
  • the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence.
  • the adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand.
  • the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • immunoglobulin such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • “Active” or “activity” for the purposes herein refers to form(s) of a PRO polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring PRO, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO and an “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO.
  • agonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO polypeptide disclosed herein.
  • agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein.
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc.
  • Methods for identifying agonists or antagonists of a PRO polypeptide may comprise contacting a PRO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI Americas Inc., Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS® (BASF Corporation, Mount Olive, N.J.).
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab′) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab fragments differ from Fab′ fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H -V L ).
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal.
  • a drug such as a PRO polypeptide or antibody thereto
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • a “small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • an “effective amount” of a polypeptide disclosed herein or an agonist or antagonist thereof is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides.
  • cDNAs encoding various PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed.
  • PRO/number the protein encoded by the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO, will be referred to as “PRO/number”, regardless of their origin or mode of preparation.
  • PRO variants can be prepared.
  • PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide.
  • amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • Variations in the native full-length sequence PRO or in various domains of the PRO described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934.
  • Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO.
  • the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO.
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • PRO polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide.
  • PRO fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5′ and 3′ primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
  • PCR polymerase chain reaction
  • conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Substantial modifications in function or immunological identity of the PRO polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • the variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)]
  • cassette mutagenesis [Wells et al., Gene, 34:315 (1985)]
  • restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO variant DNA.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)].
  • Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
  • Covalent modifications of PRO are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa.
  • crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • 1,1-bis(diazoacetyl)-2-phenylethane glutaraldehyde
  • N-hydroxysuccinimide esters for example, esters with 4-azidosalicylic acid
  • homobifunctional imidoesters including disuccinimidyl esters such as 3,3′-dithiobis(s
  • Another type of covalent modification of the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO.
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO (for O-linked glycosylation sites).
  • the PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation.
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
  • PRO polypeptide
  • nonproteinaceous polymers e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • the PRO of the present invention may also be modified in a way to form a chimeric molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence.
  • such a chimeric molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the amino- or carboxyl-terminus of the PRO. The presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • tag polypeptides and their respective antibodies are well known in the art.
  • poly-histidine poly-his
  • poly-histidine-glycine poly-his-glycine tags
  • flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]
  • c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]
  • Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)].
  • tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an ⁇ -tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al. Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
  • the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin.
  • an immunoglobulin also referred to as an “immunoadhesin”
  • a fusion could be to the Fc region of an IgG molecule.
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule.
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule.
  • PRO sequence or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid - Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)].
  • In vitro protein synthesis may be performed using manual techniques or by automation.
  • Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions.
  • Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO.
  • DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level. Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples.
  • the PRO-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
  • Probes such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
  • An alternative means to isolate the gene encoding PRO is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GENBANK® (US Department of Health and Human Services, Bethesda, Md.) or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
  • Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl 2 , CaPO 4 , liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes.
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989.
  • DNA into cells such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.
  • polycations e.g., polybrene, polyornithine.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
  • Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC® 31,446); E. coli X1776 (ATCC® 31,537); E. coli strain W3110 (ATCC® 27,325) and K5 772 (ATCC® 53,635).
  • suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting.
  • Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.
  • strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E.
  • coli W3110 strain 27C7 (ATCC® 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan r ;
  • E. coli W3110 strain 37D6 which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan r ;
  • E. coli W3110 strain 40B4 which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990.
  • in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K.
  • lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC® 12,424), K. bulgaricus (ATCC® 16,045), K. wickeramii (ATCC® 24,178), K. waltii (ATCC® 56,500), K. drosophilarum (ATCC® 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K.
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem
  • Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula.
  • yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula.
  • yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula.
  • a list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
  • Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC® CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
  • mice sertoli cells TM4, Mather, Biol. Reprod., 23:243-251 (1980)
  • human lung cells W138, ATCC® CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC® CCL51. The selection of the appropriate host cell is deemed to be within the skill in the art.
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • the PRO may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces ⁇ -factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).
  • a suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7.
  • the trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC® No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PRO transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin).
  • an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5′ or 3′ to the PRO coding sequence, but is preferably located at a site 5′ from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO.
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope.
  • PRO may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PRO can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • a suitable detergent solution e.g. Triton-X 100
  • Cells employed in expression of PRO can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, SEPHADEX® G-75(Amersham Biosciences AB Corp., Uppsala, Sweden); PROTEIN A-SEPHAROSETM (Pharmacia Biotech AB, Uppsala, Sweden) columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PRO.
  • Nucleotide sequences (or their complement) encoding PRO have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA.
  • PRO nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein.
  • the full-length native sequence PRO gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length PRO cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of PRO or PRO from other species) which have a desired sequence identity to the native PRO sequence disclosed herein.
  • the length of the probes will be about 20 to about 50 bases.
  • the hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence PRO.
  • a screening method will comprise isolating the coding region of the PRO gene using the known DNA sequence to synthesize a selected probe of about 40 bases.
  • Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32 P or 35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the PRO gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
  • antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRO DNA (antisense) sequences.
  • Antisense or sense oligonucleotides comprise a fragment of the coding region of PRO DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • Stein and Cohen Cancer Res. 48:2659, 1988
  • van der Krol et al. BioTechniques 6:958, 1988.
  • binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
  • the antisense oligonucleotides thus may be used to block expression of PRO proteins.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases.
  • Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
  • sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine).
  • intercalating agents such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO 4 -mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
  • an antisense or sense oligonucleotide is inserted into a suitable retroviral vector.
  • a cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo.
  • Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
  • the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • Antisense or sense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more.
  • the probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related PRO coding sequences.
  • Nucleotide sequences encoding a PRO can also be used to construct hybridization probes for mapping the gene which encodes that PRO and for the genetic analysis of individuals with genetic disorders.
  • the nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
  • the PRO can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor PRO can be used to isolate correlative ligand(s). Screening assays can be designed to find lead compounds that mimic the biological activity of a native PRO or a receptor for PRO. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • Small molecules contemplated include synthetic organic or inorganic compounds.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • Nucleic acids which encode PRO or its modified forms can also be used to generate either transgenic animals or “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents.
  • a transgenic animal e.g., a mouse or rat
  • a transgenic animal is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops.
  • cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding PRO.
  • Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.
  • particular cells would be targeted for PRO transgene incorporation with tissue-specific enhancers.
  • Transgenic animals that include a copy of a transgene encoding PRO introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding PRO.
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
  • an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
  • non-human homologues of PRO can be used to construct a PRO “knock out” animal which has a defective or altered gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced into an embryonic stem cell of the animal.
  • cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques. A portion of the genomic DNA encoding PRO can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors].
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)].
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152].
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a “knock out” animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO polypeptide.
  • Nucleic acid encoding the PRO polypeptides may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane.
  • oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique, of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem.
  • PRO polypeptides described herein may also be employed as molecular weight markers for protein electrophoresis purposes and the isolated nucleic acid sequences may be used for recombinantly expressing those markers.
  • nucleic acid molecules encoding the PRO polypeptides or fragments thereof described herein are useful for chromosome identification.
  • there exists an ongoing need to identify new chromosome markers since relatively few chromosome marking reagents, based upon actual sequence data are presently available.
  • Each PRO nucleic acid molecule of the present invention can be used as a chromosome marker.
  • PRO polypeptides and nucleic acid molecules of the present invention may also be used diagnostically for tissue typing, wherein the PRO polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type.
  • PRO nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
  • PRO polypeptides described herein may also be employed as therapeutic agents.
  • the PRO polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the PRO product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI Americas Inc., Bridgewater, N.J.), PLURONICS® (BASF Corporation, Mount Olive, N.J.) or PEG.
  • buffers such as phosphate,
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the route of administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems.
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The use of interspecies scaling in toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.
  • normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 ⁇ g/kg/day to 10 mg/kg/day, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
  • microencapsulation of the PRO polypeptide is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed.
  • rhGH human growth hormone
  • rhIFN- interferon-(rhIFN-)
  • interleukin-2 interleukin-2
  • MN rgp120 MN rgp120
  • the sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties.
  • PLGA poly-lactic-coglycolic acid
  • the degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body.
  • the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition.
  • Lewis “Controlled release of bioactive agents from lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.
  • This invention encompasses methods of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists).
  • Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins.
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
  • All assays for antagonists are common in that they call for contacting the drug candidate with a PRO polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
  • the interaction is binding and the complex formed can be isolated or detected in the reaction mixture.
  • the PRO polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments.
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO polypeptide and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component.
  • the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature ( London ), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci.
  • yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain.
  • yeast expression system described in the foregoing publications (generally referred to as the “two-hybrid system”) takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain.
  • GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech, Palo Alto, Calif. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products.
  • a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound.
  • a placebo may be added to a third reaction mixture, to serve as positive control.
  • the binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
  • the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide.
  • antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist.
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).
  • expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO polypeptide. Transfected cells that are grown on glass slides are exposed to labeled PRO polypeptide.
  • the PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
  • labeled PRO polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
  • potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
  • Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5′ coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of the PRO polypeptide.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988).
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide.
  • antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about ⁇ 10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Hoogsteen base-pairing rules which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • Diagnostic and therapeutic uses of the herein disclosed molecules may also be based upon the positive functional assay hits disclosed and described below.
  • the present invention further provides anti-PRO antibodies.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
  • the anti-PRO antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the anti-PRO antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PRO polypeptide or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and art sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, PROTEIN A-SEPHAROSETM (Pharmacia Biotech AB, Uppsala, Sweden), hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable, of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • the anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • the antibodies may also be affinity matured using known selection and/or mutagenesis methods as described above.
  • Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the PRO, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′) 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab′ fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′) 2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein.
  • an anti-PRO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO polypeptide.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide.
  • These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the PRO polypeptide and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
  • cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti - Cancer Drug Design, 3: 219-230 (1989).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that tan be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a receptor such streptavidin
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • the antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
  • Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
  • the PRO polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred.
  • lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • sustained release preparations may be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (TAP Pharmaceuticals, Inc., North Chicago, Ill.) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-( ⁇ )-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • anti-PRO antibodies of the invention have various utilities.
  • anti-PRO antibodies may be used in diagnostic assays for PRO, e.g., detecting its expression (and in some cases, differential expression) in specific cells, tissues, or serum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158].
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • a radioisotope such as 3 H, 14 C, 32 P, 35 S, or 125 I
  • a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et
  • Anti-PRO antibodies also are useful for the affinity purification of PRO from recombinant cell culture or natural sources.
  • the antibodies against PRO are immobilized on a suitable support, such a SEPHADEX® (Amersham Biosciences AB Corp., Uppsala, Sweden) resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the PRO to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the PRO, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the PRO from the antibody.
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases.
  • the EST databases included public databases (e.g., Dayhoff, GENBANK® (US Department of Health and Human Services, Bethesda, Md.), and proprietary databases (e.g. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.).
  • the search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program “phrap” (Phil Green, University of Washington, Seattle, Wash.).
  • consensus DNA sequences were assembled relative to the other identified EST sequences using phrap.
  • consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
  • oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide.
  • Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length.
  • the probe sequences are typically 40-55 bp in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, Calif.
  • the cDNA was primed with oligo dT containing a NotI site, linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique XhoI and NotI sites.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes
  • mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, Md. (Super Script Plasmid System). In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNA was cloned into XhoI/NotI cleaved vector.
  • pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloning sites.
  • a secondary cDNA library was generated in order to preferentially represent the 5′ ends of the primary cDNA clones.
  • Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleaved with SfiI, and cloned into SfiI/NotI cleaved vector.
  • pSST-AMY.0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites.
  • cDNAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
  • DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37° C. for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37° C.). Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCl-gradient. The purified DNA was then carried on to the yeast protocols below.
  • the yeast methods were divided into three categories: (1) Transformation of yeast with the plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis.
  • yeast strain used was HD56-5A (ATCC®-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL + , SUC + , GAL + .
  • yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in sec71, sec72, sec62, with truncated sec71 being most preferred.
  • antagonists including antisense nucleotides and/or ligands which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p or SSA1p-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
  • other proteins implicated in this post translation pathway e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p or SSA1p-4p
  • the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
  • the cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3 ), and resuspended into LiAc/TE (2.5 ml).
  • LiAc/TE 10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3
  • Transformation took place by mixing the prepared cells (100 ⁇ l) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, Md.) and transforming DNA (1 ⁇ g, vol. ⁇ 10 ⁇ l) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 ⁇ l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li 2 OOCCH 3 , pH 7.5) was added. This mixture was gently mixed and incubated at 30° C. while agitating for 30 minutes. The cells were then heat shocked at 42° C.
  • TE 500 ⁇ l, 10 mM Tris-HCl, 1 mM EDTA pH 7.5
  • the cells were then diluted into TE (1 ml) and aliquots (200 ⁇ l) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
  • the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
  • the selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 208-210 (1994). Transformants were grown at 30° C. for 2-3 days.
  • the detection of colonies secreting amylase was performed by including red starch in the selective growth media.
  • Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al., Anal. Biochem., 172: 176-179 (1988).
  • the coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).
  • the positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies.
  • Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.
  • sequence of reverse oligonucleotide 2 was: (SEQ ID NO:4) 5′-CAGGAAACAGCTATGACC ACCTGCACACCTGCAAATCCATT -3′
  • PCR was then performed as follows: a. Denature 92° C., 5 minutes b. 3 cycles of: Denature 92° C., 30 seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c. 3 cycles of: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C., 60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C., 30 seconds Extend 72° C., 60 seconds e. Hold 4° C.
  • the underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY.0 when no insert was present.
  • the first 18 nucleotides of the 5′ end of these oligonucleotides contained annealing sites for the sequencing primers.
  • the total product of the PCR reaction from an empty vector was 343 bp.
  • signal sequence-fused cDNA resulted in considerably longer nucleotide sequences.
  • polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc. (South San Francisco, Calif.) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GENBANK® (US Department of Health and Human Services, Bethesda, Md.)) and/or private (LIPESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) databases.
  • the signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5′-end of the sequence or sequence fragment under consideration.
  • the nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored.
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals. Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
  • the following method describes use of a nucleotide sequence encoding PRO as a hybridization probe.
  • DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries.
  • Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions.
  • Hybridization of radiolabeled PRO-derived probe to the filters is performed in a solution of 50% formamide, 5 ⁇ SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 ⁇ Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1 ⁇ SSC and 0.1% SDS at 42° C.
  • DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO can then be identified using standard techniques known in the art.
  • This example illustrates preparation of an unglycosylated form of PRO by recombinant expression in E. coli.
  • the DNA sequence encoding PRO is initially amplified using selected PCR primers.
  • the primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector.
  • restriction enzyme sites A variety of expression vectors may be employed.
  • An example of a suitable vector is pBR322 (derived from E. coli, see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance.
  • the vector is digested with restriction enzyme and dephosphorylated.
  • the PCR amplified sequences are then ligated into the vector.
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene.
  • the ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
  • Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics.
  • the overnight culture may subsequently be used to inoculate a larger scale culture.
  • the cells are then grown to a desired optical density, during which the expression promoter is turned on.
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation.
  • the cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PRO protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
  • PRO may be expressed in E. coli in a poly-His tagged form, using the following procedure.
  • the DNA encoding PRO is initially amplified using selected PCR primers.
  • the primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase.
  • the PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).
  • Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH 4 ) 2 SO 4 , 0.71 g sodium citrate 2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO 4 ) and grown for approximately 20-30 hours at 30° C. with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
  • CRAP media prepared by mixing 3.57 g (NH 4 ) 2 SO 4 , 0.71 g sodium citrate 2H2O, 1.07 g KCl
  • E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer.
  • Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1 M and 0.02 M, respectively, and the solution is stirred overnight at 4° C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization.
  • the solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min.
  • the supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
  • the clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer.
  • the column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4.
  • the protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4° C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
  • the proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml.
  • the refolding solution is stirred gently at 4° C. for 12-36 hours.
  • the refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3).
  • the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration.
  • the refolded protein is chromatographed on a Poros R1/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
  • Fractions containing the desired folded PRO polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 SUPERFINETM (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
  • This example illustrates preparation of a potentially glycosylated form of PRO by recombinant expression in mammalian cells.
  • the vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as the expression vector.
  • the PRO DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO DNA using ligation methods such as described in Sambrook et al., supra.
  • the resulting vector is called pRK5-PRO.
  • the selected host cells may be 293 cells.
  • Human 293 cells (ATCC® CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics.
  • About 10 ⁇ g pRK5-PRO DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 ⁇ l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl 2 .
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cysteine and 200 ⁇ Ci/ml 35 S-methionine.
  • culture medium alone
  • culture medium containing 200 ⁇ Ci/ml 35 S-cysteine and 200 ⁇ Ci/ml 35 S-methionine After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide.
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
  • PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-PRO DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours.
  • the cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin and 0.1 ⁇ g/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
  • PRO in another embodiment, can be expressed in CHO cells.
  • the pRK5-PRO can be transfected into CHO cells using known reagents such as CaPO 4 or DEAE-dextran.
  • the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35 S-methionine.
  • the culture medium may be replaced with serum free medium.
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested.
  • the medium containing the expressed PRO can then be concentrated and purified by any selected method.
  • Epitope-tagged PRO may also be expressed in host CHO cells.
  • the PRO may be subcloned out of the pRK5 vector.
  • the subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector.
  • the poly-his tagged PRO insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones.
  • the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression.
  • the culture medium containing the expressed poly-His tagged PRO can then be concentrated and purified by any selected method, such as by Ni 2+ -chelate affinity chromatography.
  • PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.
  • Stable expression in CHO cells is performed using the following procedure.
  • the proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG1 constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
  • CHO expression vectors are constructed to have compatible restriction sites 5′ and 3′ of the DNA of interest to allow the convenient shuttling of cDNA's.
  • the vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR).
  • DHFR expression permits selection for stable maintenance of the plasmid following transfection.
  • Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents SUPERFECT® (Quiagen), DOSPERTM (Roche Applied Science, Indianapolis, Ind.) or FUGENE® (Boehringer Mannheim). The cells are grown as described in Lucas et al., supra. Approximately 3 ⁇ 10 ⁇ 7 cells are frozen in an ampule for further growth and production as described below.
  • the ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing.
  • the contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes.
  • the supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 ⁇ m filtered PS20 with 5% 0.2 ⁇ m diafiltered fetal bovine serum).
  • the cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C.
  • spinners After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 ⁇ 10 5 cells/mL.
  • the cell media is exchanged with fresh media by centrifugation and resuspension in production medium.
  • any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used.
  • a 3L production spinner is seeded at 1.2 ⁇ 10 6 cells/mL. On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced.
  • the spinner On day 2, the spinner is sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 ⁇ m filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
  • 10% antifoam e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion
  • the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 SUPERFINETM (Pharmacia) column and stored at ⁇ 80° C.
  • Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows.
  • the conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ L of 1 M Tris buffer, pH 9.
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
  • the following method describes recombinant expression of PRO in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of PRO from the ADH2/GAPDH promoter.
  • DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO.
  • DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO.
  • yeast cells such as yeast strain AB110
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • the concentrate containing PRO may further be purified using selected column chromatography resins.
  • the following method describes recombinant expression of PRO in Baculovirus-infected insect cells.
  • sequence coding for PRO is fused upstream of an epitope tag contained within a baculovirus expression vector.
  • epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen).
  • the sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5′ and 3′ regions.
  • the 5′ primer may incorporate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BACULOGOLDTM virus DNA (Pharmingen Corp., San Diego, Calif.) into Spodoptera frugiperda (“Sf9”) cells (ATCC® CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28° C., the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
  • Expressed poly-his tagged PRO can then be purified, for example, by Ni 2+ -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice.
  • sonication buffer 25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl
  • the sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 ⁇ m filter.
  • loading buffer 50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8
  • a Ni 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
  • the filtered cell extract is loaded onto the column at 0.5 mL per minute.
  • the column is washed to baseline A280 with loading buffer, at which point fraction collection is started.
  • the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0
  • the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer.
  • One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni 2+ -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His 10 -tagged PRO are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
  • This example illustrates preparation of monoclonal antibodies which can specifically bind PRO.
  • Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • mice such as Balb/c are immunized with the PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads.
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, Mont.
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO antibodies.
  • the animals “positive” for antibodies can be injected with a final intravenous injection of PRO.
  • the mice Three to four days later, the mice are sacrificed and the spleen cells are harvested.
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC®, No. CRL 1597.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • hybridoma cells will be screened in an ELISA for reactivity against PRO. Determination of “positive” hybridoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art.
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies.
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
  • Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, -pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
  • a soluble PRO polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e.g. high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected.
  • a low pH buffer such as approximately pH 2-3
  • a chaotrope such as urea or thiocyanate ion
  • This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques.
  • the PRO polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
  • the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypeptide or fragment and the cell, by methods well known in the art.
  • the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e., a PRO polypeptide) or of small molecules with which they interact, e.g. agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
  • the three-dimensional structure of the PRO polypeptide, or of an PRO polypeptide-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors.
  • Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography.
  • knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
  • Nucleic acid microarrays are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts.
  • test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes.
  • the cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support.
  • the array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • hybridization signal of a probe from a test (disease tissue) sample is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified.
  • an overexpressed protein in a diseased tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition.
  • cancerous tumors derived from various human tissues were studied for PRO polypeptide-encoding gene expression relative to non-cancerous human tissue in an attempt to identify those PRO polypeptides which are overexpressed in cancerous tumors.
  • Two sets of experimental data were generated. In one set, cancerous human colon tumor tissue and matched non-cancerous human colon tumor tissue from the same patient (“matched colon control”) were obtained and analyzed for PRO polypeptide expression using the above described microarray technology.
  • cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a “universal” epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung.
  • mRNA isolated from the pooled tissues represents a mixture of expressed gene products from these different tissues.
  • Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color-analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:control detection) within each experiment. The normalized ratios from various experiments were then compared and used to identify clustering of gene expression.
  • the pooled “universal control” sample not only allowed effective relative gene expression determinations in a simple 2-sample comparison, it also allowed multi-sample comparisons across several experiments.
  • nucleic acid probes derived from the herein described PRO polypeptide-encoding nucleic acid sequences were used in the creation of the microarray and RNA from the tumor tissues listed above were used for the hybridization thereto.
  • a value based upon the normalized ratio:experimental ratio was designated as a “cutoff ratio”. Only values that were above this cutoff ratio were determined to be significant.
  • Table 8 below shows the results of these experiments, demonstrating that various PRO polypeptides of the preent invention are significantly overexpressed in various human tumor tissues as compared to a non-cancerous human tissue control.

Abstract

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

    RELATED APPLICATIONS
  • This application is a continuation of, and claims priority under 35 U.S.C. §120, to U.S. patent application Ser. No. 10/142,423, filed May 10, 2002, which is a continuation of, and claims priority under 35 U.S.C. §120, to U.S. patent application Ser. No. 10/028,072 filed Dec. 19, 2001, which is a continuation of, and claims priority under 35 U.S.C. §120 to PCT Application No. PCT/US00/32678 filed Dec. 1, 2000, which claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 60/209,832, filed Jun. 5, 2000, the entire disclosures of which are hereby expressly incorporated by reference.
  • FIELD OF THE INVENTION
  • The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides.
  • BACKGROUND OF THE INVENTION
  • Extracellular proteins play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.
  • Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts are being undertaken by both industry and academia to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].
  • Membrane-bound proteins and receptors can play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
  • Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance, can be employed as therapeutic agents to block receptor-ligand interactions. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • Efforts are being undertaken by both industry and academia to identify new, native receptor or membrane-bound proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor or membrane-bound proteins.
  • SUMMARY OF THE INVENTION
  • In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
  • In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alteratively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
  • In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
  • In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC® (American Type Culture Collection, Manassas, Va.) as disclosed herein, or (b) the complement of the DNA molecule of (a).
  • Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PRO polypeptides are contemplated.
  • Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 10 nucleotides in length, alternatively at least about 15 nucleotides in length, alternatively at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length; alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
  • In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
  • In a certain aspect, the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
  • In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC® as disclosed herein.
  • In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • Another aspect the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
  • In yet another embodiment, the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein. In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule.
  • In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
  • In a still further embodiment, the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody.
  • In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
  • In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence. Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
  • In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
  • In yet other embodiments, the invention provides oligonucleotide probes which may be useful for isolating genomic and cDNA nucleotide sequences, measuring or detecting expression of an associated gene or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences. Preferred probe lengths are described above.
  • In yet other embodiments, the present invention is directed to methods of using the PRO polypeptides of the present invention for a variety of uses based upon the functional biological assay data presented in the Examples below.
  • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequence PRO5997 cDNA, wherein SEQ ID NO:1 is a clone designated herein as “DNA97005-2687”.
  • FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • I. Definitions
  • The terms “PRO polypeptide” and “PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers, to specific polypeptide sequences as described herein. The terms “PRO/number polypeptide” and “PRO/number” wherein the term “number” is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term “PRO polypeptide” refers to each individual PRO/number polypeptide disclosed herein. All disclosures in this specification which refer to the “PRO polypeptide” refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term “PRO polypeptide” also includes variants of the PRO/number polypeptides disclosed herein.
  • A “native sequence PRO polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence PRO polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons are shown in bold font and underlined in the figures. However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides.
  • The PRO polypeptide “extracellular domain”. or “ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are comtemplated by the present invention.
  • The approximate location of the “signal peptides” of the various PRO polypeptides disclosed herein are shown in the present specification and/or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
  • “PRO polypeptide variant” means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more.
  • “Percent (%) amino acid sequence identity” with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital, UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
    100 times the fraction X/Y
    where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated “Comparison Protein” to the amino acid sequence designated “PRO”, wherein “PRO” represents the amino acid sequence of a hypothetical PRO polypeptide of interest, “Comparison Protein” represents the amino acid sequence of a polypeptide against which the “PRO” polypeptide of interest is being compared, and “X, “Y” and “Z” each represent different hypothetical amino acid residues.
  • Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, % amino acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest. For example, in the statement “a polypeptide comprising an the amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B”, the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
  • Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.
  • In situations where NCBI-BLAST2 is employed ,for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
    100 times the fraction X/Y
    where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • “PRO variant polynucleotide” or “PRO variant nucleic acid sequence” means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.
  • Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
  • “Percent (%) nucleic acid sequence identity” with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
    100 times the fraction W/Z
    where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to, C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated “Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”, wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acid sequence of interest, “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the “PRO-DNA” nucleic acid molecule of interest is being compared, and “N”, “L” and “V” each represent different hypothetical nucleotides.
  • Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, % nucleic acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest. For example, in the statement “an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B”, the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.
  • Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.
  • In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
    100 times the fraction W/Z
    where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
  • In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length PRO polypeptide as disclosed herein. PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide.
  • “Isolated,” when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • An “isolated” PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • The term “antibody” is used in the broadest sense and specifically covers, for example, single anti-PRO monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below). The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.
  • “Moderately stringent conditions” may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
  • The term “epitope tagged” when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a “tag polypeptide”. The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
  • “Active” or “activity” for the purposes herein refers to form(s) of a PRO polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring PRO, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO and an “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO.
  • The term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO polypeptide disclosed herein. In a similar manner, the term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a PRO polypeptide may comprise contacting a PRO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide.
  • “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • “Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI Americas Inc., Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS® (BASF Corporation, Mount Olive, N.J.).
  • “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab′ fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
  • The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • The word “label” when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • By “solid phase” is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • A “small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • An “effective amount” of a polypeptide disclosed herein or an agonist or antagonist thereof is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose.
    TABLE 2
    PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
    Comparison XXXXXYYYYYYY (Length = 12 amino acids)
    Protein

    % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = 5 divided by 15 = 33.3%
  • TABLE 3
    PRO XXXXXXXXXX (Length = 10 amino acids)
    Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids)
    Protein

    % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) = 5 divided by 10 = 50%
  • TABLE 4
    PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
    Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
    DNA

    % nucleic acid sequence identity = (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
  • TABLE 5
    PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
    Comparison NNNNLLLVV (Length = 9 nucleotides)
    DNA

    % nucleic acid sequence identity = (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%

    II. Compositions and Methods of the Invention
  • A. Full-Length PRO Polypeptides
  • The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides. In particular, cDNAs encoding various PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below. It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed. However, for sake of simplicity, in the present specification the protein encoded by the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO, will be referred to as “PRO/number”, regardless of their origin or mode of preparation.
  • As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC®. The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the PRO polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time.
  • B. PRO Polypeptide Variants
  • In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO variants can be prepared. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • Variations in the native full-length sequence PRO or in various domains of the PRO described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO. Optionally the variation, is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • PRO polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide.
  • PRO fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5′ and 3′ primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
  • In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
    TABLE 6
    Original Exemplary Preferred
    Residue Substitutions Substitutions
    Ala (A) val; leu; ile val
    Arg (R) lys; gln; asn lys
    Asn (N) gln; his; lys; arg gln
    Asp (D) glu glu
    Cys (C) ser ser
    Gln (Q) asn asn
    Glu (E) asp asp
    Gly (G) pro; ala ala
    His (H) asn; gln; lys; arg arg
    Ile (I) leu; val; met; ala; phe; leu
    norleucine
    Leu (L) norleucine; ile; val; ile
    met; ala; phe,
    Lys (K) arg; gln; asn arg
    Met (M) leu; phe; ile leu
    Phe (F) leu; val; ile; ala; tyr leu
    Pro (P) ala ala
    Ser (S) thr thr
    Thr (T) ser ser
    Trp (W) tyr; phe tyr
    Tyr (Y) trp; phe; thr; ser phe
    Val (V) ile; leu; met; phe; leu
    ala; norleucine
  • Substantial modifications in function or immunological identity of the PRO polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
    • (1) hydrophobic: norleucine, met, ala, val, leu, ile;
    • (2) neutral hydrophilic: cys, ser, thr;
    • (3) acidic: asp, glu;
    • (4) basic: asn, gln, his, lys, arg;
    • (5) residues that influence chain orientation: gly, pro; and
    • (6) aromatic: trp, tyr, phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO variant DNA.
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
  • C. Modifications of PRO
  • Covalent modifications of PRO are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO. Derivatization with bifunctional agents is useful, for instance, for crosslinking PRO to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
  • Another type of covalent modification of the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
  • Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO (for O-linked glycosylation sites). The PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
  • Another type of covalent modification of PRO comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • The PRO of the present invention may also be modified in a way to form a chimeric molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence.
  • In one embodiment, such a chimeric molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the PRO. The presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the PRO to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al. Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
  • In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
  • D. Preparation of PRO
  • The description below relates primarily to production of PRO by culturing cells transformed or transfected with a vector containing PRO nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO. For instance, the PRO sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO.
  • 1. Isolation of DNA Encoding PRO
  • DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level. Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The PRO-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
  • Libraries can be screened with probes (such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding PRO is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
  • The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GENBANK® (US Department of Health and Human Services, Bethesda, Md.) or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
  • 2. Selection and Transformation of Host Cells
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
  • Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaPO4, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC® 31,446); E. coli X1776 (ATCC® 31,537); E. coli strain W3110 (ATCC® 27,325) and K5 772 (ATCC® 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC® 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kanr ; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr ; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC® 12,424), K. bulgaricus (ATCC® 16,045), K. wickeramii (ATCC® 24,178), K. waltii (ATCC® 56,500), K. drosophilarum (ATCC® 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
  • Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC® CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC® CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC® CCL51). The selection of the appropriate host cell is deemed to be within the skill in the art.
  • 3. Selection and Use of a Replicable Vector
  • The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • The PRO may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7. [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC® No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.
  • Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PRO transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • Transcription of a DNA encoding the PRO by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′ or 3′ to the PRO coding sequence, but is preferably located at a site 5′ from the promoter.
  • Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO.
  • Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
  • 4. Detecting Gene Amplification/Expression
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope.
  • 5. Purification of Polypeptide
  • Forms of PRO may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PRO can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • It may be desired to purify PRO from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, SEPHADEX® G-75(Amersham Biosciences AB Corp., Uppsala, Sweden); PROTEIN A-SEPHAROSE™ (Pharmacia Biotech AB, Uppsala, Sweden) columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PRO. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO produced.
  • E. Uses for PRO
  • Nucleotide sequences (or their complement) encoding PRO have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. PRO nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein.
  • The full-length native sequence PRO gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length PRO cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of PRO or PRO from other species) which have a desired sequence identity to the native PRO sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence PRO. By way of example, a screening method will comprise isolating the coding region of the PRO gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the PRO gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
  • Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
  • Other useful fragments of the PRO nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRO DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of PRO DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).
  • Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of PRO proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
  • Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • Antisense or sense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more.
  • The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related PRO coding sequences.
  • Nucleotide sequences encoding a PRO can also be used to construct hybridization probes for mapping the gene which encodes that PRO and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
  • When the coding sequences for PRO encode a protein which binds to another protein (example, where the PRO is a receptor), the PRO can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor PRO can be used to isolate correlative ligand(s). Screening assays can be designed to find lead compounds that mimic the biological activity of a native PRO or a receptor for PRO. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • Nucleic acids which encode PRO or its modified forms can also be used to generate either transgenic animals or “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding PRO. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for PRO transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding PRO introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding PRO. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
  • Alternatively, non-human homologues of PRO can be used to construct a PRO “knock out” animal which has a defective or altered gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced into an embryonic stem cell of the animal. For example, cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with established techniques. A portion of the genomic DNA encoding PRO can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO polypeptide.
  • Nucleic acid encoding the PRO polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. “Gene therapy” includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique, of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262,4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science 256, 808-813 (1992).
  • The PRO polypeptides described herein may also be employed as molecular weight markers for protein electrophoresis purposes and the isolated nucleic acid sequences may be used for recombinantly expressing those markers.
  • The nucleic acid molecules encoding the PRO polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each PRO nucleic acid molecule of the present invention can be used as a chromosome marker.
  • The PRO polypeptides and nucleic acid molecules of the present invention may also be used diagnostically for tissue typing, wherein the PRO polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type. PRO nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
  • The PRO polypeptides described herein may also be employed as therapeutic agents. The PRO polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the PRO product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI Americas Inc., Bridgewater, N.J.), PLURONICS® (BASF Corporation, Mount Olive, N.J.) or PEG.
  • The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • The route of administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems.
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The use of interspecies scaling in toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.
  • When in vivo administration of a PRO polypeptide or agonist or antagonist thereof is employed, normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
  • Where sustained-release administration of a PRO polypeptide is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of the PRO polypeptide, microencapsulation of the PRO polypeptide is contemplated. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio/Technology, 8:755-758 (1990); Cleland, “Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), p. 439462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.
  • The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, “Controlled release of bioactive agents from lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.
  • This invention encompasses methods of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the PRO polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
  • All assays for antagonists are common in that they call for contacting the drug candidate with a PRO polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
  • In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the PRO polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • If the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the “two-hybrid system”) takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech, Palo Alto, Calif. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • Compounds that interfere with the interaction of a gene encoding a PRO polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
  • To assay for antagonists, the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide. Alternatively, antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the PRO polypeptide. Transfected cells that are grown on glass slides are exposed to labeled PRO polypeptide. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
  • As an alternative approach for receptor identification, labeled PRO polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
  • More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
  • Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of the PRO polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about −10 and +10 positions of the target gene nucleotide sequence, are preferred.
  • Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra.
  • These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
  • Diagnostic and therapeutic uses of the herein disclosed molecules may also be based upon the positive functional assay hits disclosed and described below.
  • F. Anti-PRO Antibodies
  • The present invention further provides anti-PRO antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
  • 1. Polyclonal Antibodies
  • The anti-PRO antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
  • 2. Monoclonal Antibodies
  • The anti-PRO antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
  • The immunizing agent will typically include the PRO polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and art sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
  • The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, PROTEIN A-SEPHAROSE™ (Pharmacia Biotech AB, Uppsala, Sweden), hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable, of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • 3. Human and Humanized Antibodies
  • The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
  • The antibodies may also be affinity matured using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared
  • 4. Bispecific Antibodies
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
  • Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
  • According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′)2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab′ fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
  • Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • Exemplary bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein. Alternatively, an anti-PRO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide. These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the PRO polypeptide and further binds tissue factor (TF).
  • 5. Heteroconjugate Antibodies
  • Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
  • 6. Effector Function Engineering
  • It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
  • 7. Immunoconjugates
  • The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that tan be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • In another embodiment, the antibody may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • 8. Immunoliposomes
  • The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
  • 9. Pharmaceutical Compositions of Antibodies
  • Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
  • If the PRO polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
  • The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
  • The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (TAP Pharmaceuticals, Inc., North Chicago, Ill.) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • G. Uses for Anti-PRO Antibodies
  • The anti-PRO antibodies of the invention have various utilities. For example, anti-PRO antibodies may be used in diagnostic assays for PRO, e.g., detecting its expression (and in some cases, differential expression) in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
  • Anti-PRO antibodies also are useful for the affinity purification of PRO from recombinant cell culture or natural sources. In this process, the antibodies against PRO are immobilized on a suitable support, such a SEPHADEX® (Amersham Biosciences AB Corp., Uppsala, Sweden) resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the PRO to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the PRO, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the PRO from the antibody.
  • The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
  • All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
  • EXAMPLES
  • Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC® accession numbers is the American Type Culture Collection, Manassas, Va.
  • Example 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encoding Therefor
  • The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST databases included public databases (e.g., Dayhoff, GENBANK® (US Department of Health and Human Services, Bethesda, Md.), and proprietary databases (e.g. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program “phrap” (Phil Green, University of Washington, Seattle, Wash.).
  • Using this extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequences using phrap. In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
  • Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
  • The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, Calif. The cDNA was primed with oligo dT containing a NotI site, linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique XhoI and NotI sites.
  • Example 2 Isolation of cDNA clones by Amylase Screening
  • 1. Preparation of Oligo dT Primed cDNA Library
  • mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, Md. (Super Script Plasmid System). In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNA was cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloning sites. 2. Preparation of Random Primed cDNA Library
  • A secondary cDNA library was generated in order to preferentially represent the 5′ ends of the primary cDNA clones. Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleaved with SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites. Thus, cDNAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
  • 3. Transformation and Detection
  • DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37° C. for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37° C.). Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCl-gradient. The purified DNA was then carried on to the yeast protocols below.
  • The yeast methods were divided into three categories: (1) Transformation of yeast with the plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis.
  • The yeast strain used was HD56-5A (ATCC®-90785). This strain has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL+, SUC+, GAL+. Preferably, yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in sec71, sec72, sec62, with truncated sec71 being most preferred. Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p or SSA1p-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
  • Transformation was performed based on the protocol outlined by Gietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30° C. The YEPD broth was prepared as described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 207 (1994). The overnight culture was then diluted to about 2×106 cells/ml (approx. OD600=0.1) into fresh YEPD broth (500 ml) and regrown to 1×107 cells/ml (approx. OD600=0.4-0.5).
  • The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li2OOCCH3), and resuspended into LiAc/TE (2.5 ml).
  • Transformation took place by mixing the prepared cells (100 μl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg, Md.) and transforming DNA (1 μg, vol. <10 μl) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li2OOCCH3, pH 7.5) was added. This mixture was gently mixed and incubated at 30° C. while agitating for 30 minutes. The cells were then heat shocked at 42° C. for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were then diluted into TE (1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
  • Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
  • The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 208-210 (1994). Transformants were grown at 30° C. for 2-3 days.
  • The detection of colonies secreting amylase was performed by including red starch in the selective growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al., Anal. Biochem., 172: 176-179 (1988). The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration).
  • The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies. Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.
  • 4. Isolation of DNA by PCR Amplification
  • When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate. At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified. An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing: 0.5 μl KLENTAQ® (Clontech, Palo Alto, Calif.); 4.0 μl 10 mM dNTP's (Perkin Elmer-Cetus); 2.5 μl KLENTAQ® buffer (Clontech); 0.25 μl forward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. The sequence of the forward oligonucleotide 1 was:
    (SEQ ID NO:3)
    5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′
  • The sequence of reverse oligonucleotide 2 was:
    (SEQ ID NO:4)
    5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′
  • PCR was then performed as follows:
    a. Denature 92° C., 5 minutes
    b.  3 cycles of: Denature 92° C., 30 seconds
    Anneal 59° C., 30 seconds
    Extend 72° C., 60 seconds
    c.  3 cycles of: Denature 92° C., 30 seconds
    Anneal 57° C., 30 seconds
    Extend 72° C., 60 seconds
    d. 25 cycles of: Denature 92° C., 30 seconds
    Anneal 55° C., 30 seconds
    Extend 72° C., 60 seconds
    e. Hold  4° C.
  • The underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY.0 when no insert was present. Typically, the first 18 nucleotides of the 5′ end of these oligonucleotides contained annealing sites for the sequencing primers. Thus, the total product of the PCR reaction from an empty vector was 343 bp. However, signal sequence-fused cDNA resulted in considerably longer nucleotide sequences.
  • Following the PCR, an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et al., supra. Clones resulting in a single strong PCR product larger than 400 bp were further analyzed by DNA sequencing after purification with a 96 QIAQUICK® PCR clean-up column (Qiagen Inc., Chatsworth, Calif.).
  • Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis
  • Various polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc. (South San Francisco, Calif.) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GENBANK® (US Department of Health and Human Services, Bethesda, Md.)) and/or private (LIPESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5′-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals. Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
  • Example 4 Isolation of cDNA clones Encoding Human PRO Polypeptides
  • Using the techniques described in Examples 1 to 3 above, numerous full-length cDNA clones were identified as encoding PRO polypeptides as disclosed herein. These cDNAs were then deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC®) as shown in Table 7, which is disclosed in the parent application U.S. application Ser. No. 10/142,423, filed May 10, 2002 and is expressly and specifically incorporated herein by reference for the disclosure describing and disclosed in Table 7. In addition, DNA97005-2687 is ATCC Deposit No: PTA-378, deposited Jul. 20, 1999.
  • These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations there under (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit and for at least five (5) years after the most recent request for the furnishing of a sample of the deposit received by the depository. The deposits will be made available by ATCC® under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC®, which assures that all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the pertinent U.S. patent, assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638).
  • The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
  • Example 5 Use of PRO as a Hybridization Probe
  • The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe.
  • DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries.
  • Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled PRO-derived probe to the filters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.
  • DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO can then be identified using standard techniques known in the art.
  • Example 6 Expression of PRO in E. coli
  • This example illustrates preparation of an unglycosylated form of PRO by recombinant expression in E. coli.
  • The DNA sequence encoding PRO is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli, see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene.
  • The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
  • Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
  • After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized PRO protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
  • PRO may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PRO is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2SO4, 0.71 g sodium citrate 2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO4) and grown for approximately 20-30 hours at 30° C. with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
  • E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1 M and 0.02 M, respectively, and the solution is stirred overnight at 4° C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4° C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
  • The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4° C. for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros R1/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
  • Fractions containing the desired folded PRO polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 SUPERFINE™ (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
  • Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
  • Example 7 Expression of PRO in Mammalian Cells
  • This example illustrates preparation of a potentially glycosylated form of PRO by recombinant expression in mammalian cells.
  • The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as the expression vector. Optionally, the PRO DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-PRO.
  • In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC® CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO4, and a precipitate is allowed to form for 10 minutes at 25° C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37° C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
  • Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
  • In an alternative technique, PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PRO can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
  • In another embodiment, PRO can be expressed in CHO cells. The pRK5-PRO can be transfected into CHO cells using known reagents such as CaPO4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methionine. After determining the presence of PRO polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PRO can then be concentrated and purified by any selected method.
  • Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged PRO insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged PRO can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography.
  • PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.
  • Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG1 constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
  • Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5′ and 3′ of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
  • Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents SUPERFECT® (Quiagen), DOSPER™ (Roche Applied Science, Indianapolis, Ind.) or FUGENE® (Boehringer Mannheim). The cells are grown as described in Lucas et al., supra. Approximately 3×10−7 cells are frozen in an ampule for further growth and production as described below.
  • The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3×105 cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3L production spinner is seeded at 1.2×106 cells/mL. On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
  • For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 SUPERFINE™ (Pharmacia) column and stored at −80° C.
  • Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
  • Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
  • Example 8 Expression of PRO in Yeast
  • The following method describes recombinant expression of PRO in yeast.
  • First, yeast expression vectors are constructed for intracellular production or secretion of PRO from the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO. For secretion, DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO.
  • Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO may further be purified using selected column chromatography resins.
  • Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
  • Example 9 Expression of PRO in Baculovirus-Infected Insect Cells
  • The following method describes recombinant expression of PRO in Baculovirus-infected insect cells.
  • The sequence coding for PRO is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5′ and 3′ regions. The 5′ primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BACULOGOLD™ virus DNA (Pharmingen Corp., San Diego, Calif.) into Spodoptera frugiperda (“Sf9”) cells (ATCC® CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28° C., the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
  • Expressed poly-his tagged PRO can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His10-tagged PRO are pooled and dialyzed against loading buffer.
  • Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
  • Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
  • Example 10 Preparation of Antibodies that Bind PRO
  • This example illustrates preparation of monoclonal antibodies which can specifically bind PRO.
  • Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO antibodies.
  • After a suitable antibody titer has been detected, the animals “positive” for antibodies can be injected with a final intravenous injection of PRO. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC®, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • The hybridoma cells will be screened in an ELISA for reactivity against PRO. Determination of “positive” hybridoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art.
  • The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
  • Example 11 Purification of PRO Polypeptides Using Specific Antibodies
  • Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, -pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such an immunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
  • A soluble PRO polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e.g. high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected.
  • Example 12 Drug Screening
  • This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a variety of drug screening techniques. The PRO polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
  • Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypeptide or fragment and the cell, by methods well known in the art. In such competitive binding assays, the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known in the art. Purified PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
  • Example 13 Rational Drug Design
  • The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e., a PRO polypeptide) or of small molecules with which they interact, e.g. agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
  • In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).
  • It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
  • By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
  • Example 14 Microarray Analysis to Detect Overexpression of PRO Polypeptides in Cancerous Tumors
  • Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sample is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified. The implication of this result is that an overexpressed protein in a diseased tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition.
  • The methodology of hybridization of nucleic acids and microarray technology is well known in the art. In the present example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in U.S. Provisional Patent Application Serial No. 60/193,767, filed on Mar. 31, 2000 and which is herein incorporated by reference.
  • In the present example, cancerous tumors derived from various human tissues were studied for PRO polypeptide-encoding gene expression relative to non-cancerous human tissue in an attempt to identify those PRO polypeptides which are overexpressed in cancerous tumors. Two sets of experimental data were generated. In one set, cancerous human colon tumor tissue and matched non-cancerous human colon tumor tissue from the same patient (“matched colon control”) were obtained and analyzed for PRO polypeptide expression using the above described microarray technology. In the second set of data, cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a “universal” epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung. mRNA isolated from the pooled tissues represents a mixture of expressed gene products from these different tissues. Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color-analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:control detection) within each experiment. The normalized ratios from various experiments were then compared and used to identify clustering of gene expression. Thus, the pooled “universal control” sample not only allowed effective relative gene expression determinations in a simple 2-sample comparison, it also allowed multi-sample comparisons across several experiments.
  • In the present experiments, nucleic acid probes derived from the herein described PRO polypeptide-encoding nucleic acid sequences were used in the creation of the microarray and RNA from the tumor tissues listed above were used for the hybridization thereto. A value based upon the normalized ratio:experimental ratio was designated as a “cutoff ratio”. Only values that were above this cutoff ratio were determined to be significant. Table 8 below shows the results of these experiments, demonstrating that various PRO polypeptides of the preent invention are significantly overexpressed in various human tumor tissues as compared to a non-cancerous human tissue control. As described above, these data demonstrate that the PRO polypeptides of the present invention are useful not only as diagnostic markers for the presence of one or more cancerous tumors, but also serve as therapeutic targets for the treatment of those tumors. Additional data for Table 8 is present in Table 8 as disclosed in parent U.S. application Ser. No. 10/142,423, filed May 10, 2002 and is specifically and expressly incorporated herein by reference for disclosure describing and data presented in Table 8.
    TABLE 8
    Molecule Is overexpressed in: as compared to:
    PRO5997 colon tumor universal normal control
    PRO5997 lung tumor universal normal control
  • The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims (19)

1. A diagnostic method, comprising determining, in a test biological sample obtained from a mammalian subject, the level of nucleic acid encoding a PRO5997 polypeptide of SEQ ID NO:2 relative to the level of said nucleic acid in a corresponding normal biological sample wherein increased level of said nucleic acid in the test biological sample is indicative that the test biological sample contains cancerous cells.
2. The method of claim 1 wherein the test and normal biological samples are tissue samples.
3. The method of claim 2 wherein the test tissue sample is from colon or lung tissue.
4. The method of claim 3 wherein the normal biological sample is from the same type of tissue as the test biological sample.
5. The method of claim 2 wherein the normal biological sample is an epithelial tissue sample from a combination of tissues.
6. The method of claim 5 wherein epithelial tissue sample includes epithelial cells from colon or lung tissue.
7. The method of claim 1 wherein the nucleic acid levels are determined by hybridization of nucleic acid obtained from the test and normal biological samples to one or more probes specific for the nucleic acid encoding PRO5997.
8. The method of claim 7 wherein hybridization is performed under stringent conditions.
9. The method of claim 8 wherein said stringent conditions use 50% formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 50% formamide at 55° C., followed by a wash comprising of 0.1×SSC containing EDTA at 55° C.
10. The method of claim 7 wherein the nucleic acids obtained from the test and normal biological samples are cDNAs.
11. The method of claim 10 wherein the nucleic acids obtained from the test and normal biological samples are placed on microarrays.
12. A diagnostic method comprising determining the expression level of the PRO5997 polypeptide of SEQ ID NO: 2 in test biological sample relative to a normal biological sample, wherein overexpression of said polypeptide in the test biological sample is indicative that the sample contains cancerous cells.
13. The method of claim 12 wherein the test and normal biological samples are tissue samples.
14. The method of claim 13 wherein the test tissue sample is from colon or lung tissue.
15. The method of claim 14 wherein overexpression is detected with an antibody that specifically binds to the PRO5997 polypeptide.
16. The method of claim 15 wherein said antibody is a monoclonal antibody.
17. The method of claim 16 wherein said antibody is a humanized antibody.
18. The method of claim 17 wherein said antibody is an antibody fragment.
19. The method of claim 18 wherein said antibody is labeled.
US11/110,520 2000-06-05 2005-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same Abandoned US20050202496A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/110,520 US20050202496A1 (en) 2000-06-05 2005-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US20983200P 2000-06-05 2000-06-05
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/110,520 US20050202496A1 (en) 2000-06-05 2005-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/142,423 Continuation US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Publications (1)

Publication Number Publication Date
US20050202496A1 true US20050202496A1 (en) 2005-09-15

Family

ID=21841414

Family Applications (468)

Application Number Title Priority Date Filing Date
US09/990,456 Abandoned US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 Abandoned US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 Abandoned US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 Abandoned US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 Abandoned US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 Abandoned US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 Abandoned US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 Abandoned US20030096386A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 Abandoned US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 Abandoned US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 Abandoned US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 Abandoned US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,053 Abandoned US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,061 Abandoned US20030082761A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,062 Abandoned US20030077779A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,050 Abandoned US20030054516A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 Abandoned US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 Abandoned US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 Abandoned US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 Abandoned US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 Abandoned US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 Abandoned US20030190722A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 Abandoned US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 Expired - Lifetime US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/123,154 Abandoned US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 Abandoned US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 Abandoned US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 Abandoned US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,214 Expired - Fee Related US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,108 Expired - Fee Related US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 Expired - Lifetime US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,322 Granted US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,291 Abandoned US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 Granted US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 Granted US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 Expired - Lifetime US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,771 Abandoned US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 Abandoned US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,235 Abandoned US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 Abandoned US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 Abandoned US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 Abandoned US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 Expired - Lifetime US7700736B2 (en) 2000-05-22 2002-04-15 PRO350 antibodies
US10/123,910 Expired - Lifetime US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,902 Abandoned US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 Expired - Lifetime US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,913 Abandoned US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,911 Expired - Lifetime US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,907 Expired - Fee Related US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,906 Abandoned US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,908 Expired - Lifetime US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,912 Abandoned US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 Abandoned US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 Abandoned US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 Expired - Lifetime US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,815 Expired - Lifetime US7342096B2 (en) 1999-12-09 2002-04-17 PRO1879 polypeptide
US10/124,822 Expired - Lifetime US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,813 Expired - Lifetime US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,824 Abandoned US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 Abandoned US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 Abandoned US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 Expired - Lifetime US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,823 Abandoned US20030199062A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 Abandoned US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,817 Abandoned US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 Expired - Lifetime US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 Expired - Lifetime US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,820 Abandoned US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,816 Abandoned US20030190728A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,927 Abandoned US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,928 Abandoned US20030087349A1 (en) 1998-06-19 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,923 Abandoned US20030087348A1 (en) 2000-06-05 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 Expired - Lifetime US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,921 Expired - Lifetime US7312313B2 (en) 1998-08-17 2002-04-19 Anti-PRO1309 antibodies
US10/125,926 Abandoned US20030082686A1 (en) 2000-06-05 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 Expired - Lifetime US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,931 Abandoned US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,930 Expired - Lifetime US7291700B2 (en) 2000-06-05 2002-04-19 PRO4985 polypeptide
US10/125,932 Expired - Fee Related US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/127,900 Abandoned US20030203429A1 (en) 2000-06-05 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,824 Abandoned US20030087352A1 (en) 1998-08-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,836 Expired - Lifetime US7432345B2 (en) 1998-11-17 2002-04-22 PRO1475 polypeptide
US10/127,833 Abandoned US20030087358A1 (en) 1998-09-01 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,901 Expired - Fee Related US7342098B2 (en) 1998-06-17 2002-04-22 PRO1154 polypeptide
US10/127,837 Abandoned US20030082690A1 (en) 1998-09-01 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,852 Abandoned US20030203428A1 (en) 1999-12-09 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,822 Expired - Lifetime US7371827B2 (en) 1998-06-17 2002-04-22 Antibodies against the PRO1126 polypeptide
US10/127,839 Expired - Lifetime US7449554B2 (en) 2000-06-05 2002-04-22 Antibodies against the PRO4977 polypeptides
US10/127,825 Abandoned US20030077710A1 (en) 1998-10-22 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,844 Expired - Lifetime US7566774B2 (en) 2000-06-05 2002-04-22 PRO4977 polypeptides
US10/127,830 Expired - Lifetime US7351793B2 (en) 1998-09-15 2002-04-22 PRO1286 polypeptide
US10/127,850 Abandoned US20030082698A1 (en) 1998-08-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,851 Expired - Fee Related US7348414B2 (en) 1998-06-17 2002-04-22 Antibodies against the PRO1154 polypeptide
US10/127,835 Abandoned US20030077712A1 (en) 1998-10-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,843 Abandoned US20030082693A1 (en) 2000-06-05 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,842 Abandoned US20030082692A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,847 Abandoned US20030119103A1 (en) 1998-08-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,838 Abandoned US20030082691A1 (en) 1998-11-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,849 Abandoned US20030082697A1 (en) 1998-10-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,821 Abandoned US20030087350A1 (en) 1998-08-04 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,840 Abandoned US20030153033A1 (en) 1998-09-10 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,845 Abandoned US20030082694A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,841 Abandoned US20030087361A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,826 Expired - Lifetime US7309763B2 (en) 1998-06-17 2002-04-22 PRO1126 polypeptide
US10/127,832 Abandoned US20030087357A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,834 Abandoned US20030087359A1 (en) 1998-09-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,829 Abandoned US20030077711A1 (en) 1998-10-22 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,827 Abandoned US20030087354A1 (en) 1998-08-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,848 Abandoned US20030082696A1 (en) 1998-11-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,831 Abandoned US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,828 Abandoned US20030087355A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,846 Abandoned US20030082695A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,691 Expired - Lifetime US7319135B2 (en) 1999-12-09 2002-04-23 PRO1341 polypeptides
US10/128,689 Abandoned US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,684 Abandoned US20030082700A1 (en) 2000-06-05 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,693 Expired - Lifetime US7355006B2 (en) 1998-08-31 2002-04-23 Antibodies against the PRO1271 polypeptides
US10/128,687 Abandoned US20030087363A1 (en) 1998-09-10 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,694 Expired - Lifetime US7189813B2 (en) 2000-03-02 2002-04-23 PRO1338 polypeptides
US10/128,686 Expired - Fee Related US7345146B2 (en) 1998-08-31 2002-04-23 PRO1271 Polypeptides
US10/128,692 Active 2024-06-24 US7704496B2 (en) 1999-12-09 2002-04-23 Antibodies against PRO1341 polypeptide
US10/128,688 Expired - Fee Related US7323544B2 (en) 1999-02-09 2002-04-23 PRO1434 polypeptides
US10/128,685 Abandoned US20030203430A1 (en) 1998-08-11 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,690 Abandoned US20030082702A1 (en) 2000-03-02 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,824 Expired - Fee Related US7326413B2 (en) 1999-02-09 2002-04-24 Antibodies against the PRO1434 polypeptide
US10/131,820 Expired - Lifetime US7312314B2 (en) 1998-10-28 2002-04-24 Antibody that binds a pro1693 polypeptide
US10/131,815 Abandoned US20030092103A1 (en) 1998-12-22 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,828 Expired - Lifetime US7309777B2 (en) 1998-10-07 2002-04-24 Antibodies against the PRO1556 polypeptide
US10/131,837 Expired - Lifetime US7294495B2 (en) 1999-12-09 2002-04-24 PR03580 polypeptides
US10/131,819 Expired - Lifetime US7273926B2 (en) 1999-12-09 2002-04-24 Antibody to PRO1779 polypeptides
US10/131,817 Expired - Lifetime US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,829 Abandoned US20030082705A1 (en) 1999-12-09 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,830 Abandoned US20030077720A1 (en) 1999-12-09 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,818 Expired - Fee Related US7166703B2 (en) 1998-10-07 2002-04-24 PRO1561 polypeptides
US10/131,823 Expired - Lifetime US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,833 Expired - Fee Related US7141652B1 (en) 1998-10-07 2002-04-24 Antibodies to PRO1561 polypeptide
US10/131,816 Expired - Lifetime US7361337B2 (en) 1999-12-09 2002-04-24 Antibodies against the PRO1754 polypeptides
US10/131,836 Expired - Lifetime US7351794B2 (en) 1999-12-09 2002-04-24 PRO1754 polypeptides
US10/131,826 Expired - Lifetime US7202345B2 (en) 1998-08-19 2002-04-24 PRO 1384 antibodies
US10/131,813 Expired - Lifetime US7279551B2 (en) 1998-10-07 2002-04-24 Pro1556 Polypeptide
US10/131,835 Expired - Lifetime US7294705B2 (en) 2000-12-01 2002-04-24 Anti-PRO3580 antibodies
US10/131,821 Abandoned US20030092105A1 (en) 1999-12-09 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,825 Expired - Lifetime US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,822 Expired - Lifetime US7189806B2 (en) 1998-08-19 2002-04-24 Pro 1384 polypeptides
US10/137,869 Expired - Lifetime US7282558B2 (en) 2000-03-03 2002-05-03 PRO4329 polypeptide
US10/137,871 Expired - Lifetime US7323545B2 (en) 1999-12-09 2002-05-03 PRO1885 polypeptides
US10/137,868 Abandoned US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,866 Abandoned US20030129689A1 (en) 2000-03-01 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,872 Abandoned US20030077722A1 (en) 2000-03-03 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,867 Abandoned US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,865 Abandoned US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,870 Abandoned US20030138883A1 (en) 2000-03-01 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,864 Expired - Lifetime US7291715B2 (en) 2000-03-03 2002-05-03 Antibodies to the PRO4329 polypeptide
US10/137,873 Abandoned US20060084138A1 (en) 2000-03-03 2002-05-03 The pro4979 polypeptide
US10/140,474 Abandoned US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,019 Abandoned US20030148423A1 (en) 1999-12-09 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 Expired - Lifetime US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,470 Granted US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,022 Expired - Lifetime US7285627B2 (en) 2000-06-05 2002-05-06 PRO4989 polypeptides
US10/140,274 Abandoned US20030143674A1 (en) 2000-03-03 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,471 Expired - Lifetime US7291702B2 (en) 1999-12-09 2002-05-06 PRO4326 polypeptides
US10/140,473 Abandoned US20030207351A1 (en) 2000-12-01 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 Expired - Lifetime US7521539B2 (en) 2000-12-01 2002-05-06 Anti-PRO4989 antibodies
US10/140,024 Abandoned US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 Abandoned US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,021 Expired - Lifetime US7279552B2 (en) 1999-12-09 2002-05-06 Pro1782 polypeptides
US10/140,472 Abandoned US20030138888A1 (en) 1999-12-09 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 Abandoned US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,018 Abandoned US20030138885A1 (en) 2000-03-03 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 Expired - Lifetime US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,928 Abandoned US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,862 Expired - Lifetime US7285628B2 (en) 1999-12-09 2002-05-07 PRO1867 polypeptides
US10/140,805 Abandoned US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,809 Abandoned US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,926 Abandoned US20030134356A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,810 Abandoned US20030207353A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 Expired - Lifetime US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,806 Active 2024-05-10 US7309764B2 (en) 2000-03-03 2002-05-07 PRO4401 polypeptide
US10/140,922 Expired - Lifetime US7309765B2 (en) 2000-03-03 2002-05-07 PRO4349 polypeptides
US10/140,861 Abandoned US20030148425A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,807 Expired - Lifetime US7335745B2 (en) 2000-03-03 2002-05-07 Antibodies against the PRO4403 polypeptides
US10/140,864 Abandoned US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,927 Expired - Lifetime US7495082B2 (en) 2000-03-03 2002-05-07 Antibodies to PRO4349 polypeptides
US10/140,865 Abandoned US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,924 Abandoned US20030134355A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,925 Abandoned US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 Expired - Lifetime US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,863 Expired - Lifetime US7288626B2 (en) 2000-03-03 2002-05-07 PRO4403 polypeptides
US10/140,923 Expired - Lifetime US7282559B2 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 Expired - Fee Related US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/141,757 Expired - Fee Related US7314920B2 (en) 2000-03-03 2002-05-08 Antibodies against the PRO4340 polypeptide
US10/141,704 Expired - Lifetime US7396916B2 (en) 2000-06-05 2002-05-08 Antibody to a polypeptide overexpressed in tumors
US10/141,697 Expired - Lifetime US7417123B2 (en) 2000-03-03 2002-05-08 PRO4399 Antibodies
US10/141,698 Expired - Lifetime US7282560B2 (en) 2000-03-03 2002-05-08 PRO 4326 polypeptides
US10/141,701 Abandoned US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,703 Abandoned US20030207357A1 (en) 1999-03-10 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,706 Expired - Lifetime US7425609B2 (en) 2000-03-03 2002-05-08 PRO4330 polypeptides
US10/141,761 Expired - Fee Related US7335729B2 (en) 2000-03-03 2002-05-08 PRO4320 polypeptide
US10/141,760 Expired - Fee Related US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,699 Expired - Lifetime US7390887B2 (en) 2000-03-03 2002-05-08 Antibodies against PRO4330
US10/141,754 Expired - Lifetime US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,759 Expired - Lifetime US7291716B2 (en) 2000-03-03 2002-05-08 PRO4400 antibodies
US10/141,702 Expired - Lifetime US7294692B2 (en) 2000-06-05 2002-05-08 Pro6014 polypeptides
US10/141,700 Expired - Lifetime US7312308B2 (en) 2000-03-03 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 Expired - Lifetime US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,753 Expired - Lifetime US7323550B2 (en) 2000-03-03 2002-05-08 Anti-PRO4318 antibodies
US10/141,758 Expired - Lifetime US7301007B2 (en) 2000-03-03 2002-05-08 PRO4340 polypeptides
US10/141,755 Expired - Lifetime US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,705 Expired - Lifetime US7396906B2 (en) 2000-03-03 2002-05-08 Pro4399 Polypeptides
US10/141,762 Abandoned US20030207362A1 (en) 2000-03-03 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,117 Expired - Lifetime US7318922B2 (en) 2000-06-05 2002-05-09 Anti-PRO5337 antibodies
US10/142,422 Expired - Lifetime US7385030B2 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,425 Abandoned US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,426 Abandoned US20040048333A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,027 Abandoned US20030207366A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,421 Expired - Lifetime US7311909B2 (en) 2000-03-03 2002-05-09 PRO4348 antibodies
US10/142,888 Abandoned US20030157606A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,761 Expired - Lifetime US7306795B2 (en) 2000-06-05 2002-05-09 PRO5774 antibodies
US10/142,432 Expired - Lifetime US7390877B2 (en) 2000-03-03 2002-05-09 PRO4381 polypeptides
US10/142,428 Abandoned US20030207363A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,113 Expired - Lifetime US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,417 Expired - Lifetime US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,116 Expired - Lifetime US7390486B2 (en) 2000-03-03 2002-05-09 PRO4381 antibodies
US10/143,034 Expired - Lifetime US7378502B2 (en) 2000-03-03 2002-05-09 PRO4389 antibodies
US10/142,430 Expired - Lifetime US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,427 Expired - Lifetime US7294702B2 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,887 Expired - Lifetime US7291717B2 (en) 1999-12-09 2002-05-09 PRO3446 antibodies
US10/143,035 Expired - Fee Related US7317081B2 (en) 2000-03-03 2002-05-09 PRO4371 polypeptides
US10/143,115 Expired - Lifetime US7309767B2 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,114 Abandoned US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,118 Expired - Lifetime US7309768B2 (en) 2000-03-03 2002-05-09 PRO4347 polypeptides
US10/142,420 Abandoned US20030148434A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,889 Abandoned US20030194765A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,762 Expired - Lifetime US7288627B2 (en) 2000-03-03 2002-05-09 PRO4322 polypeptides
US10/142,884 Abandoned US20030207365A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,760 Abandoned US20030148437A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,767 Expired - Lifetime US7294693B2 (en) 2000-03-03 2002-05-10 Pro4428 Polypeptides
US10/142,886 Abandoned US20030203432A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,424 Expired - Lifetime US7297766B2 (en) 2000-06-05 2002-05-10 PRO5337 polypeptides
US10/143,033 Abandoned US20030134363A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,431 Expired - Lifetime US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,419 Expired - Lifetime US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,423 Abandoned US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,763 Expired - Lifetime US7189807B2 (en) 2000-06-05 2002-05-10 PRO4994 polypeptides
US10/142,765 Abandoned US20030157603A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 Expired - Lifetime US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,418 Expired - Lifetime US7297765B2 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,764 Abandoned US20030180865A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,885 Expired - Lifetime US7718173B2 (en) 2000-06-05 2002-05-10 Anti-pro6094 antibodies
US10/142,429 Abandoned US20030207364A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,766 Expired - Lifetime US7390883B2 (en) 2000-03-03 2002-05-10 PRO4428 antibodies
US10/145,090 Abandoned US20030157613A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,015 Expired - Lifetime US7439325B2 (en) 2000-06-05 2002-05-13 PRO6001 polypeptides
US10/145,127 Expired - Lifetime US7355007B2 (en) 2000-06-05 2002-05-13 Antibodies that bind PRO5005 Polypeptides
US10/144,958 Expired - Lifetime US7479545B2 (en) 2000-06-05 2002-05-13 PRO6001 antibodies
US10/144,956 Abandoned US20030207368A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,091 Abandoned US20030157614A1 (en) 2000-06-05 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,993 Abandoned US20040038336A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,992 Abandoned US20030157611A1 (en) 2000-06-05 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,957 Abandoned US20030157610A1 (en) 2000-06-05 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,994 Abandoned US20030134364A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,754 Abandoned US20030157620A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,632 Expired - Lifetime US7385031B2 (en) 2000-06-05 2002-05-14 PRO6097 polypeptides
US10/145,752 Expired - Lifetime US7319136B2 (en) 2000-06-05 2002-05-14 PRO7434 polypeptides
US10/145,961 Abandoned US20040235092A1 (en) 1999-12-09 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,818 Abandoned US20030157622A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,747 Abandoned US20030157618A1 (en) 1999-03-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,877 Expired - Lifetime US7411040B2 (en) 2000-06-05 2002-05-14 PRO6182 polypeptides
US10/145,825 Expired - Lifetime US7335740B2 (en) 2000-06-05 2002-05-14 Antibodies against the PRO7434 polypeptides
US10/145,755 Expired - Lifetime US7312312B2 (en) 2000-03-03 2002-05-14 PRO6242 polypeptides
US10/145,872 Abandoned US20030157624A1 (en) 1999-10-08 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,749 Abandoned US20030207371A1 (en) 2000-05-22 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,870 Expired - Lifetime US7294703B2 (en) 2000-06-05 2002-05-14 PRO6093 Antibodies
US10/145,631 Expired - Lifetime US7371824B2 (en) 2000-06-05 2002-05-14 PRO6093 polypeptides
US10/145,628 Expired - Lifetime US7285636B2 (en) 2000-06-05 2002-05-14 PRO6095 polypeptides
US10/145,821 Abandoned US20030148438A1 (en) 2000-09-15 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,751 Abandoned US20030166074A1 (en) 2000-05-22 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,748 Expired - Lifetime US7354998B2 (en) 2000-06-05 2002-05-14 PRO7171 polypeptides
US10/145,878 Expired - Lifetime US7317088B2 (en) 2000-06-05 2002-05-14 PRO5993 polypeptides
US10/145,869 Abandoned US20030166078A1 (en) 1999-10-08 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,634 Abandoned US20030170788A1 (en) 2000-09-15 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,958 Expired - Lifetime US7408042B2 (en) 2000-06-05 2002-05-14 Anti-PRO6181 antibodies
US10/145,629 Expired - Lifetime US7285644B2 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,626 Expired - Lifetime US7429640B2 (en) 2000-06-05 2002-05-14 PRO6181 polypeptides
US10/145,625 Abandoned US20030180868A1 (en) 2000-03-03 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,750 Expired - Lifetime US7417115B2 (en) 2000-06-05 2002-05-14 PRO6027 polypeptides
US10/145,746 Abandoned US20030134366A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,959 Expired - Lifetime US7390878B2 (en) 2000-06-05 2002-05-14 PRO6090 polypeptides
US10/145,874 Abandoned US20030194766A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,630 Expired - Lifetime US7309778B2 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,824 Expired - Lifetime US7411047B2 (en) 2000-06-05 2002-05-14 PRO6097 antibodies
US10/145,827 Expired - Lifetime US7304140B2 (en) 2000-03-03 2002-05-14 PRO4503 polypeptides
US10/145,823 Abandoned US20030134368A1 (en) 2000-03-03 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,820 Abandoned US20030157623A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,960 Expired - Lifetime US7317074B2 (en) 2000-03-03 2002-05-14 Pro4976 polypeptides
US10/145,633 Abandoned US20030138892A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,826 Expired - Lifetime US7375195B2 (en) 2000-06-05 2002-05-14 Anti-PRO7171 polypeptides
US10/145,822 Abandoned US20030166075A1 (en) 2000-12-01 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,962 Expired - Lifetime US7335746B2 (en) 2000-03-03 2002-05-14 Anti-pro4976 antibodies
US10/145,871 Expired - Lifetime US7396907B2 (en) 2000-06-05 2002-05-14 Antibodies that bind PRO6182 polypeptides
US10/146,724 Abandoned US20030134373A1 (en) 2000-06-05 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,793 Abandoned US20030166084A1 (en) 2000-09-15 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,791 Abandoned US20030082709A1 (en) 1998-08-17 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,725 Abandoned US20030134374A1 (en) 1999-12-09 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,726 Abandoned US20030129690A1 (en) 2000-06-05 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,731 Abandoned US20030129692A1 (en) 1998-05-07 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,790 Abandoned US20030166083A1 (en) 1998-04-09 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,787 Abandoned US20030166082A1 (en) 2000-05-22 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,792 Abandoned US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 Abandoned US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,788 Abandoned US20030129693A1 (en) 1998-06-23 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,795 Abandoned US20030134375A1 (en) 2000-12-01 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,728 Abandoned US20030203437A1 (en) 1998-07-01 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,729 Abandoned US20030082708A1 (en) 2000-06-05 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,500 Abandoned US20030077723A1 (en) 1998-08-12 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,503 Abandoned US20030157628A1 (en) 1998-06-24 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,517 Abandoned US20030077726A1 (en) 1998-06-24 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,508 Abandoned US20030082711A1 (en) 1998-07-02 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,484 Abandoned US20030082710A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,512 Expired - Fee Related US7087428B2 (en) 1998-05-15 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,520 Abandoned US20030170789A1 (en) 1998-08-17 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,502 Abandoned US20030077724A1 (en) 2000-06-05 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,509 Abandoned US20030134380A1 (en) 1998-05-13 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,523 Abandoned US20030092113A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,524 Abandoned US20030166093A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 Abandoned US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,497 Abandoned US20030194767A1 (en) 1998-08-26 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,529 Abandoned US20030134383A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,518 Abandoned US20040214269A1 (en) 1998-07-07 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,485 Expired - Fee Related US7098003B2 (en) 1998-08-19 2002-05-17 PRO 1384 nucleic acids
US10/147,536 Abandoned US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,498 Abandoned US20030166091A1 (en) 1999-02-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,481 Abandoned US20030157626A1 (en) 1998-09-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,507 Abandoned US20030207377A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,495 Abandoned US20030134376A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,493 Abandoned US20040029217A1 (en) 1998-06-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,483 Abandoned US20030180873A1 (en) 1998-08-11 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 Abandoned US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,526 Abandoned US20030077727A1 (en) 2000-05-30 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,496 Abandoned US20030180874A1 (en) 1998-09-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,488 Abandoned US20040253666A1 (en) 1998-09-01 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,510 Abandoned US20030134381A1 (en) 2000-03-03 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,522 Abandoned US20030157629A1 (en) 1998-09-15 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,537 Abandoned US20030207379A1 (en) 1998-09-10 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,516 Abandoned US20030180876A1 (en) 1998-08-20 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,515 Abandoned US20030077725A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,491 Abandoned US20030175865A1 (en) 1998-10-20 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,506 Abandoned US20030134379A1 (en) 2000-12-01 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 Abandoned US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,487 Abandoned US20030166088A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,513 Expired - Fee Related US7220568B2 (en) 1998-10-07 2002-05-17 Nucleic acids encoding the PRO1561 polypeptide
US10/147,490 Abandoned US20030166089A1 (en) 1998-11-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,480 Abandoned US20030166085A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,511 Abandoned US20030134382A1 (en) 1999-07-26 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,501 Abandoned US20030134377A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,489 Abandoned US20030207376A1 (en) 1998-10-28 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,514 Abandoned US20030166092A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,535 Abandoned US20030207378A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,504 Abandoned US20030134378A1 (en) 2000-12-01 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,482 Abandoned US20030157627A1 (en) 1998-08-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,494 Abandoned US20030166090A1 (en) 1998-10-07 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,486 Abandoned US20030166087A1 (en) 2000-03-02 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,527 Abandoned US20030077728A1 (en) 2000-03-03 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,499 Abandoned US20030203439A1 (en) 1998-08-04 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,531 Abandoned US20040253667A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,505 Abandoned US20030180875A1 (en) 1998-06-19 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,373 Abandoned US20030186367A1 (en) 1998-12-22 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,397 Abandoned US20030134384A1 (en) 2000-03-01 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,400 Abandoned US20030207383A1 (en) 2000-06-05 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,396 Abandoned US20030199027A1 (en) 2000-03-01 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,405 Abandoned US20030211571A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,401 Abandoned US20030157630A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,406 Abandoned US20030166096A1 (en) 1999-12-09 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,393 Abandoned US20030199026A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,531 Abandoned US20030148439A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,391 Abandoned US20030194773A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,385 Abandoned US20030199025A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,378 Abandoned US20030175866A1 (en) 2000-06-05 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,381 Abandoned US20030207382A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,399 Abandoned US20030194774A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 Expired - Lifetime US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/152,384 Abandoned US20030175869A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,375 Expired - Lifetime US7160993B2 (en) 2000-03-03 2002-05-21 Nucleic acids encoding PRO4400 polypeptides
US10/152,374 Abandoned US20030194769A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,392 Abandoned US20030175873A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,403 Abandoned US20030175874A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,379 Abandoned US20030166094A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,398 Expired - Fee Related US7074910B2 (en) 2000-03-03 2002-05-21 PRO4340 nucleic acids
US10/152,380 Abandoned US20030129694A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,371 Abandoned US20030194768A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,370 Abandoned US20060084139A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,382 Abandoned US20030175867A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,387 Abandoned US20030175870A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,376 Abandoned US20030207381A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,372 Abandoned US20040126839A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,386 Abandoned US20030194772A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,390 Abandoned US20030175872A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,377 Abandoned US20030194771A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,394 Abandoned US20030166095A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,389 Abandoned US20030175871A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,383 Abandoned US20030175868A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,552 Abandoned US20030199028A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,756 Abandoned US20030175875A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,840 Abandoned US20030199029A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,934 Abandoned US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,586 Abandoned US20030134385A1 (en) 1999-12-09 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,585 Abandoned US20030207384A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,847 Abandoned US20030166098A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,842 Abandoned US20030199031A1 (en) 2000-06-05 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,844 Abandoned US20030199032A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,848 Abandoned US20030194775A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,841 Abandoned US20030199030A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,845 Abandoned US20030199033A1 (en) 2000-06-05 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,799 Abandoned US20030166101A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,797 Abandoned US20030175879A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,796 Abandoned US20030194778A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,462 Abandoned US20030158104A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,802 Abandoned US20030207388A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 Abandoned US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,801 Abandoned US20030207387A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,800 Abandoned US20030207386A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 Abandoned US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,784 Abandoned US20030175878A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,783 Abandoned US20030157631A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,778 Abandoned US20030166100A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,785 Abandoned US20030194776A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,779 Abandoned US20030175877A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,491 Abandoned US20030175880A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,780 Abandoned US20030207385A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,798 Abandoned US20030203440A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,781 Abandoned US20030170790A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,794 Abandoned US20030194777A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,504 Abandoned US20030166102A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,787 Abandoned US20040039164A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,792 Abandoned US20030157632A1 (en) 1999-10-08 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,500 Abandoned US20030194779A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,788 Abandoned US20050074837A1 (en) 2000-09-15 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,789 Abandoned US20030207390A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,785 Abandoned US20030092115A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,784 Abandoned US20030207389A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,783 Abandoned US20030138893A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 Abandoned US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,503 Abandoned US20040033559A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,786 Abandoned US20030134791A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,790 Abandoned US20030180879A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 Abandoned US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 Abandoned US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/230,417 Expired - Lifetime US7297768B2 (en) 1999-12-09 2002-08-28 PRO3574 polypeptide
US10/931,886 Abandoned US20050019823A1 (en) 2000-12-01 2004-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/955,952 Abandoned US20050153396A1 (en) 1998-09-16 2004-09-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/964,241 Abandoned US20070026487A1 (en) 1997-11-24 2004-10-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/973,115 Abandoned US20060040351A1 (en) 1999-03-05 2004-10-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/020,604 Abandoned US20050153348A1 (en) 2000-05-22 2004-12-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/036,869 Abandoned US20050170396A1 (en) 2000-12-01 2005-01-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/040,809 Abandoned US20050118635A1 (en) 1998-04-09 2005-01-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/057,268 Abandoned US20060084144A1 (en) 1997-09-17 2005-02-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/056,802 Abandoned US20050136515A1 (en) 2000-12-01 2005-02-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/057,966 Abandoned US20050187379A1 (en) 1997-09-17 2005-02-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/060,240 Abandoned US20050164279A1 (en) 2000-03-03 2005-02-16 Secreted and transmembrane polypetides and nucleic acids encoding the same
US11/060,652 Abandoned US20050136475A1 (en) 1998-10-22 2005-02-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/110,520 Abandoned US20050202496A1 (en) 2000-06-05 2005-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/117,757 Abandoned US20050214846A1 (en) 2000-09-15 2005-04-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/290,153 Expired - Fee Related US7524497B2 (en) 1998-07-01 2005-11-30 Antibodies to PRO 1120 polypeptide
US11/341,175 Expired - Fee Related US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/537,235 Expired - Fee Related US7504484B2 (en) 1998-10-07 2006-09-29 Antibodies to the PRO1561 polypeptide
US11/538,714 Expired - Fee Related US8106156B2 (en) 1998-07-01 2006-10-04 PRO1120 polypeptides
US11/553,810 Abandoned US20070264686A1 (en) 2000-06-05 2006-10-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/929,190 Abandoned US20080050758A1 (en) 1998-09-09 2007-10-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US12/364,329 Abandoned US20090142786A1 (en) 1998-10-07 2009-02-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Family Applications Before (459)

Application Number Title Priority Date Filing Date
US09/990,456 Abandoned US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 Abandoned US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 Abandoned US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 Abandoned US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 Abandoned US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 Abandoned US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 Abandoned US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 Abandoned US20030096386A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 Abandoned US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 Abandoned US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 Abandoned US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 Abandoned US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,053 Abandoned US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,061 Abandoned US20030082761A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,062 Abandoned US20030077779A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,050 Abandoned US20030054516A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 Abandoned US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 Abandoned US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 Abandoned US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 Abandoned US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 Abandoned US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 Abandoned US20030190722A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 Abandoned US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 Expired - Lifetime US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/123,154 Abandoned US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 Abandoned US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 Abandoned US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 Abandoned US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,214 Expired - Fee Related US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,108 Expired - Fee Related US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 Expired - Lifetime US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,322 Granted US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,291 Abandoned US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 Granted US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 Granted US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 Expired - Lifetime US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,771 Abandoned US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,109 Abandoned US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,235 Abandoned US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 Abandoned US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 Abandoned US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 Abandoned US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 Expired - Lifetime US7700736B2 (en) 2000-05-22 2002-04-15 PRO350 antibodies
US10/123,910 Expired - Lifetime US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,902 Abandoned US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 Expired - Lifetime US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,913 Abandoned US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,911 Expired - Lifetime US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,907 Expired - Fee Related US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,906 Abandoned US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,908 Expired - Lifetime US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,912 Abandoned US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 Abandoned US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 Abandoned US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 Expired - Lifetime US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/124,815 Expired - Lifetime US7342096B2 (en) 1999-12-09 2002-04-17 PRO1879 polypeptide
US10/124,822 Expired - Lifetime US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,813 Expired - Lifetime US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,824 Abandoned US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 Abandoned US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 Abandoned US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 Expired - Lifetime US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,823 Abandoned US20030199062A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,805 Abandoned US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,817 Abandoned US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 Expired - Lifetime US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 Expired - Lifetime US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,820 Abandoned US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,816 Abandoned US20030190728A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,927 Abandoned US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,928 Abandoned US20030087349A1 (en) 1998-06-19 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,923 Abandoned US20030087348A1 (en) 2000-06-05 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 Expired - Lifetime US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/125,921 Expired - Lifetime US7312313B2 (en) 1998-08-17 2002-04-19 Anti-PRO1309 antibodies
US10/125,926 Abandoned US20030082686A1 (en) 2000-06-05 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 Expired - Lifetime US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,931 Abandoned US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,930 Expired - Lifetime US7291700B2 (en) 2000-06-05 2002-04-19 PRO4985 polypeptide
US10/125,932 Expired - Fee Related US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/127,900 Abandoned US20030203429A1 (en) 2000-06-05 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,824 Abandoned US20030087352A1 (en) 1998-08-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,836 Expired - Lifetime US7432345B2 (en) 1998-11-17 2002-04-22 PRO1475 polypeptide
US10/127,833 Abandoned US20030087358A1 (en) 1998-09-01 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,901 Expired - Fee Related US7342098B2 (en) 1998-06-17 2002-04-22 PRO1154 polypeptide
US10/127,837 Abandoned US20030082690A1 (en) 1998-09-01 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,852 Abandoned US20030203428A1 (en) 1999-12-09 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,822 Expired - Lifetime US7371827B2 (en) 1998-06-17 2002-04-22 Antibodies against the PRO1126 polypeptide
US10/127,839 Expired - Lifetime US7449554B2 (en) 2000-06-05 2002-04-22 Antibodies against the PRO4977 polypeptides
US10/127,825 Abandoned US20030077710A1 (en) 1998-10-22 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,844 Expired - Lifetime US7566774B2 (en) 2000-06-05 2002-04-22 PRO4977 polypeptides
US10/127,830 Expired - Lifetime US7351793B2 (en) 1998-09-15 2002-04-22 PRO1286 polypeptide
US10/127,850 Abandoned US20030082698A1 (en) 1998-08-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,851 Expired - Fee Related US7348414B2 (en) 1998-06-17 2002-04-22 Antibodies against the PRO1154 polypeptide
US10/127,835 Abandoned US20030077712A1 (en) 1998-10-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,843 Abandoned US20030082693A1 (en) 2000-06-05 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,842 Abandoned US20030082692A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,847 Abandoned US20030119103A1 (en) 1998-08-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,838 Abandoned US20030082691A1 (en) 1998-11-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,849 Abandoned US20030082697A1 (en) 1998-10-20 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,821 Abandoned US20030087350A1 (en) 1998-08-04 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,840 Abandoned US20030153033A1 (en) 1998-09-10 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,845 Abandoned US20030082694A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,841 Abandoned US20030087361A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,826 Expired - Lifetime US7309763B2 (en) 1998-06-17 2002-04-22 PRO1126 polypeptide
US10/127,832 Abandoned US20030087357A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,834 Abandoned US20030087359A1 (en) 1998-09-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,829 Abandoned US20030077711A1 (en) 1998-10-22 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,827 Abandoned US20030087354A1 (en) 1998-08-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,848 Abandoned US20030082696A1 (en) 1998-11-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,831 Abandoned US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,828 Abandoned US20030087355A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,846 Abandoned US20030082695A1 (en) 2000-03-03 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,691 Expired - Lifetime US7319135B2 (en) 1999-12-09 2002-04-23 PRO1341 polypeptides
US10/128,689 Abandoned US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,684 Abandoned US20030082700A1 (en) 2000-06-05 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,693 Expired - Lifetime US7355006B2 (en) 1998-08-31 2002-04-23 Antibodies against the PRO1271 polypeptides
US10/128,687 Abandoned US20030087363A1 (en) 1998-09-10 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,694 Expired - Lifetime US7189813B2 (en) 2000-03-02 2002-04-23 PRO1338 polypeptides
US10/128,686 Expired - Fee Related US7345146B2 (en) 1998-08-31 2002-04-23 PRO1271 Polypeptides
US10/128,692 Active 2024-06-24 US7704496B2 (en) 1999-12-09 2002-04-23 Antibodies against PRO1341 polypeptide
US10/128,688 Expired - Fee Related US7323544B2 (en) 1999-02-09 2002-04-23 PRO1434 polypeptides
US10/128,685 Abandoned US20030203430A1 (en) 1998-08-11 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/128,690 Abandoned US20030082702A1 (en) 2000-03-02 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,824 Expired - Fee Related US7326413B2 (en) 1999-02-09 2002-04-24 Antibodies against the PRO1434 polypeptide
US10/131,820 Expired - Lifetime US7312314B2 (en) 1998-10-28 2002-04-24 Antibody that binds a pro1693 polypeptide
US10/131,815 Abandoned US20030092103A1 (en) 1998-12-22 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,828 Expired - Lifetime US7309777B2 (en) 1998-10-07 2002-04-24 Antibodies against the PRO1556 polypeptide
US10/131,837 Expired - Lifetime US7294495B2 (en) 1999-12-09 2002-04-24 PR03580 polypeptides
US10/131,819 Expired - Lifetime US7273926B2 (en) 1999-12-09 2002-04-24 Antibody to PRO1779 polypeptides
US10/131,817 Expired - Lifetime US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,829 Abandoned US20030082705A1 (en) 1999-12-09 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,830 Abandoned US20030077720A1 (en) 1999-12-09 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,818 Expired - Fee Related US7166703B2 (en) 1998-10-07 2002-04-24 PRO1561 polypeptides
US10/131,823 Expired - Lifetime US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,833 Expired - Fee Related US7141652B1 (en) 1998-10-07 2002-04-24 Antibodies to PRO1561 polypeptide
US10/131,816 Expired - Lifetime US7361337B2 (en) 1999-12-09 2002-04-24 Antibodies against the PRO1754 polypeptides
US10/131,836 Expired - Lifetime US7351794B2 (en) 1999-12-09 2002-04-24 PRO1754 polypeptides
US10/131,826 Expired - Lifetime US7202345B2 (en) 1998-08-19 2002-04-24 PRO 1384 antibodies
US10/131,813 Expired - Lifetime US7279551B2 (en) 1998-10-07 2002-04-24 Pro1556 Polypeptide
US10/131,835 Expired - Lifetime US7294705B2 (en) 2000-12-01 2002-04-24 Anti-PRO3580 antibodies
US10/131,821 Abandoned US20030092105A1 (en) 1999-12-09 2002-04-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,825 Expired - Lifetime US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,822 Expired - Lifetime US7189806B2 (en) 1998-08-19 2002-04-24 Pro 1384 polypeptides
US10/137,869 Expired - Lifetime US7282558B2 (en) 2000-03-03 2002-05-03 PRO4329 polypeptide
US10/137,871 Expired - Lifetime US7323545B2 (en) 1999-12-09 2002-05-03 PRO1885 polypeptides
US10/137,868 Abandoned US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,866 Abandoned US20030129689A1 (en) 2000-03-01 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,872 Abandoned US20030077722A1 (en) 2000-03-03 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,867 Abandoned US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,865 Abandoned US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,870 Abandoned US20030138883A1 (en) 2000-03-01 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,864 Expired - Lifetime US7291715B2 (en) 2000-03-03 2002-05-03 Antibodies to the PRO4329 polypeptide
US10/137,873 Abandoned US20060084138A1 (en) 2000-03-03 2002-05-03 The pro4979 polypeptide
US10/140,474 Abandoned US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,019 Abandoned US20030148423A1 (en) 1999-12-09 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 Expired - Lifetime US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,470 Granted US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,022 Expired - Lifetime US7285627B2 (en) 2000-06-05 2002-05-06 PRO4989 polypeptides
US10/140,274 Abandoned US20030143674A1 (en) 2000-03-03 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,471 Expired - Lifetime US7291702B2 (en) 1999-12-09 2002-05-06 PRO4326 polypeptides
US10/140,473 Abandoned US20030207351A1 (en) 2000-12-01 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 Expired - Lifetime US7521539B2 (en) 2000-12-01 2002-05-06 Anti-PRO4989 antibodies
US10/140,024 Abandoned US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 Abandoned US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,021 Expired - Lifetime US7279552B2 (en) 1999-12-09 2002-05-06 Pro1782 polypeptides
US10/140,472 Abandoned US20030138888A1 (en) 1999-12-09 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 Abandoned US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,018 Abandoned US20030138885A1 (en) 2000-03-03 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 Expired - Lifetime US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,928 Abandoned US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,862 Expired - Lifetime US7285628B2 (en) 1999-12-09 2002-05-07 PRO1867 polypeptides
US10/140,805 Abandoned US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,809 Abandoned US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,926 Abandoned US20030134356A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,810 Abandoned US20030207353A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 Expired - Lifetime US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/140,806 Active 2024-05-10 US7309764B2 (en) 2000-03-03 2002-05-07 PRO4401 polypeptide
US10/140,922 Expired - Lifetime US7309765B2 (en) 2000-03-03 2002-05-07 PRO4349 polypeptides
US10/140,861 Abandoned US20030148425A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,807 Expired - Lifetime US7335745B2 (en) 2000-03-03 2002-05-07 Antibodies against the PRO4403 polypeptides
US10/140,864 Abandoned US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,927 Expired - Lifetime US7495082B2 (en) 2000-03-03 2002-05-07 Antibodies to PRO4349 polypeptides
US10/140,865 Abandoned US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,924 Abandoned US20030134355A1 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,925 Abandoned US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 Expired - Lifetime US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,863 Expired - Lifetime US7288626B2 (en) 2000-03-03 2002-05-07 PRO4403 polypeptides
US10/140,923 Expired - Lifetime US7282559B2 (en) 2000-03-03 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 Expired - Fee Related US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/141,757 Expired - Fee Related US7314920B2 (en) 2000-03-03 2002-05-08 Antibodies against the PRO4340 polypeptide
US10/141,704 Expired - Lifetime US7396916B2 (en) 2000-06-05 2002-05-08 Antibody to a polypeptide overexpressed in tumors
US10/141,697 Expired - Lifetime US7417123B2 (en) 2000-03-03 2002-05-08 PRO4399 Antibodies
US10/141,698 Expired - Lifetime US7282560B2 (en) 2000-03-03 2002-05-08 PRO 4326 polypeptides
US10/141,701 Abandoned US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,703 Abandoned US20030207357A1 (en) 1999-03-10 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,706 Expired - Lifetime US7425609B2 (en) 2000-03-03 2002-05-08 PRO4330 polypeptides
US10/141,761 Expired - Fee Related US7335729B2 (en) 2000-03-03 2002-05-08 PRO4320 polypeptide
US10/141,760 Expired - Fee Related US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,699 Expired - Lifetime US7390887B2 (en) 2000-03-03 2002-05-08 Antibodies against PRO4330
US10/141,754 Expired - Lifetime US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,759 Expired - Lifetime US7291716B2 (en) 2000-03-03 2002-05-08 PRO4400 antibodies
US10/141,702 Expired - Lifetime US7294692B2 (en) 2000-06-05 2002-05-08 Pro6014 polypeptides
US10/141,700 Expired - Lifetime US7312308B2 (en) 2000-03-03 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 Expired - Lifetime US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/141,753 Expired - Lifetime US7323550B2 (en) 2000-03-03 2002-05-08 Anti-PRO4318 antibodies
US10/141,758 Expired - Lifetime US7301007B2 (en) 2000-03-03 2002-05-08 PRO4340 polypeptides
US10/141,755 Expired - Lifetime US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,705 Expired - Lifetime US7396906B2 (en) 2000-03-03 2002-05-08 Pro4399 Polypeptides
US10/141,762 Abandoned US20030207362A1 (en) 2000-03-03 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,117 Expired - Lifetime US7318922B2 (en) 2000-06-05 2002-05-09 Anti-PRO5337 antibodies
US10/142,422 Expired - Lifetime US7385030B2 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,425 Abandoned US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,426 Abandoned US20040048333A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,027 Abandoned US20030207366A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,421 Expired - Lifetime US7311909B2 (en) 2000-03-03 2002-05-09 PRO4348 antibodies
US10/142,888 Abandoned US20030157606A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,761 Expired - Lifetime US7306795B2 (en) 2000-06-05 2002-05-09 PRO5774 antibodies
US10/142,432 Expired - Lifetime US7390877B2 (en) 2000-03-03 2002-05-09 PRO4381 polypeptides
US10/142,428 Abandoned US20030207363A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,113 Expired - Lifetime US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,417 Expired - Lifetime US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,116 Expired - Lifetime US7390486B2 (en) 2000-03-03 2002-05-09 PRO4381 antibodies
US10/143,034 Expired - Lifetime US7378502B2 (en) 2000-03-03 2002-05-09 PRO4389 antibodies
US10/142,430 Expired - Lifetime US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,427 Expired - Lifetime US7294702B2 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,887 Expired - Lifetime US7291717B2 (en) 1999-12-09 2002-05-09 PRO3446 antibodies
US10/143,035 Expired - Fee Related US7317081B2 (en) 2000-03-03 2002-05-09 PRO4371 polypeptides
US10/143,115 Expired - Lifetime US7309767B2 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,114 Abandoned US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,118 Expired - Lifetime US7309768B2 (en) 2000-03-03 2002-05-09 PRO4347 polypeptides
US10/142,420 Abandoned US20030148434A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,889 Abandoned US20030194765A1 (en) 2000-03-03 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,762 Expired - Lifetime US7288627B2 (en) 2000-03-03 2002-05-09 PRO4322 polypeptides
US10/142,884 Abandoned US20030207365A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,760 Abandoned US20030148437A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,767 Expired - Lifetime US7294693B2 (en) 2000-03-03 2002-05-10 Pro4428 Polypeptides
US10/142,886 Abandoned US20030203432A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,424 Expired - Lifetime US7297766B2 (en) 2000-06-05 2002-05-10 PRO5337 polypeptides
US10/143,033 Abandoned US20030134363A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,431 Expired - Lifetime US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,419 Expired - Lifetime US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/142,423 Abandoned US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,763 Expired - Lifetime US7189807B2 (en) 2000-06-05 2002-05-10 PRO4994 polypeptides
US10/142,765 Abandoned US20030157603A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,032 Expired - Lifetime US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,418 Expired - Lifetime US7297765B2 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,764 Abandoned US20030180865A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,885 Expired - Lifetime US7718173B2 (en) 2000-06-05 2002-05-10 Anti-pro6094 antibodies
US10/142,429 Abandoned US20030207364A1 (en) 2000-06-05 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,766 Expired - Lifetime US7390883B2 (en) 2000-03-03 2002-05-10 PRO4428 antibodies
US10/145,090 Abandoned US20030157613A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,015 Expired - Lifetime US7439325B2 (en) 2000-06-05 2002-05-13 PRO6001 polypeptides
US10/145,127 Expired - Lifetime US7355007B2 (en) 2000-06-05 2002-05-13 Antibodies that bind PRO5005 Polypeptides
US10/144,958 Expired - Lifetime US7479545B2 (en) 2000-06-05 2002-05-13 PRO6001 antibodies
US10/144,956 Abandoned US20030207368A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,091 Abandoned US20030157614A1 (en) 2000-06-05 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,993 Abandoned US20040038336A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,992 Abandoned US20030157611A1 (en) 2000-06-05 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,957 Abandoned US20030157610A1 (en) 2000-06-05 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/144,994 Abandoned US20030134364A1 (en) 2000-03-03 2002-05-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,754 Abandoned US20030157620A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,632 Expired - Lifetime US7385031B2 (en) 2000-06-05 2002-05-14 PRO6097 polypeptides
US10/145,752 Expired - Lifetime US7319136B2 (en) 2000-06-05 2002-05-14 PRO7434 polypeptides
US10/145,961 Abandoned US20040235092A1 (en) 1999-12-09 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,818 Abandoned US20030157622A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,747 Abandoned US20030157618A1 (en) 1999-03-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,877 Expired - Lifetime US7411040B2 (en) 2000-06-05 2002-05-14 PRO6182 polypeptides
US10/145,825 Expired - Lifetime US7335740B2 (en) 2000-06-05 2002-05-14 Antibodies against the PRO7434 polypeptides
US10/145,755 Expired - Lifetime US7312312B2 (en) 2000-03-03 2002-05-14 PRO6242 polypeptides
US10/145,872 Abandoned US20030157624A1 (en) 1999-10-08 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,749 Abandoned US20030207371A1 (en) 2000-05-22 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,870 Expired - Lifetime US7294703B2 (en) 2000-06-05 2002-05-14 PRO6093 Antibodies
US10/145,631 Expired - Lifetime US7371824B2 (en) 2000-06-05 2002-05-14 PRO6093 polypeptides
US10/145,628 Expired - Lifetime US7285636B2 (en) 2000-06-05 2002-05-14 PRO6095 polypeptides
US10/145,821 Abandoned US20030148438A1 (en) 2000-09-15 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,751 Abandoned US20030166074A1 (en) 2000-05-22 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,748 Expired - Lifetime US7354998B2 (en) 2000-06-05 2002-05-14 PRO7171 polypeptides
US10/145,878 Expired - Lifetime US7317088B2 (en) 2000-06-05 2002-05-14 PRO5993 polypeptides
US10/145,869 Abandoned US20030166078A1 (en) 1999-10-08 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,634 Abandoned US20030170788A1 (en) 2000-09-15 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,958 Expired - Lifetime US7408042B2 (en) 2000-06-05 2002-05-14 Anti-PRO6181 antibodies
US10/145,629 Expired - Lifetime US7285644B2 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,626 Expired - Lifetime US7429640B2 (en) 2000-06-05 2002-05-14 PRO6181 polypeptides
US10/145,625 Abandoned US20030180868A1 (en) 2000-03-03 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,750 Expired - Lifetime US7417115B2 (en) 2000-06-05 2002-05-14 PRO6027 polypeptides
US10/145,746 Abandoned US20030134366A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,959 Expired - Lifetime US7390878B2 (en) 2000-06-05 2002-05-14 PRO6090 polypeptides
US10/145,874 Abandoned US20030194766A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,630 Expired - Lifetime US7309778B2 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,824 Expired - Lifetime US7411047B2 (en) 2000-06-05 2002-05-14 PRO6097 antibodies
US10/145,827 Expired - Lifetime US7304140B2 (en) 2000-03-03 2002-05-14 PRO4503 polypeptides
US10/145,823 Abandoned US20030134368A1 (en) 2000-03-03 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,820 Abandoned US20030157623A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,960 Expired - Lifetime US7317074B2 (en) 2000-03-03 2002-05-14 Pro4976 polypeptides
US10/145,633 Abandoned US20030138892A1 (en) 2000-06-05 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,826 Expired - Lifetime US7375195B2 (en) 2000-06-05 2002-05-14 Anti-PRO7171 polypeptides
US10/145,822 Abandoned US20030166075A1 (en) 2000-12-01 2002-05-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,962 Expired - Lifetime US7335746B2 (en) 2000-03-03 2002-05-14 Anti-pro4976 antibodies
US10/145,871 Expired - Lifetime US7396907B2 (en) 2000-06-05 2002-05-14 Antibodies that bind PRO6182 polypeptides
US10/146,724 Abandoned US20030134373A1 (en) 2000-06-05 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,793 Abandoned US20030166084A1 (en) 2000-09-15 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,791 Abandoned US20030082709A1 (en) 1998-08-17 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,725 Abandoned US20030134374A1 (en) 1999-12-09 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,726 Abandoned US20030129690A1 (en) 2000-06-05 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,731 Abandoned US20030129692A1 (en) 1998-05-07 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,790 Abandoned US20030166083A1 (en) 1998-04-09 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,787 Abandoned US20030166082A1 (en) 2000-05-22 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,792 Abandoned US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 Abandoned US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,788 Abandoned US20030129693A1 (en) 1998-06-23 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,795 Abandoned US20030134375A1 (en) 2000-12-01 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,728 Abandoned US20030203437A1 (en) 1998-07-01 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,729 Abandoned US20030082708A1 (en) 2000-06-05 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,500 Abandoned US20030077723A1 (en) 1998-08-12 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,503 Abandoned US20030157628A1 (en) 1998-06-24 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,517 Abandoned US20030077726A1 (en) 1998-06-24 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,508 Abandoned US20030082711A1 (en) 1998-07-02 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,484 Abandoned US20030082710A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,512 Expired - Fee Related US7087428B2 (en) 1998-05-15 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,520 Abandoned US20030170789A1 (en) 1998-08-17 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,502 Abandoned US20030077724A1 (en) 2000-06-05 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,509 Abandoned US20030134380A1 (en) 1998-05-13 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,523 Abandoned US20030092113A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,524 Abandoned US20030166093A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 Abandoned US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,497 Abandoned US20030194767A1 (en) 1998-08-26 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,529 Abandoned US20030134383A1 (en) 1999-12-09 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,518 Abandoned US20040214269A1 (en) 1998-07-07 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,485 Expired - Fee Related US7098003B2 (en) 1998-08-19 2002-05-17 PRO 1384 nucleic acids
US10/147,536 Abandoned US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,498 Abandoned US20030166091A1 (en) 1999-02-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,481 Abandoned US20030157626A1 (en) 1998-09-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,507 Abandoned US20030207377A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,495 Abandoned US20030134376A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,493 Abandoned US20040029217A1 (en) 1998-06-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,483 Abandoned US20030180873A1 (en) 1998-08-11 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 Abandoned US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,526 Abandoned US20030077727A1 (en) 2000-05-30 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,496 Abandoned US20030180874A1 (en) 1998-09-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,488 Abandoned US20040253666A1 (en) 1998-09-01 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,510 Abandoned US20030134381A1 (en) 2000-03-03 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,522 Abandoned US20030157629A1 (en) 1998-09-15 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,537 Abandoned US20030207379A1 (en) 1998-09-10 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,516 Abandoned US20030180876A1 (en) 1998-08-20 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,515 Abandoned US20030077725A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,491 Abandoned US20030175865A1 (en) 1998-10-20 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,506 Abandoned US20030134379A1 (en) 2000-12-01 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 Abandoned US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,487 Abandoned US20030166088A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,513 Expired - Fee Related US7220568B2 (en) 1998-10-07 2002-05-17 Nucleic acids encoding the PRO1561 polypeptide
US10/147,490 Abandoned US20030166089A1 (en) 1998-11-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,480 Abandoned US20030166085A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,511 Abandoned US20030134382A1 (en) 1999-07-26 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,501 Abandoned US20030134377A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,489 Abandoned US20030207376A1 (en) 1998-10-28 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,514 Abandoned US20030166092A1 (en) 1999-12-09 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,535 Abandoned US20030207378A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,504 Abandoned US20030134378A1 (en) 2000-12-01 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,482 Abandoned US20030157627A1 (en) 1998-08-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,494 Abandoned US20030166090A1 (en) 1998-10-07 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,486 Abandoned US20030166087A1 (en) 2000-03-02 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,527 Abandoned US20030077728A1 (en) 2000-03-03 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,499 Abandoned US20030203439A1 (en) 1998-08-04 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,531 Abandoned US20040253667A1 (en) 2000-06-05 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,505 Abandoned US20030180875A1 (en) 1998-06-19 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,373 Abandoned US20030186367A1 (en) 1998-12-22 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,397 Abandoned US20030134384A1 (en) 2000-03-01 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,400 Abandoned US20030207383A1 (en) 2000-06-05 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,396 Abandoned US20030199027A1 (en) 2000-03-01 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,405 Abandoned US20030211571A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,401 Abandoned US20030157630A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,406 Abandoned US20030166096A1 (en) 1999-12-09 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,393 Abandoned US20030199026A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,531 Abandoned US20030148439A1 (en) 2000-03-03 2002-05-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,391 Abandoned US20030194773A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,385 Abandoned US20030199025A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,378 Abandoned US20030175866A1 (en) 2000-06-05 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,381 Abandoned US20030207382A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,399 Abandoned US20030194774A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 Expired - Lifetime US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/152,384 Abandoned US20030175869A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,375 Expired - Lifetime US7160993B2 (en) 2000-03-03 2002-05-21 Nucleic acids encoding PRO4400 polypeptides
US10/152,374 Abandoned US20030194769A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,392 Abandoned US20030175873A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,403 Abandoned US20030175874A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,379 Abandoned US20030166094A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,398 Expired - Fee Related US7074910B2 (en) 2000-03-03 2002-05-21 PRO4340 nucleic acids
US10/152,380 Abandoned US20030129694A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,371 Abandoned US20030194768A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,370 Abandoned US20060084139A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,382 Abandoned US20030175867A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,387 Abandoned US20030175870A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,376 Abandoned US20030207381A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,372 Abandoned US20040126839A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,386 Abandoned US20030194772A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,390 Abandoned US20030175872A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,377 Abandoned US20030194771A1 (en) 1999-12-09 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,394 Abandoned US20030166095A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,389 Abandoned US20030175871A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,383 Abandoned US20030175868A1 (en) 2000-03-03 2002-05-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,552 Abandoned US20030199028A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,756 Abandoned US20030175875A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,840 Abandoned US20030199029A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,934 Abandoned US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,586 Abandoned US20030134385A1 (en) 1999-12-09 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/153,585 Abandoned US20030207384A1 (en) 2000-03-03 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,847 Abandoned US20030166098A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,842 Abandoned US20030199031A1 (en) 2000-06-05 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,844 Abandoned US20030199032A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,848 Abandoned US20030194775A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,841 Abandoned US20030199030A1 (en) 2000-03-03 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,845 Abandoned US20030199033A1 (en) 2000-06-05 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,799 Abandoned US20030166101A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,797 Abandoned US20030175879A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,796 Abandoned US20030194778A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,462 Abandoned US20030158104A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,802 Abandoned US20030207388A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 Abandoned US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,801 Abandoned US20030207387A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,800 Abandoned US20030207386A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 Abandoned US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,784 Abandoned US20030175878A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,783 Abandoned US20030157631A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,778 Abandoned US20030166100A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,785 Abandoned US20030194776A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,779 Abandoned US20030175877A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,491 Abandoned US20030175880A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,780 Abandoned US20030207385A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,798 Abandoned US20030203440A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,781 Abandoned US20030170790A1 (en) 2000-03-03 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,794 Abandoned US20030194777A1 (en) 2000-06-05 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,504 Abandoned US20030166102A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,787 Abandoned US20040039164A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,792 Abandoned US20030157632A1 (en) 1999-10-08 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,500 Abandoned US20030194779A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,788 Abandoned US20050074837A1 (en) 2000-09-15 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,789 Abandoned US20030207390A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,785 Abandoned US20030092115A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,784 Abandoned US20030207389A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,783 Abandoned US20030138893A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 Abandoned US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,503 Abandoned US20040033559A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,786 Abandoned US20030134791A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,790 Abandoned US20030180879A1 (en) 2000-06-05 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 Abandoned US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 Abandoned US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/230,417 Expired - Lifetime US7297768B2 (en) 1999-12-09 2002-08-28 PRO3574 polypeptide
US10/931,886 Abandoned US20050019823A1 (en) 2000-12-01 2004-08-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/955,952 Abandoned US20050153396A1 (en) 1998-09-16 2004-09-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/964,241 Abandoned US20070026487A1 (en) 1997-11-24 2004-10-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/973,115 Abandoned US20060040351A1 (en) 1999-03-05 2004-10-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/020,604 Abandoned US20050153348A1 (en) 2000-05-22 2004-12-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/036,869 Abandoned US20050170396A1 (en) 2000-12-01 2005-01-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/040,809 Abandoned US20050118635A1 (en) 1998-04-09 2005-01-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/057,268 Abandoned US20060084144A1 (en) 1997-09-17 2005-02-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/056,802 Abandoned US20050136515A1 (en) 2000-12-01 2005-02-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/057,966 Abandoned US20050187379A1 (en) 1997-09-17 2005-02-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/060,240 Abandoned US20050164279A1 (en) 2000-03-03 2005-02-16 Secreted and transmembrane polypetides and nucleic acids encoding the same
US11/060,652 Abandoned US20050136475A1 (en) 1998-10-22 2005-02-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Family Applications After (8)

Application Number Title Priority Date Filing Date
US11/117,757 Abandoned US20050214846A1 (en) 2000-09-15 2005-04-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/290,153 Expired - Fee Related US7524497B2 (en) 1998-07-01 2005-11-30 Antibodies to PRO 1120 polypeptide
US11/341,175 Expired - Fee Related US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/537,235 Expired - Fee Related US7504484B2 (en) 1998-10-07 2006-09-29 Antibodies to the PRO1561 polypeptide
US11/538,714 Expired - Fee Related US8106156B2 (en) 1998-07-01 2006-10-04 PRO1120 polypeptides
US11/553,810 Abandoned US20070264686A1 (en) 2000-06-05 2006-10-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/929,190 Abandoned US20080050758A1 (en) 1998-09-09 2007-10-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US12/364,329 Abandoned US20090142786A1 (en) 1998-10-07 2009-02-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Country Status (5)

Country Link
US (468) US20020137890A1 (en)
KR (1) KR20040074090A (en)
AT (1) ATE408419T1 (en)
DE (1) DE60228997D1 (en)
ZA (1) ZA200404849B (en)

Families Citing this family (170)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6300487B1 (en) * 1996-03-19 2001-10-09 Cell Therapuetics, Inc. Mammalian lysophosphatidic acid acyltransferase
US20020137890A1 (en) * 1997-03-31 2002-09-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20060122373A1 (en) * 1997-04-04 2006-06-08 Millennium Pharmaceuticals, Inc. Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 and integrin alpha subunit protein and nucleic acid molecules and uses thereof
US20040141972A1 (en) 1997-11-21 2004-07-22 Genentech, Inc. Compounds, compositions and methods for the treatment of diseases characterized by A-33 related antigens
JP2002502605A (en) * 1998-02-09 2002-01-29 ジェンセット CDNA encoding secreted protein
US20030175778A1 (en) * 1998-06-05 2003-09-18 Jian Ni Interferon Receptor HKAEF92
US20030211578A1 (en) * 1998-06-05 2003-11-13 Jian Ni Interferon receptor HKAEF92
US6699688B1 (en) * 1998-09-16 2004-03-02 The Research Foundation Of The State University Of New York Human platelet F11 receptor
AU6280899A (en) * 1998-09-30 2000-04-17 Millennium Biotherapeutics, Inc. Secreted proteins and nucleic acids encoding them
US7396810B1 (en) * 2000-08-14 2008-07-08 Oregon Health Sciences University Compositions and methods for treating cancer by modulating HER-2 and EGF receptors
US7393823B1 (en) 1999-01-20 2008-07-01 Oregon Health And Science University HER-2 binding antagonists
US7625859B1 (en) * 2000-02-16 2009-12-01 Oregon Health & Science University HER-2 binding antagonists
US7144996B1 (en) * 1999-03-05 2006-12-05 Genentech, Inc. Nucleic acid encoding human suppressor of fused
AU4462600A (en) * 1999-04-12 2000-11-14 Agensys, Inc. Novel prostate-restricted gene expressed in prostate cancer
US6630142B2 (en) * 1999-05-03 2003-10-07 Zymogenetics, Inc. Method of treating fibroproliferative disorders
US6468543B1 (en) * 1999-05-03 2002-10-22 Zymogenetics, Inc. Methods for promoting growth of bone using ZVEGF4
AUPQ206399A0 (en) * 1999-08-06 1999-08-26 Imr Worldwide Pty Ltd. Network user measurement system and method
EP1200590B1 (en) * 1999-08-12 2009-01-07 Agensys, Inc. C-type lectin transmembrane antigen expressed in human prostate cancer and uses thereof
US7842458B2 (en) 1999-08-12 2010-11-30 Agensys, Inc. Nucleic acids and corresponding proteins entitled 58P1D12 useful in treatment and detection of cancer
US20030228288A1 (en) 1999-10-15 2003-12-11 Scarborough Nelson L. Volume maintaining osteoinductive/osteoconductive compositions
IL149046A0 (en) 1999-10-28 2002-11-10 Agensys Inc 36p6d5: secreted tumor antigen
US6639041B2 (en) * 1999-12-03 2003-10-28 Dupont-Toray Co. Ltd. Spandex having low set at low temperatures
US20010044525A1 (en) * 2000-01-05 2001-11-22 Conklin Darrell C. Novel FGF Homolog zFGF12
US20060160181A1 (en) * 2000-02-15 2006-07-20 Amgen Inc. Fibroblast Growth Factor-23 molecules and uses thereof
CA2407746A1 (en) * 2000-05-04 2001-11-08 Incyte Genomics, Inc. Proteases
US20040081961A1 (en) * 2001-05-04 2004-04-29 Delegeane Angelo M Proteases
MXPA02012717A (en) * 2000-06-21 2003-09-22 Amgen Inc Secreted epithelial colon stromal-1 polypeptides, nucleic acids encoding the same and uses thereof.
US9387094B2 (en) 2000-07-19 2016-07-12 Warsaw Orthopedic, Inc. Osteoimplant and method of making same
US6984522B2 (en) 2000-08-03 2006-01-10 Regents Of The University Of Michigan Isolation and use of solid tumor stem cells
US8044259B2 (en) 2000-08-03 2011-10-25 The Regents Of The University Of Michigan Determining the capability of a test compound to affect solid tumor stem cells
EP1191097A1 (en) * 2000-09-21 2002-03-27 Leids Universitair Medisch Centrum Induction of exon skipping in eukaryotic cells
US7323193B2 (en) 2001-12-14 2008-01-29 Osteotech, Inc. Method of making demineralized bone particles
CA2443123A1 (en) * 2001-04-10 2002-10-24 Agensys, Inc. Nuleic acids and corresponding proteins useful in the detection and treatment of various cancers
US7927597B2 (en) * 2001-04-10 2011-04-19 Agensys, Inc. Methods to inhibit cell growth
EP1434608B1 (en) 2001-10-12 2018-08-22 Warsaw Orthopedic, Inc. Improved bone graft
ES2302845T3 (en) * 2001-10-19 2008-08-01 Zymogenetics, Inc. DIMERIZED GROWTH FACTOR AND MATERIALS AND METHODS TO PRODUCE IT.
US20050123925A1 (en) 2002-11-15 2005-06-09 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US6935922B2 (en) * 2002-02-04 2005-08-30 Kla-Tencor Technologies Corp. Methods and systems for generating a two-dimensional map of a characteristic at relative or absolute locations of measurement spots on a specimen during polishing
US7241593B2 (en) * 2002-02-11 2007-07-10 Zymogenetics, Inc. Materials and methods for preparing dimeric growth factors
US6993539B2 (en) 2002-03-19 2006-01-31 Network Appliance, Inc. System and method for determining changes in two snapshots and for transmitting changes to destination snapshot
EP1541677A4 (en) * 2002-07-10 2007-06-27 Takeda Pharmaceutical Novel proteins and use thereof
US7126148B2 (en) * 2002-07-17 2006-10-24 The Johns Hopkins University Neutron detection based on boron activated liquid scintillation
DE10232329A1 (en) * 2002-07-17 2004-02-05 Daimlerchrysler Ag Car body with a strut assembly
AU2003279084A1 (en) * 2002-09-25 2004-04-19 Genentech, Inc. Novel compositions and methods for the treatment of psoriasis
US6890175B2 (en) * 2002-12-18 2005-05-10 Ultradent Products, Inc. Cooling system for hand-held curing light
JP2004267015A (en) * 2003-03-05 2004-09-30 National Institute Of Advanced Industrial & Technology Nucleic acid and method for testing canceration using the same nucleic acid
MXPA05009913A (en) * 2003-03-19 2005-11-04 Biogen Idec Inc Nogo receptor binding protein.
AU2003225410A1 (en) 2003-03-21 2004-10-11 Academisch Ziekenhuis Leiden Modulation of exon recognition in pre-mrna by interfering with the secondary rna structure
NZ544050A (en) * 2003-06-11 2009-03-31 Osteotech Inc Osteoimplants and methods for their manufacture
JP4054727B2 (en) * 2003-07-14 2008-03-05 株式会社リコー Output buffer circuit and interface circuit using output buffer circuit
US20070178458A1 (en) * 2003-09-05 2007-08-02 O'brien Philippa Methods of diagnosis and prognosis of ovarian cancer II
EP1560025A3 (en) * 2003-10-03 2011-09-07 F. Hoffmann-La Roche AG Specific markers for diabetes
EP1694864A2 (en) * 2003-11-20 2006-08-30 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US20070249530A1 (en) * 2004-01-29 2007-10-25 Genentech, Inc. Bcma Polypeptides and Uses Thereof
EP1730189A2 (en) * 2004-02-20 2006-12-13 DeveloGen Aktiengesellschaft Use of secreted protein products for preventing and treating pancreatic diseases and/or obesity and/or metabolic syndrome
DE102004010203B4 (en) * 2004-03-02 2007-05-10 Siemens Ag Method, device and computer program for creating a configuration for an operating device of an automation component
US20050214303A1 (en) * 2004-03-24 2005-09-29 Oncotherapy Science, Inc. Methods for damaging cells using effector functions of anti FAM3D antibodies
WO2005089799A1 (en) * 2004-03-24 2005-09-29 Oncotherapy Science, Inc. Cytotoxicity method using effector function of anti-fam3d antibody
US7659915B2 (en) * 2004-04-02 2010-02-09 K-Nfb Reading Technology, Inc. Portable reading device with mode processing
CN1997394B (en) 2004-04-14 2012-11-28 健泰科生物技术公司 Compositions and methods comprising an EGFL7 antagonist for modulating vascular development
WO2005108999A2 (en) * 2004-05-10 2005-11-17 Evotec Neurosciences Gmbh Diagnostic and therapeutic use of kcnj6 for alzheimer’s disease
EP1751188A1 (en) * 2004-06-04 2007-02-14 Applied Research Systems ARS Holding N.V. Splice variant of unc5h2
PT1776136E (en) 2004-06-24 2012-12-05 Biogen Idec Inc Treatment of conditions involving demyelination
WO2006005459A2 (en) * 2004-07-15 2006-01-19 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with thyrotropin-releasing hormone degrading ectoenzyme (trhde)
US8604185B2 (en) 2004-07-20 2013-12-10 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
CN101080419B (en) 2004-07-20 2014-07-02 健泰科生物技术公司 Compositions and methods of using angiopoietin-like 4 protein
DE602005019167D1 (en) * 2004-07-20 2010-03-18 Genentech Inc ANGIOPOIETIN-LIKE 4 PROTEIN HEMMER COMBINATIONS AND THEIR USE
JP5076058B2 (en) * 2004-08-04 2012-11-21 独立行政法人理化学研究所 Bone and joint disease susceptibility genes and their uses
ATE555124T1 (en) * 2004-08-16 2012-05-15 Agensys Inc NUCLEIC ACIDS AND CORRESPONDING PROTEINS 58P1D12 FOR DETECTING CANCER
AU2005294347A1 (en) * 2004-10-05 2006-04-20 Oregon Health And Science University Compositions and methods for treating disease
US7938307B2 (en) * 2004-10-18 2011-05-10 Tyco Healthcare Group Lp Support structures and methods of using the same
US20060155862A1 (en) * 2005-01-06 2006-07-13 Hari Kathi Data traffic load balancing based on application layer messages
MX2007008561A (en) * 2005-01-14 2008-02-21 Osteotech Inc Expandable osteoimplant.
WO2006081272A2 (en) * 2005-01-27 2006-08-03 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
EP2366726A3 (en) 2005-04-22 2015-05-06 Mitsubishi Chemical Corporation Biomass-resource-derived polyester and production process thereof
WO2006115295A1 (en) * 2005-04-25 2006-11-02 University Of Yamanashi Compositions and methods for treating hemostasis disorders associated with clec-2 signal transduction
PT1904104E (en) 2005-07-08 2013-11-21 Biogen Idec Inc Sp35 antibodies and uses thereof
EP1995321A2 (en) * 2005-08-15 2008-11-26 Genentech, Inc. Gene disruptions, compositions and methods relating thereto
ES2376479T3 (en) * 2005-08-16 2012-03-14 Genentech, Inc. APOPTOTIC SENSITIVITY TO APO2L / TRAIL THROUGH THE GALNAC-T14 EXPRESSION TEST IN CELLS AND FABRICS.
JP2007063225A (en) 2005-09-01 2007-03-15 Takeda Chem Ind Ltd Imidazopyridine compound
WO2007041216A2 (en) 2005-09-30 2007-04-12 Seattle Biomedical Research Institute Plasmodium liver stage antigens
US7723477B2 (en) 2005-10-31 2010-05-25 Oncomed Pharmaceuticals, Inc. Compositions and methods for inhibiting Wnt-dependent solid tumor cell growth
JP2009513708A (en) 2005-10-31 2009-04-02 オンコメッド ファーマシューティカルズ インコーポレイテッド Compositions and methods for diagnosis and treatment of cancer
WO2007056671A1 (en) * 2005-11-02 2007-05-18 Osteotech, Inc. Hemostatic bone graft
US7293487B2 (en) * 2005-11-15 2007-11-13 3M Innovative Properties Company Cutting tool having variable and independent movement in an x-direction and a z-direction into and laterally along a work piece for making microstructures
EP1948614A2 (en) * 2005-11-18 2008-07-30 Takeda San Diego, Inc. Glucokinase activators
US20090175833A1 (en) * 2006-01-13 2009-07-09 Paula Dore-Duffy Pericytes for use as stem cells
EP2001875A2 (en) 2006-03-08 2008-12-17 Takeda San Diego, Inc. Glucokinase activators
WO2007123391A1 (en) * 2006-04-20 2007-11-01 Academisch Ziekenhuis Leiden Therapeutic intervention in a genetic disease in an individual by modifying expression of an aberrantly expressed gene.
EP1857548A1 (en) * 2006-05-19 2007-11-21 Academisch Ziekenhuis Leiden Means and method for inducing exon-skipping
JP5386350B2 (en) 2006-05-31 2014-01-15 タケダ カリフォルニア インコーポレイテッド Indazole and isoindole derivatives as glucokinase activators
ATE524547T1 (en) 2006-08-11 2011-09-15 Prosensa Technologies Bv SINGLE STRANDED OLIGONUCLEOTIDES COMPLEMENTARY TO REPETITIVE ELEMENTS FOR THE TREATMENT OF DNA REPEATS-INSTABILITY-ASSOCIATED DISEASES
DK2066694T3 (en) * 2006-09-29 2016-02-08 Oncomed Pharm Inc Compositions and Methods for Diagnosing and Treating Cancer
US7875747B2 (en) * 2006-10-10 2011-01-25 Afton Chemical Corporation Branched succinimide dispersant compounds and methods of making the compounds
JP2010507340A (en) * 2006-10-20 2010-03-04 シュレイダー エレクトロニクス リミテッド Data error detection and correction method in RF data link
JP2010515428A (en) * 2006-11-14 2010-05-13 ジェネンテック, インコーポレイテッド Novel gene disruption, composition, and related methods
JP5419706B2 (en) 2006-12-20 2014-02-19 タケダ カリフォルニア インコーポレイテッド Glucokinase activator
US7980000B2 (en) * 2006-12-29 2011-07-19 Applied Materials, Inc. Vapor dryer having hydrophilic end effector
US8128926B2 (en) 2007-01-09 2012-03-06 Biogen Idec Ma Inc. Sp35 antibodies and uses thereof
WO2008092002A2 (en) 2007-01-24 2008-07-31 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing pancreatic cancer
EP1961849A1 (en) * 2007-02-22 2008-08-27 ALBIS Spa Pre-consolidated spunbonded web, composite nonwowen comprising said pre-consolidated spunbonded web, method and continuous system for producing said composite
GB0704396D0 (en) * 2007-03-07 2007-04-11 Royal Bank Of Scotland Plc The Cash dispensing methods and systems
WO2008116107A2 (en) 2007-03-21 2008-09-25 Takeda San Diego, Inc. Piperazine derivatives as glucokinase activators
US20080240081A1 (en) * 2007-03-30 2008-10-02 Texas Instruments Incorporated Method, system and apparatus for providing rules-based restriction of incoming calls
US7705085B2 (en) * 2007-04-06 2010-04-27 3M Innovative Properties Company Fluoroelastomer composition for cold shrink articles
EP2167135A2 (en) * 2007-07-12 2010-03-31 Prosensa Technologies B.V. Molecules for targeting compounds to various selected organs, tissues or tumor cells
NZ582521A (en) * 2007-07-12 2011-09-30 Prosensa Technologies Bv A conjugate comprising the amino acid sequence LGAQSNF for targeting compounds to muscle tissue
US8103302B2 (en) * 2007-09-11 2012-01-24 Telefonaktiebolaget Lm Ericsson (Publ) Power-aware link adaptation with variable bandwidth allocation
ES2502217T3 (en) * 2007-10-02 2014-10-03 Genentech, Inc. NLRR-1 antagonists and their uses
US20110020786A1 (en) * 2007-10-08 2011-01-27 Baylor College Of Medicine Peptide dendrimers: affinity reagents for binding noroviruses
CA2701189C (en) * 2007-10-11 2017-05-16 Biogen Idec Ma Inc. Methods for treating pressure induced optic neuropathy, preventing neuronal degeneration and promoting neuronal cell survival via administration of lingo-1 antagonists and trkb agonists
USRE48468E1 (en) 2007-10-26 2021-03-16 Biomarin Technologies B.V. Means and methods for counteracting muscle disorders
JP5600064B2 (en) 2007-10-26 2014-10-01 アカデミシュ ジーケンハウス ライデン Means and methods for offsetting myopathy
WO2009061500A1 (en) * 2007-11-08 2009-05-14 Biogen Idec Ma Inc. Use of lingo-4 antagonists in the treatment of conditions involving demyelination
CA2714120A1 (en) * 2008-02-08 2009-08-13 Prosensa Holding Bv Methods and means for treating dna repeat instability associated genetic disorders
EP2119783A1 (en) 2008-05-14 2009-11-18 Prosensa Technologies B.V. Method for efficient exon (44) skipping in Duchenne Muscular Dystrophy and associated means
WO2010005570A2 (en) 2008-07-09 2010-01-14 Biogen Idec Ma Inc. Compositions comprising antibodies to lingo or fragments thereof
US20100047166A1 (en) * 2008-08-20 2010-02-25 Kanner Steven B Antibodies and related molecules that bind to 58p1d12 proteins
WO2010026473A2 (en) * 2008-09-04 2010-03-11 Oxford Biotherapeutics Ltd. Pta072 protein
BRPI0919473A2 (en) 2008-09-26 2017-08-29 Oncomed Pharm Inc FRIZZLED BINDING AGENTS AND THEIR USES
WO2010048610A2 (en) 2008-10-24 2010-04-29 Osteotech, Inc. Compositions and methods for promoting bone formation
JP2010225758A (en) * 2009-03-23 2010-10-07 Fuji Xerox Co Ltd Organic semiconductor transistor
ES2593836T3 (en) 2009-04-24 2016-12-13 Biomarin Technologies B.V. Oligonucleotide comprising an inosine to treat DMD
PE20120902A1 (en) 2009-05-08 2012-08-08 Genentech Inc HUMANIZED ANTI-EGFL7 ANTIBODIES
JP2012526563A (en) * 2009-05-15 2012-11-01 インサイト ジェネティクス インコーポレイテッド Methods and compositions relating to fusion of ALK for diagnosing and treating cancer
ES2784555T3 (en) 2009-08-28 2020-09-28 Solenis Technologies Cayman Lp Stable Acid Denatured Soybean / Urea Adhesives and Preparation Methods
ES2567030T3 (en) 2009-09-03 2016-04-19 Cancer Research Technology Limited CLEC14A inhibitors
US20110086806A1 (en) * 2009-10-09 2011-04-14 Anaphore, Inc. Polypeptides that Bind IL-23R
DK2488204T3 (en) 2009-10-16 2016-06-06 Oncomed Pharm Inc Therapeutic combination and use of DLL4 antagonist antibodies and blood pressure lowering agents
WO2011051276A1 (en) 2009-10-26 2011-05-05 Externautics S.P.A. Colon and rectal tumor markers and methods of use thereof
WO2011051277A1 (en) 2009-10-26 2011-05-05 Externautics S.P.A. Breast tumor markers and methods of use thereof
EP2493916B1 (en) 2009-10-26 2016-06-08 Externautics S.p.A. Lung tumor markers and methods of use thereof
WO2011068801A2 (en) 2009-12-01 2011-06-09 The Rockefeller University Methods for identifying compounds that modulate ion channel activity of a kir channel
CA2782299A1 (en) * 2009-12-01 2011-06-09 Oncomed Pharmaceuticals, Inc. Methods for treating cancers comprising k-ras mutations
GB0922085D0 (en) * 2009-12-17 2010-02-03 Cambridge Entpr Ltd Cancer diagnosis and treatment
AU2010335039B2 (en) 2009-12-24 2015-03-26 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Molecule for treating an inflammatory disorder
TWI535445B (en) 2010-01-12 2016-06-01 安可美德藥物股份有限公司 Wnt antagonists and methods of treatment and screening
CA2794674A1 (en) 2010-04-01 2011-10-06 Oncomed Pharmaceuticals, Inc. Frizzled-binding agents and uses thereof
EP2927242A1 (en) * 2010-04-09 2015-10-07 Amgen, Inc Btnl9 proteins, nucleic acids, and antibodies and uses thereof
US9139629B2 (en) 2010-06-05 2015-09-22 The Uab Research Foundation Methods for treatment of nephrotic syndrome and related conditions
US20110301103A1 (en) 2010-06-05 2011-12-08 Chugh Sumant S Methods of Treatment
US8551479B2 (en) 2010-09-10 2013-10-08 Oncomed Pharmaceuticals, Inc. Methods for treating melanoma
CN102569428B (en) * 2010-12-21 2015-06-03 上海华虹宏力半导体制造有限公司 Longitudinal voltage-controlled varactor and preparation method thereof
PL3485903T3 (en) 2011-09-23 2023-06-12 Mereo Biopharma 5, Inc. Vegf/dll4 binding agents and uses thereof
WO2013059426A1 (en) * 2011-10-21 2013-04-25 The Regents Of The University Of California Human endogenous retrovirus peptides, antibodies to the peptides, and methods of use thereof
US9443007B2 (en) 2011-11-02 2016-09-13 Salesforce.Com, Inc. Tools and techniques for extracting knowledge from unstructured data retrieved from personal data sources
ITUD20110196A1 (en) * 2011-12-02 2013-06-03 Asoltech S R L COMPOSITION BASED ON UBIDECARENONE
WO2013112053A1 (en) 2012-01-27 2013-08-01 Prosensa Technologies B.V. Rna modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy
EA030716B1 (en) 2012-05-14 2018-09-28 Байоджен Ма Инк. Lingo-2 antagonists for treatment of conditions involving motor neurons
US20140035949A1 (en) * 2012-08-03 2014-02-06 Tempo Ai, Inc. Method and apparatus for enhancing a calendar view on a device
JP2015536933A (en) 2012-10-23 2015-12-24 オンコメッド ファーマシューティカルズ インコーポレイテッド Methods of treating neuroendocrine tumors using Wnt pathway binding agents
WO2014071018A1 (en) 2012-10-31 2014-05-08 Oncomed Pharmaceuticals, Inc. Methods and monitoring of treatment with a dll4 antagonist
CN105073195A (en) 2013-02-04 2015-11-18 昂科梅德制药有限公司 Methods and monitoring of treatment with a Wnt pathway inhibitor
US9168300B2 (en) 2013-03-14 2015-10-27 Oncomed Pharmaceuticals, Inc. MET-binding agents and uses thereof
EP2970484B2 (en) 2013-03-15 2022-09-21 Amgen Inc. Heterodimeric bispecific antibodies
US20160257748A1 (en) 2013-09-25 2016-09-08 Amgen Inc. V-c-fc-v-c antibody
US10367649B2 (en) 2013-11-13 2019-07-30 Salesforce.Com, Inc. Smart scheduling and reporting for teams
CA2960128A1 (en) 2014-09-25 2016-03-31 Amgen Inc Protease-activatable bispecific proteins
EP3212233B1 (en) 2014-10-31 2020-06-24 Oncomed Pharmaceuticals, Inc. Combination therapy for treatment of disease
EP3242893A1 (en) 2015-01-08 2017-11-15 Biogen MA Inc. Lingo-1 antagonists and uses for treatment of demyelinating disorders
EP3896080A1 (en) 2015-09-07 2021-10-20 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Novel igfr-like receptor and uses thereof
AU2016326609B2 (en) 2015-09-23 2023-03-09 Mereo Biopharma 5, Inc. Methods and compositions for treatment of cancer
US20180061660A1 (en) * 2016-08-26 2018-03-01 Infineon Technologies Ag Barrier Layer Formation Using Thermal Processing
US11667697B2 (en) 2016-10-19 2023-06-06 Vanderbilt University Human orthopoxvirus antibodies and methods of use therefor
CN110121507A (en) 2016-12-23 2019-08-13 蓝鳍生物医药公司 Anti- SEZ6L2 antibody and antibody drug conjugate
EP3346271A1 (en) * 2017-01-10 2018-07-11 Chang Gung Memorial Hospital, Linkou Methods and kits for detecting cancer
MX2021005008A (en) * 2018-10-31 2021-06-15 Delinia Inc Multivalent regulatory t cell modulators.
WO2020247852A1 (en) 2019-06-07 2020-12-10 Amgen Inc. Bispecific binding constructs
CA3184351A1 (en) 2020-06-04 2021-12-09 Amgen Inc. Bispecific binding constructs
JP2023553384A (en) 2020-12-03 2023-12-21 アムジエン・インコーポレーテツド Immunoglobulin constructs with multiple binding domains

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536637A (en) * 1993-04-07 1996-07-16 Genetics Institute, Inc. Method of screening for cDNA encoding novel secreted mammalian proteins in yeast
US20030194797A1 (en) * 1999-01-22 2003-10-16 Young Paul E. Metalloproteinase ADAM 22

Family Cites Families (361)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US553637A (en) * 1896-01-28 bagnall
US2929866A (en) * 1953-10-30 1960-03-22 Westinghouse Electric Corp Television pickup tube
GB937358A (en) * 1961-11-13 1963-09-18 Marconi Wireless Telegraph Co Improvements in or relating to television scanning systems
JPS4844279B1 (en) * 1965-05-08 1973-12-24
US3515800A (en) * 1966-05-10 1970-06-02 Victor Company Of Japan Television camera for selectively picking up scenes in color or monochromatically
US3654090A (en) * 1968-09-24 1972-04-04 Organon Method for the determination of antigens and antibodies
US3783190A (en) * 1971-04-08 1974-01-01 Gte Sylvania Inc Aperture correction circuit for low light level television camera
JPS5110986B2 (en) * 1972-10-05 1976-04-08
GB1415827A (en) * 1973-03-31 1975-11-26 Schumacher Klimsch Co E Photomechanical reproduction
US4256833A (en) * 1979-01-08 1981-03-17 Majid Ali Enzyme immunoassay for allergic disorders
JPS57108866A (en) * 1980-12-26 1982-07-07 Fuji Xerox Co Ltd Dichromatic copying machine
US4554101A (en) * 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US4433346A (en) * 1982-03-31 1984-02-21 Xerox Corporation Raster input scanner
US4491865A (en) * 1982-09-29 1985-01-01 Welch Allyn, Inc. Image sensor assembly
US4516153A (en) * 1983-03-30 1985-05-07 Rca Corporation Composite video signal processing apparatus providing amplitude equalization
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4668978A (en) * 1983-08-24 1987-05-26 Kabushiki Kaisha Toshiba Thermal transfer color image forming apparatus with image color and image color density control functions
KR850004274A (en) * 1983-12-13 1985-07-11 원본미기재 Method for preparing erythropoietin
US4608606A (en) * 1984-03-15 1986-08-26 Rca Corporation CCD floating-element output stages providing low reset noise with single sampling
US4658298A (en) * 1984-05-30 1987-04-14 El Planning System Ltd. Portable type audio-visual sensory apparatus
JPS612957A (en) * 1984-06-18 1986-01-08 Toyota Motor Corp Hydraulic controller for power transmission gear with continuously variable transmission
JPS6147919A (en) * 1984-08-15 1986-03-08 Olympus Optical Co Ltd Optical system of endoscope
JPS6142513U (en) * 1984-08-23 1986-03-19 富士写真光機株式会社 Endoscope
US4599116A (en) * 1984-11-08 1986-07-08 Parker Chemical Company Alkaline cleaning process
US4879224A (en) * 1985-01-10 1989-11-07 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing human phospholipase inhibitor polypeptides
GB2199690A (en) * 1985-08-13 1988-07-13 Robert Joseph Mears Fibre-optic lasers and amplifiers
JPH0522897Y2 (en) * 1985-08-16 1993-06-11
JP2679014B2 (en) * 1986-03-19 1997-11-19 オリンパス光学工業株式会社 Electronic endoscope device
JPS6365840A (en) * 1986-04-04 1988-03-24 オリンパス光学工業株式会社 Endoscope
US4735501A (en) * 1986-04-21 1988-04-05 Identechs Corporation Method and apparatus for fluid propelled borescopes
JPH0664243B2 (en) * 1986-04-30 1994-08-22 オリンパス光学工業株式会社 Endoscope
DE3715417A1 (en) * 1986-05-13 1987-11-19 Olympus Optical Co SEMICONDUCTOR IMAGE GENERATION DEVICE, AND ENDOSCOPE HERE EQUIPPED WITH IT
JPS635722A (en) * 1986-06-25 1988-01-11 オリンパス光学工業株式会社 Endoscope
JP2812940B2 (en) * 1986-09-01 1998-10-22 オリンパス光学工業株式会社 Endoscope
US4946778A (en) * 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5013652A (en) * 1986-10-14 1991-05-07 Genex Corporation Composite yeast vectors
US4733937A (en) * 1986-10-17 1988-03-29 Welch Allyn, Inc. Illuminating system for endoscope or borescope
US4758891A (en) * 1986-10-20 1988-07-19 North American Philips Consumer Electronics Corp. Method and apparatus for improving the rise and fall time of a video signal
US4895431A (en) * 1986-11-13 1990-01-23 Olympus Optical Co., Ltd. Method of processing endoscopic images
US5011912A (en) * 1986-12-19 1991-04-30 Immunex Corporation Hybridoma and monoclonal antibody for use in an immunoaffinity purification system
US4727859A (en) * 1986-12-29 1988-03-01 Welch Allyn, Inc. Right angle detachable prism assembly for borescope
US4918521A (en) * 1987-01-20 1990-04-17 Olympus Optical Co., Ltd. Solid state imaging apparatus
JPS63316476A (en) * 1987-06-18 1988-12-23 Seiko Instr & Electronics Ltd Semiconductor device and manufacture thereof
US4796607A (en) * 1987-07-28 1989-01-10 Welch Allyn, Inc. Endoscope steering section
US4794912A (en) * 1987-08-17 1989-01-03 Welch Allyn, Inc. Borescope or endoscope with fluid dynamic muscle
US4786609A (en) * 1987-10-05 1988-11-22 North American Philips Corporation, Signetics Division Method of fabricating field-effect transistor utilizing improved gate sidewall spacers
US5021888A (en) * 1987-12-18 1991-06-04 Kabushiki Kaisha Toshiba Miniaturized solid state imaging device
JP2594627B2 (en) * 1988-02-26 1997-03-26 オリンパス光学工業株式会社 Electronic endoscope device
US5654270A (en) * 1988-06-28 1997-08-05 La Jolla Cancer Research Foundation Use of fibromodulin to prevent or reduce dermal scarring
US5583103A (en) * 1988-06-28 1996-12-10 La Jolla Cancer Research Foundation Inhibition of transforming growth factor beta activity
US4862253A (en) * 1988-07-20 1989-08-29 Welch Allyn, Inc. Apparatus for converting a video processor
US5089473A (en) * 1988-08-29 1992-02-18 Monsanto Company Somatotropin variants and their use
US4909600A (en) * 1988-10-28 1990-03-20 Welch Allyn, Inc. Light chopper assembly
US5530101A (en) * 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5968778A (en) * 1989-01-12 1999-10-19 Jurgen Hoppe PDGF-AB, preparation process and pharmaceuticals containing them
JP2751320B2 (en) * 1989-02-21 1998-05-18 日本電気株式会社 Emitter follower circuit
US5116964A (en) * 1989-02-23 1992-05-26 Genentech, Inc. Hybrid immunoglobulins
US4993405A (en) * 1989-05-15 1991-02-19 Olympus Optical Co., Ltd. Imaging apparatus
US5014515A (en) * 1989-05-30 1991-05-14 Welch Allyn, Inc. Hydraulic muscle pump
US4913369A (en) * 1989-06-02 1990-04-03 Welch Allyn, Inc. Reel for borescope insertion tube
JPH0323832A (en) * 1989-06-22 1991-01-31 Toshiba Corp Electric endoscope apparatus
US5194596A (en) * 1989-07-27 1993-03-16 California Biotechnology Inc. Production of vascular endothelial cell growth factor
US5395760A (en) * 1989-09-05 1995-03-07 Immunex Corporation DNA encoding tumor necrosis factor-α and -β receptors
US4941454A (en) * 1989-10-05 1990-07-17 Welch Allyn, Inc. Servo actuated steering mechanism for borescope or endoscope
US4941456A (en) * 1989-10-05 1990-07-17 Welch Allyn, Inc. Portable color imager borescope
FR2654258A1 (en) * 1989-11-03 1991-05-10 Philips Nv METHOD FOR MANUFACTURING A MITTED TRANSISTOR DEVICE HAVING A REVERSE "T" SHAPE ELECTRODE ELECTRODE
JP2579372B2 (en) * 1989-12-04 1997-02-05 日本テキサス・インスツルメンツ株式会社 Low power imaging device
US4971655A (en) * 1989-12-26 1990-11-20 Micron Technology, Inc. Protection of a refractory metal silicide during high-temperature processing using a dual-layer cap of silicon dioxide and silicon nitride
WO1991010727A1 (en) 1990-01-22 1991-07-25 La Jolla Cancer Research Foundation Inhibitors of cell regulatory factors
US5014600A (en) * 1990-02-06 1991-05-14 Welch Allyn, Inc. Bistep terminator for hydraulic or pneumatic muscle
US4998182A (en) * 1990-02-08 1991-03-05 Welch Allyn, Inc. Connector for optical sensor
US4981810A (en) * 1990-02-16 1991-01-01 Micron Technology, Inc. Process for creating field effect transistors having reduced-slope, staircase-profile sidewall spacers
US5536638A (en) * 1990-04-18 1996-07-16 N.V. Innogenetics S.A. Hybridization probes derived from the spacer region between the 16S and 23S rRNA genes for the detection of Neisseria gonorrhoeae
US5126283A (en) * 1990-05-21 1992-06-30 Motorola, Inc. Process for the selective encapsulation of an electrically conductive structure in a semiconductor device
US5019121A (en) * 1990-05-25 1991-05-28 Welch Allyn, Inc. Helical fluid-actuated torsional motor
US4989581A (en) * 1990-06-01 1991-02-05 Welch Allyn, Inc. Torsional strain relief for borescope
US5203319A (en) * 1990-06-18 1993-04-20 Welch Allyn, Inc. Fluid controlled biased bending neck
US5018506A (en) * 1990-06-18 1991-05-28 Welch Allyn, Inc. Fluid controlled biased bending neck
JPH0456341A (en) * 1990-06-26 1992-02-24 Matsushita Electric Ind Co Ltd Semiconductor integrated circuit layout method
US5306655A (en) * 1990-07-24 1994-04-26 Matsushita Electric Industrial Co., Ltd. Structure and method of manufacture for MOS field effect transistor having lightly doped drain and source diffusion regions
US5018436A (en) * 1990-07-31 1991-05-28 Welch Allyn, Inc. Folded bladder for fluid dynamic muscle
US5114636A (en) * 1990-07-31 1992-05-19 Welch Allyn, Inc. Process for reducing the internal cross section of elastomeric tubing
JP3216650B2 (en) * 1990-08-27 2001-10-09 オリンパス光学工業株式会社 Solid-state imaging device
JPH0817235B2 (en) * 1990-08-29 1996-02-21 株式会社東芝 Offset gate structure transistor and manufacturing method thereof
US5418566A (en) * 1990-09-10 1995-05-23 Kabushiki Kaisha Toshiba Compact imaging apparatus for electronic endoscope with improved optical characteristics
DE4037837A1 (en) * 1990-11-28 1992-06-04 Behringwerke Ag CELL-FREE RECEPTOR BINDING TESTS, THEIR PRODUCTION AND USE
DE69121535T2 (en) * 1990-12-07 1997-01-02 At & T Corp Field effect transistor with inverse T-shaped silicide gate electrode
WO1992010518A1 (en) 1990-12-07 1992-06-25 Yale University Purified slit protein and sequence elements thereof
US5291151A (en) * 1991-01-19 1994-03-01 Canon Kabushiki Kaisha Sensor amplifier
US5994088A (en) * 1991-03-08 1999-11-30 Board Of Trustees Of The University Of Illinois Methods and reagents for preparing and using immunological agents specific for P-glycoprotein
JP3078085B2 (en) * 1991-03-26 2000-08-21 オリンパス光学工業株式会社 Image processing apparatus and image processing method
JPH0595900A (en) * 1991-04-11 1993-04-20 Olympus Optical Co Ltd Endoscope image processing device
US5417210A (en) * 1992-05-27 1995-05-23 International Business Machines Corporation System and method for augmentation of endoscopic surgery
KR940006702B1 (en) * 1991-06-14 1994-07-25 금성일렉트론 주식회사 Manufacturing method of mosfet
US5191879A (en) * 1991-07-24 1993-03-09 Welch Allyn, Inc. Variable focus camera for borescope or endoscope
KR960000225B1 (en) * 1991-08-26 1996-01-03 가부시키가이샤 한도오따이 에네루기 겐큐쇼 Making method of insulated gate type semiconductor device
US5314834A (en) * 1991-08-26 1994-05-24 Motorola, Inc. Field effect transistor having a gate dielectric with variable thickness
US5445940A (en) * 1991-08-28 1995-08-29 Brigham & Women's Hospital Methods and compositions for detecting and treating a subset of human patients having an autoimmune disease
US5202758A (en) * 1991-09-16 1993-04-13 Welch Allyn, Inc. Fluorescent penetrant measurement borescope
ES2136092T3 (en) * 1991-09-23 1999-11-16 Medical Res Council PROCEDURES FOR THE PRODUCTION OF HUMANIZED ANTIBODIES.
JP3067347B2 (en) * 1991-10-30 2000-07-17 株式会社島津製作所 Gel-like bead sorting equipment
US5187759A (en) * 1991-11-07 1993-02-16 At&T Bell Laboratories High gain multi-mode optical amplifier
US5932211A (en) * 1991-11-12 1999-08-03 Women's And Children's Hospital Glycosylation variants of iduronate 2-sulfatase
US5817310A (en) * 1991-12-02 1998-10-06 Cor Therapeutics, Inc. Inhibitory immunoglobulin polypeptides to human PDGF beta receptor
US5614943A (en) * 1991-12-19 1997-03-25 Olympus Optical Co., Ltd. Dissimilar endoscopes usable with a common control unit
US5278642A (en) * 1992-02-26 1994-01-11 Welch Allyn, Inc. Color imaging system
US5441883A (en) * 1992-03-03 1995-08-15 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University A3 adenosine receptor, DNA, and uses
GB2264948B (en) * 1992-03-13 1996-10-16 Merck & Co Inc Human adenosine receptors
US5569662A (en) * 1992-03-23 1996-10-29 Pfizer Inc. Quinuclidine derivatives as substance P antagonists
DE4211547C2 (en) * 1992-04-06 1994-08-11 Henke Sass Wolf Gmbh Protective cover for the distal end of endoscopes
US5306951A (en) * 1992-05-14 1994-04-26 Micron Technology, Inc. Sidewall silicidation for improved reliability and conductivity
AU4397593A (en) * 1992-05-29 1993-12-30 Vivorx, Inc. Microencapsulation of cells
US5429821A (en) * 1992-05-29 1995-07-04 The Regents Of The University Of California Non-fibrogenic high mannuronate alginate coated transplants, processes for their manufacture, and methods for their use
WO1994000573A1 (en) 1992-06-22 1994-01-06 Matritech, Inc. Novel malignant cell type markers of the interior nuclear matrix
US5874082A (en) * 1992-07-09 1999-02-23 Chiron Corporation Humanized anti-CD40 monoclonal antibodies and fragments capable of blocking B cell proliferation
GB9214857D0 (en) * 1992-07-13 1992-08-26 Medical Res Council Human nucleic acid fragments and their use
US5275152A (en) * 1992-07-27 1994-01-04 Welch Allyn, Inc. Insertion tube terminator
CA2142007C (en) * 1992-08-11 2007-10-30 Robert Glen Urban Immunomodulatory peptides
US5480805A (en) * 1992-08-12 1996-01-02 Amoco Corporation Composition for modulating sterols in yeast
US5322807A (en) * 1992-08-19 1994-06-21 At&T Bell Laboratories Method of making thin film transistors including recrystallization and high pressure oxidation
JP3472587B2 (en) * 1992-08-28 2003-12-02 アベンティス ファーマ株式会社 Bone-related carboxypeptidase-like protein and method for producing the same
JPH06121678A (en) * 1992-08-28 1994-05-06 Hoechst Japan Ltd Bone-related sulfatase-like protein and its production
US5262352A (en) * 1992-08-31 1993-11-16 Motorola, Inc. Method for forming an interconnection structure for conductive layers
DE69333366T2 (en) * 1992-10-30 2004-09-16 The General Hospital Corp., Boston A NEW CELL CYCLE CONTROL PROTEIN
KR970011744B1 (en) * 1992-11-04 1997-07-15 마쯔시다덴기산교 가부시기가이샤 Mosfet of ldd type and a method for fabricating the same
KR950011983B1 (en) * 1992-11-23 1995-10-13 삼성전자주식회사 Fabricating method of semiconductor device
US5371026A (en) * 1992-11-30 1994-12-06 Motorola Inc. Method for fabricating paired MOS transistors having a current-gain differential
US5314070A (en) * 1992-12-16 1994-05-24 Welch Allyn, Inc. Case for flexible borescope and endoscope insertion tubes
JP3220538B2 (en) * 1992-12-24 2001-10-22 オリンパス光学工業株式会社 Stereoscopic endoscope and stereoscopic endoscope device
US5798224A (en) * 1992-12-29 1998-08-25 Doheny Eye Institute Nucleic acids encoding protocadherin
JPH06214354A (en) * 1993-01-14 1994-08-05 Fuji Photo Film Co Ltd Silver halide color photographic sensitive material and its processing method
US5293836A (en) * 1993-01-15 1994-03-15 Ziggity Systems, Inc. Water retaining trigger pin
US5386344A (en) * 1993-01-26 1995-01-31 International Business Machines Corporation Flex circuit card elastomeric cable connector assembly
US5633675A (en) * 1993-02-16 1997-05-27 Welch Allyn, Inc, Shadow probe
US5674182A (en) * 1993-02-26 1997-10-07 Olympus Optical Co., Ltd. Endoscope system including endoscope and protection cover
USD358471S (en) * 1993-03-11 1995-05-16 Welch Allyn, Inc. Combined control handle and viewing screen for an endoscope
US5334556A (en) * 1993-03-23 1994-08-02 Texas Instruments Incorporated Method for improving gate oxide integrity using low temperature oxidation during source/drain anneal
DE69434931T2 (en) * 1993-04-02 2007-11-22 Rigel Pharmaceuticals, Inc., South San Francisco METHOD FOR THE SELECTIVE INACTIVATION OF VIRAL REPLICATION
US5323899A (en) * 1993-06-01 1994-06-28 Welch Allyn, Inc. Case for video probe
US5435296A (en) * 1993-06-11 1995-07-25 Welch Allyn, Inc. Endoscope having crimped and soldered cable terminator
US5382533A (en) * 1993-06-18 1995-01-17 Micron Semiconductor, Inc. Method of manufacturing small geometry MOS field-effect transistors having improved barrier layer to hot electron injection
GB9315011D0 (en) * 1993-07-20 1993-09-01 British Telecomm Dispersion compensation
US5439483A (en) * 1993-10-21 1995-08-08 Ventritex, Inc. Method of quantifying cardiac fibrillation using wavelet transform
US6983051B1 (en) * 1993-11-18 2006-01-03 Digimarc Corporation Methods for audio watermarking and decoding
US5439846A (en) * 1993-12-17 1995-08-08 Sgs-Thomson Microelectronics, Inc. Self-aligned method for forming contact with zero offset to gate
US5556767A (en) 1993-12-22 1996-09-17 Human Genome Sciences, Inc. Polynucleotide encoding macrophage inflammatory protein γ
US5552329A (en) * 1994-01-05 1996-09-03 Lg Semicon Co., Ltd. Method of making metal oxide semiconductor transistors
US5519003A (en) * 1994-02-01 1996-05-21 Board Of Trustees Of The Leland Stanford Junior University WD-40-derived peptides and uses thereof
JP3423761B2 (en) * 1994-03-02 2003-07-07 東北パイオニア株式会社 Optical wavelength converter
US5712111A (en) * 1994-04-15 1998-01-27 Merck & Co., Inc. DNA encoding bradykinin B1 receptor
TW387560U (en) * 1994-05-13 2000-04-11 Prec Optics Coroporation Viewing scope with image intensification
KR0141195B1 (en) * 1994-06-08 1998-07-15 김광호 Fabrication method of semiconductor device having low-resistance gate electrod
US5622701A (en) * 1994-06-14 1997-04-22 Protein Design Labs, Inc. Cross-reacting monoclonal antibodies specific for E- and P-selectin
JPH0829701A (en) * 1994-07-18 1996-02-02 Olympus Optical Co Ltd Stereoscopic viewing endoscope system
US5853975A (en) * 1994-08-23 1998-12-29 Millennium Pharmaceuticals, Inc. Methods for identifying compositions for the treatment of body weight disorders, including obesity
US6068867A (en) * 1994-11-02 2000-05-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Protective coatings for food and agricultural products
US6221839B1 (en) * 1994-11-14 2001-04-24 Helsinki University Licensing Ltd. Oy FIt4 ligand and methods of use
US5888774A (en) * 1994-12-19 1999-03-30 Cangene Corporation Recombinant DNA molecules and expression vectors for erythropoietin
JPH08243078A (en) * 1995-03-07 1996-09-24 Fuji Photo Optical Co Ltd Image pickup element assembly body of electronic endoscope
US5730129A (en) * 1995-04-03 1998-03-24 General Electric Company Imaging of interventional devices in a non-stationary subject
GB9506954D0 (en) * 1995-04-04 1995-05-24 Street Graham S B Method and apparatus for image enhancement
US6008325A (en) * 1995-04-19 1999-12-28 Orion Diagnostica Antibody to aminoterminal propeptide of type 1 procollagen
ATE256184T1 (en) * 1995-04-24 2003-12-15 Medical Res Council PROMOTER OF THE UTROPHINE GENE
US5684126A (en) * 1995-06-06 1997-11-04 The Johns Hopkins University School Of Medicine Ebnerin: a secreted von Ebner's gland protein associated with taste buds
CA2221798A1 (en) * 1995-06-06 1996-12-12 Human Genome Sciences, Inc. Colon specific genes and proteins
US5627848A (en) * 1995-09-05 1997-05-06 Imra America, Inc. Apparatus for producing femtosecond and picosecond pulses from modelocked fiber lasers cladding pumped with broad area diode laser arrays
US6060879A (en) * 1995-09-07 2000-05-09 Core Engineering Inc. Current magnitude sensing circuit
CA2230957A1 (en) * 1995-09-29 1997-04-10 Universita'degli Studi Di Siena Regulated genes and uses thereof
KR0169376B1 (en) * 1995-10-10 1999-03-20 김광호 Multi-media ccd camera system
JPH09124697A (en) * 1995-11-01 1997-05-13 Toagosei Co Ltd Peptide and monoclonal antibody
KR970049406A (en) * 1995-12-15 1997-07-29 김광호 Image processing device with graphic overlay speed improvement
US6613544B1 (en) * 1995-12-22 2003-09-02 Amgen Inc. Osteoprotegerin
US6586570B1 (en) * 1996-01-11 2003-07-01 Corixa Corporation Compositions and methods for the treatment and diagnosis of breast cancer
US5867305A (en) * 1996-01-19 1999-02-02 Sdl, Inc. Optical amplifier with high energy levels systems providing high peak powers
US6300487B1 (en) * 1996-03-19 2001-10-09 Cell Therapuetics, Inc. Mammalian lysophosphatidic acid acyltransferase
WO1997034997A1 (en) * 1996-03-21 1997-09-25 Human Genome Sciences, Inc. Human endometrial specific steroid-binding factor i, ii and iii
US6004780A (en) * 1996-03-26 1999-12-21 Human Genome Sciences, Inc. Growth factor HTTER36
US5861248A (en) * 1996-03-29 1999-01-19 Urocor, Inc. Biomarkers for detection of prostate cancer
US5874254A (en) * 1996-03-29 1999-02-23 Director-General Of Agency Of Industrial Science And Technology FGF-5 analogous protein, and pharmaceutical composition containing the same
US6828426B1 (en) * 1996-07-15 2004-12-07 Chugai Seiyaku Kabushiki Kaisha VEGF-like factor
US5857963A (en) * 1996-07-17 1999-01-12 Welch Allyn, Inc. Tab imager assembly for use in an endoscope
US5734418A (en) * 1996-07-17 1998-03-31 Welch Allyn, Inc. Endoscope with tab imager package
US5754313A (en) * 1996-07-17 1998-05-19 Welch Allyn, Inc. Imager assembly
US6111090A (en) * 1996-08-16 2000-08-29 Schering Corporation Mammalian cell surface antigens; related reagents
US6586210B1 (en) * 1996-08-23 2003-07-01 Human Genome Sciences, Inc. Polynucleotides encoding T1 receptor like ligand II
DE69739469D1 (en) * 1996-08-23 2009-07-30 Vegenics Ltd Recombinant vascular endothelial growth factor D (VEGF-D)
US6329197B2 (en) * 1996-10-09 2001-12-11 Synaptic Pharmaceutical Corporation DNA encoding galanin GALR3 receptors and uses thereof
WO1998017810A2 (en) * 1996-10-25 1998-04-30 G.D. Searle & Co. Multi-functional chimeric hematopoietic receptor agonists
WO1999063088A2 (en) 1998-06-02 1999-12-09 Genentech, Inc. Membrane-bound proteins and nucleic acids encoding the same
US6632923B1 (en) * 1996-11-27 2003-10-14 Boston Heart Foundation, Inc. Low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis
US6084461A (en) * 1996-11-29 2000-07-04 Varian Medical Systems, Inc. Charge sensitive amplifier with high common mode signal rejection
AU5902398A (en) * 1996-12-31 1998-07-31 Human Genome Sciences, Inc. Cortistatin polypeptides
WO1998031818A2 (en) * 1997-01-21 1998-07-23 Human Genome Sciences, Inc. Tace-like and matrilysin-like polypeptides
US5840532A (en) * 1997-01-21 1998-11-24 Board Of Regents, The University Of Texas System Neuronal bHLH-PAS domain proteins
US5965397A (en) * 1997-01-31 1999-10-12 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
US6130325A (en) * 1997-02-14 2000-10-10 Incyte Pharmaceuticals, Inc. Human P24 vesicle proteins
US5939271A (en) * 1997-02-19 1999-08-17 The Regents Of The University Of California Netrin receptor
JPH10295388A (en) * 1997-02-28 1998-11-10 Japan Tobacco Inc Mammalian-derived, physiologically active protein
WO2001021658A1 (en) * 1999-09-24 2001-03-29 Human Genome Sciences, Inc. 32 human secreted proteins
US6420526B1 (en) * 1997-03-07 2002-07-16 Human Genome Sciences, Inc. 186 human secreted proteins
US7053190B2 (en) * 1997-03-07 2006-05-30 Human Genome Sciences, Inc. Secreted protein HRGDF73
US20030065139A1 (en) * 1998-03-19 2003-04-03 Craig A. Rosen Secreted protein hmmbd35
WO1998039446A2 (en) * 1997-03-07 1998-09-11 Human Genome Sciences, Inc. 70 human secreted proteins
CA2283299A1 (en) 1997-03-07 1998-09-11 Human Genome Sciences, Inc. 186 human secreted proteins
WO1998056804A1 (en) 1997-06-13 1998-12-17 Human Genome Sciences, Inc. 86 human secreted proteins
US5856139A (en) * 1997-03-11 1999-01-05 Incyte Pharmaceuticals, Inc. Proline-rich acidic protein
US5989909A (en) * 1997-09-26 1999-11-23 Millennium Biotherapeutics, Inc. Huchordin and uses thereof
CA2284550A1 (en) * 1997-03-21 1998-10-01 Human Genome Sciences, Inc. 87 human secreted proteins
US5925521A (en) * 1997-03-31 1999-07-20 Incyte Pharmaceuticals, Inc. Human serine carboxypeptidase
US20030004311A1 (en) * 1997-06-18 2003-01-02 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20020137890A1 (en) 1997-03-31 2002-09-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US6088612A (en) * 1997-04-04 2000-07-11 Medtech Research Corporation Method and apparatus for reflective glare removal in digital photography useful in cervical cancer detection
US5877803A (en) * 1997-04-07 1999-03-02 Tritech Mircoelectronics International, Ltd. 3-D image detector
US20030162956A1 (en) * 1997-04-07 2003-08-28 Human Genome Sciences, Inc. Leukocyte regulatory factors 1 and 2
US20010039335A1 (en) 1997-04-10 2001-11-08 Kenneth Jacobs Secreted proteins and polynucleotides encoding them
EP0991757A1 (en) * 1997-05-06 2000-04-12 ZymoGenetics Novel tumor antigens
DE69837707T2 (en) * 1997-05-15 2008-01-10 Chugai Seiyaku K.K. CURE MEASURES
US5922567A (en) * 1997-06-03 1999-07-13 Incyte Pharmaceuticals, Inc. Two new human DNAJ-like proteins
US6525174B1 (en) * 1997-06-06 2003-02-25 Human Genome Sciences, Inc. Precerebellin-like protein
DE19726796A1 (en) * 1997-06-24 1999-01-07 Basf Ag Propylene polymers
US5818630A (en) * 1997-06-25 1998-10-06 Imra America, Inc. Single-mode amplifiers and compressors based on multi-mode fibers
AU8389498A (en) * 1997-07-11 1999-02-08 Eli Lilly And Company Combinatorial process for preparing fused substituted pyrimidine libraries
DE69834643T2 (en) * 1997-07-18 2007-05-10 Zymogenetics, Inc., Seattle ADIPOCYTE-SPECIFIC PROTEIN HOMOLOGIST
US6060284A (en) * 1997-07-25 2000-05-09 Schering Corporation DNA encoding interleukin-B30
WO1999006548A2 (en) * 1997-08-01 1999-02-11 Genset 5'ESTs FOR NON TISSUE SPECIFIC SECRETED PROTEINS
US6380370B1 (en) * 1997-08-14 2002-04-30 Genome Therapeutics Corporation Nucleic acid and amino acid sequences relating to Staphylococcus epidermidis for diagnostics and therapeutics
AU9115898A (en) 1997-08-21 1999-03-08 Genetics Institute Inc. Secreted proteins
US5876963A (en) * 1997-08-27 1999-03-02 Mitchell; Peter Human nucleotide pyrophosphohydrolase
US6211904B1 (en) * 1997-09-11 2001-04-03 Edwin L. Adair Surgical devices incorporating reduced area imaging devices
IL134968A0 (en) 1997-09-17 2001-05-20 Genentech Inc Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU1069399A (en) * 1997-10-06 1999-04-27 Zymogenetics Inc. A human 2-19 protein homologue, z219a
US20030176681A1 (en) * 1997-10-24 2003-09-18 Ping Feng 148 human secreted proteins
EP1042674A4 (en) * 1997-10-24 2005-03-23 Human Genome Sciences Inc 148 human secreted proteins
US5932445A (en) * 1997-11-07 1999-08-03 Incyte Pharmaceuticals, Inc. Signal peptide-containing proteins
US6548633B1 (en) * 1998-12-22 2003-04-15 Genset, S.A. Complementary DNA's encoding proteins with signal peptides
US6573068B1 (en) * 1997-11-13 2003-06-03 Genset, S. A. Claudin-50 protein
US6025194A (en) * 1997-11-19 2000-02-15 Geron Corporation Nucleic acid sequence of senescence asssociated gene
US5972684A (en) * 1997-11-25 1999-10-26 Incyte Pharmaceuticals, Inc. Carbonic anhydrase VIII
US6194556B1 (en) * 1997-12-11 2001-02-27 Millennium Pharmaceuticals, Inc. Angiotensin converting enzyme homolog and therapeutic and diagnostic uses therfor
WO1999031241A1 (en) * 1997-12-17 1999-06-24 Immunex Corporation Cell surface glycoproteins associated with human b cell lymphomas - ulbp, dna and polypeptides
US6124095A (en) * 1997-12-22 2000-09-26 Incyte Pharmaceuticals, Inc. Human nucleotide pyrophosphohydrolase-2
AU1726199A (en) * 1997-12-31 1999-07-19 Chiron Corporation Metastatic cancer regulated gene
US6191809B1 (en) * 1998-01-15 2001-02-20 Vista Medical Technologies, Inc. Method and apparatus for aligning stereo images
JP2002502605A (en) 1998-02-09 2002-01-29 ジェンセット CDNA encoding secreted protein
US6607879B1 (en) * 1998-02-09 2003-08-19 Incyte Corporation Compositions for the detection of blood cell and immunological response gene expression
AU2783899A (en) * 1998-02-26 1999-09-15 Human Genome Sciences, Inc. 36 human secreted proteins
US6034975A (en) * 1998-03-09 2000-03-07 Imra America, Inc. High power, passively modelocked fiber laser, and method of construction
DK1490386T3 (en) 1998-03-10 2008-12-15 Genentech Inc New polypeptide and nucleic acids encoding this
US6174687B1 (en) * 1999-02-26 2001-01-16 The Burnham Institute Methods of identifying lung homing molecules using membrane dipeptidase
US6245550B1 (en) * 1998-03-20 2001-06-12 Smithkline Beecham Corporation Cytokine family member EF-7
US5945308A (en) * 1998-04-03 1999-08-31 Incyte Pharmaceuticals, Inc. Human oxidized LDL receptor
DE69942607D1 (en) * 1998-04-14 2010-09-02 Chugai Pharmaceutical Co Ltd NEW CYTOKINIC PROTEIN
DE19817948A1 (en) * 1998-04-17 1999-10-21 Metagen Gesellschaft Fuer Genomforschung Mbh New nucleic acid sequences expressed in uterine cancer tissues, and derived polypeptides, for treatment of uterine and endometrial cancer and identification of therapeutic agents
US6150502A (en) * 1998-04-29 2000-11-21 Genesis Research & Development Corporation Limited Polypeptides expressed in skin cells
US6573095B1 (en) * 1998-04-29 2003-06-03 Genesis Research & Development Corporation Limited Polynucleotides isolated from skin cells
US6800473B1 (en) * 1998-06-05 2004-10-05 Daiichi Fine Chemical Co., Ltd. Human cathepsin L2 protein, gene encoding said protein and use thereof
AU5134799A (en) * 1998-07-30 2000-02-21 Human Genome Sciences, Inc. 98 human secreted proteins
US20020182677A1 (en) * 1998-08-03 2002-12-05 Zymogenetics, Inc. Pancreatic and ovarian polypeptide, zsig58
US6547721B1 (en) * 1998-08-07 2003-04-15 Olympus Optical Co., Ltd. Endoscope capable of being autoclaved
US6168920B1 (en) * 1998-08-10 2001-01-02 Incyte Genomics, Inc. Extracellular adhesive proteins
AU5475199A (en) * 1998-08-10 2000-03-06 Genetics Institute Inc. Human chordin-related proteins and polynucleotides encoding them
US20030096951A1 (en) * 1998-08-14 2003-05-22 Kenneth Jacobs Secreted proteins and polynucleotides encoding them
WO2000011015A1 (en) 1998-08-24 2000-03-02 Alphagene, Inc. Secreted proteins and polynucleotides encoding them
AU5583799A (en) * 1998-08-25 2000-03-14 Human Genome Sciences, Inc. 49 human secreted proteins
NZ510464A (en) * 1998-09-01 2004-05-28 Genentech Inc Further pro polypeptides and sequences thereof
US7081514B2 (en) * 1998-09-01 2006-07-25 Genentech, Inc. PRO1347 polypeptides
AU6142899A (en) 1998-09-10 2000-03-27 Incyte Pharmaceuticals, Inc. Human transferase proteins
US6808895B1 (en) * 1999-10-06 2004-10-26 Incyte Corporation DNA encoding oxidoreductase and polypeptide encoded thereby
US6174311B1 (en) * 1998-10-28 2001-01-16 Sdgi Holdings, Inc. Interbody fusion grafts and instrumentation
EP1124850A4 (en) * 1998-10-28 2005-10-19 Human Genome Sciences Inc 12 human secreted proteins
US6214582B1 (en) * 1998-11-16 2001-04-10 The Research Foundation Of State University Of Ny Y2H35 a strong IKK binding protein
DE69933647T2 (en) * 1998-11-19 2007-08-23 Azwell Inc. RECOMBINANT LYSOPHOSPHACIDIC ACID - PHOSPHATASE.
US6275512B1 (en) * 1998-11-25 2001-08-14 Imra America, Inc. Mode-locked multimode fiber laser pulse source
WO2001053455A2 (en) 1999-12-23 2001-07-26 Hyseq, Inc. Novel nucleic acids and polypeptides
WO2000034486A1 (en) * 1998-12-09 2000-06-15 Shionogi & Co., Ltd. Human secretory phospholipase a¿2?
JP2003521229A (en) 1998-12-16 2003-07-15 ジェネンテック・インコーポレーテッド Secreted and transmembrane polypeptides and nucleic acids encoding them
US6278042B1 (en) * 1998-12-16 2001-08-21 E.I. Du Pont De Nemours And Company Plant arsenic transporters
US6184514B1 (en) * 1998-12-18 2001-02-06 Eastman Kodak Company Plastic cover for image sensors
JP4632543B2 (en) * 1998-12-21 2011-02-16 ルードヴィッヒ インスティテュート フォー キャンサー リサーチ Cleaved VEGF-D antibody and use thereof
US6172361B1 (en) * 1998-12-29 2001-01-09 Cirrus Logic, Inc. Methods for mounting an imager to a support structure and circuitry and systems embodying the same
CA2360464A1 (en) * 1999-01-11 2000-07-20 Incyte Pharmaceuticals, Inc. Human peptidases
US20040132158A1 (en) * 1999-01-11 2004-07-08 Incyte Corporation Human peptidases
US6083152A (en) * 1999-01-11 2000-07-04 Welch Allyn, Inc. Endoscopic insertion tube
CA2361272A1 (en) 1999-01-19 2000-07-27 Human Genome Sciences, Inc. 33 human secreted proteins
US6492505B1 (en) * 1999-02-01 2002-12-10 Incyte Genomics, Inc. Composition for detection of genes encoding membrane-associated proteins
US6331427B1 (en) * 1999-03-26 2001-12-18 Millennium Pharmaceuticals, Inc. Protease homologs
IL145048A0 (en) 1999-03-26 2002-06-30 Smithkline Beecham Biolog Casb619 involved in colon cancers
AU3774500A (en) 1999-03-31 2000-10-16 Curagen Corporation Nucleic acids including open reading frames encoding polypeptides; "orfx"
US6538732B1 (en) * 1999-05-04 2003-03-25 Everest Vit, Inc. Inspection system and method
EP1179066A2 (en) * 1999-05-19 2002-02-13 Incyte Genomics, Inc. Extracellular signaling molecules
AU5143700A (en) * 1999-05-20 2000-12-12 Human Genome Sciences, Inc. Seven transmembrane receptor genes
US6186834B1 (en) * 1999-06-08 2001-02-13 Avaya Technology Corp. Enhanced communication connector assembly with crosstalk compensation
US20030082586A1 (en) * 1999-06-29 2003-05-01 Millennium Pharmaceuticals, Inc. Antibodies having diagnostic, preventive, therapeutic, and other uses
US6696279B1 (en) * 1999-07-01 2004-02-24 University Of Tennessee Research Foundation Purified and isolated Mat II β subunit nucleic acids and polypeptides and therapeutic and screening methods using same
EP1396543A3 (en) 1999-07-08 2004-03-31 Research Association for Biotechnology Primers for synthesizing full length cDNA clones and their use
US6951738B2 (en) * 1999-07-16 2005-10-04 Human Genome Sciences, Inc. Human tumor necrosis factor receptors TR13 and TR14
JP2003505083A (en) * 1999-07-21 2003-02-12 インサイト・ゲノミックス・インコーポレイテッド Receptors and related proteins
US20050048623A1 (en) * 1999-07-21 2005-03-03 Incyte Corporation Cell cycle and proliferation proteins
EP1074617A3 (en) 1999-07-29 2004-04-21 Research Association for Biotechnology Primers for synthesising full-length cDNA and their use
AU6181300A (en) * 1999-07-29 2001-02-19 Helix Research Institute Liver cancer-associated genes
CA2311201A1 (en) * 1999-08-05 2001-02-05 Genset S.A. Ests and encoded human proteins
EP1200590B1 (en) * 1999-08-12 2009-01-07 Agensys, Inc. C-type lectin transmembrane antigen expressed in human prostate cancer and uses thereof
CA2381985A1 (en) * 1999-08-16 2001-02-22 Universita'degli Studi Di Siena Vegf-d and angiogenic use thereof
US7090847B1 (en) * 1999-09-09 2006-08-15 Schering Corporation Mammalian cytokines; related reagents and methods
WO2001022920A2 (en) 1999-09-29 2001-04-05 Human Genome Sciences, Inc. Colon and colon cancer associated polynucleotides and polypeptides
WO2001025268A1 (en) * 1999-10-04 2001-04-12 Schrotz King Petra Human seizure related proteins
WO2001025444A2 (en) 1999-10-07 2001-04-12 Zymogenetics, Inc. Novel human phosphodiesterase zcytor13
US6992170B2 (en) * 1999-10-15 2006-01-31 Curagen Corporation Polypeptides and polynucleotides homologous to thymosin, ephrin a receptors, and fibromodulin
AU1449001A (en) 1999-11-05 2001-06-06 Human Genome Sciences, Inc. 28 human secreted proteins
AUPQ434899A0 (en) * 1999-11-26 1999-12-23 University Of Queensland, The Novel polynucleotide and polypeptide
EP1690872A3 (en) 1999-12-01 2006-08-23 Genentech, Inc. Composition and methods for the diagnosis of tumours
US6569662B1 (en) * 2000-01-21 2003-05-27 Hyseq, Inc. Nucleic acids and polypeptides
WO2001053312A1 (en) 1999-12-23 2001-07-26 Hyseq, Inc. Novel nucleic acids and polypeptides
GB0001898D0 (en) * 2000-01-27 2000-03-22 Smithkline Beecham Plc Novel compounds
CA2398003A1 (en) * 2000-01-28 2001-08-02 Incyte Genomics, Inc. Phosphodiesterases
WO2001055317A2 (en) 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
CA2394039A1 (en) 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
WO2001057190A2 (en) 2000-02-03 2001-08-09 Hyseq, Inc. Novel nucleic acids and polypeptides
WO2001059127A2 (en) 2000-02-11 2001-08-16 Incyte Genomics, Inc. Drug metabolizing enzymes
WO2001060850A1 (en) 2000-02-14 2001-08-23 Smithkline Beecham Corporation Novel compounds
US20060160181A1 (en) * 2000-02-15 2006-07-20 Amgen Inc. Fibroblast Growth Factor-23 molecules and uses thereof
WO2001073049A2 (en) * 2000-03-24 2001-10-04 Millennium Pharmaceuticals, Inc. 33877 and 47179, human glycosyl transferase family members and uses thereof
WO2001066595A2 (en) * 2000-03-08 2001-09-13 Chiron Corporation Human fgf-23 gene and gene expression products
US6800455B2 (en) * 2000-03-31 2004-10-05 Scios Inc. Secreted factors
ES2529300T3 (en) 2000-04-12 2015-02-18 Novozymes Biopharma Dk A/S Albumin fusion proteins
US7264926B2 (en) * 2000-04-18 2007-09-04 Millennium Pharmaceuticals, Inc. Nucleoside Phosphatase
WO2001085942A2 (en) * 2000-05-05 2001-11-15 Incyte Genomics, Inc. Cytoskeleton-associated proteins
US7422743B2 (en) * 2000-05-10 2008-09-09 Schering Corporation Mammalian receptor protein DCRS5;methods of treatment
AU2001274888A1 (en) 2000-05-19 2001-12-03 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
WO2001090326A2 (en) * 2000-05-22 2001-11-29 Pharmacia & Upjohn Company Novel matrix metalloproteinases
US7190705B2 (en) * 2000-05-23 2007-03-13 Imra America. Inc. Pulsed laser sources
US6429127B1 (en) * 2000-06-08 2002-08-06 Micron Technology, Inc. Methods for forming rough ruthenium-containing layers and structures/methods using same
US6590470B1 (en) * 2000-06-13 2003-07-08 Welch Allyn, Inc. Cable compensator circuit for CCD video probe
EP1176200A3 (en) * 2000-06-20 2005-01-12 Switch Biotech Aktiengesellschaft Use of polyeptides or their encoding nucleic acids for the diagnosis or treatment of skin diseases or wound healing and their use in indentifying pharmacologically acitve substances
CA2413186A1 (en) 2000-06-30 2002-01-10 Incyte Genomics, Inc. Extracellular matrix and cell adhesion molecules
ATE461213T1 (en) * 2000-07-19 2010-04-15 Advanced Res & Tech Inst NEW FIBROBLAST GROWTH FACTOR (FGF23) AND METHODS OF USE
US6812339B1 (en) * 2000-09-08 2004-11-02 Applera Corporation Polymorphisms in known genes associated with human disease, methods of detection and uses thereof
US6492281B1 (en) * 2000-09-22 2002-12-10 Advanced Micro Devices, Inc. Method of fabricating conductor structures with metal comb bridging avoidance
US20030143686A1 (en) * 2000-09-29 2003-07-31 Incyte Genomics, Inc. Transferases
US6531297B2 (en) * 2000-10-20 2003-03-11 Applera Corporation Isolated human drug-metabolizing proteins, nucleic acid molecules encoding human drug-metabolizing proteins, and uses thereof
US20030130485A1 (en) * 2000-11-14 2003-07-10 Meyers Rachel E. Novel human genes and methods of use thereof
EP1661986A1 (en) 2000-11-17 2006-05-31 Nuvelo, Inc. Nucleic acids and polypeptides enocoded thereby that are member of the kallikrein gene familyNovel nucleic acids and polypeptides
US20030166283A1 (en) 2000-12-21 2003-09-04 Millennium Pharmaceuticals, Inc. 22437, a novel human sulfatase and uses therefor
US20030148920A1 (en) 2000-12-27 2003-08-07 Steven Rosen Sulfatases and methods of use thereof
US7262797B2 (en) * 2001-02-22 2007-08-28 Ge Inspection Technologies Lp Method and system for storing calibration data within image files
CA2433474A1 (en) * 2001-02-23 2002-09-06 Human Genome Sciences, Inc. 70 human secreted proteins
US6536637B1 (en) 2001-03-16 2003-03-25 Mclaughlin Marty E. Combination backpack and water container
JP2005506049A (en) * 2001-03-30 2005-03-03 インサイト・ゲノミックス・インコーポレイテッド Secreted protein
US7436435B2 (en) * 2001-10-01 2008-10-14 Minolta Co., Ltd. Image taking device having image-blur compensator
US6841087B2 (en) * 2002-04-19 2005-01-11 Korea Institute Of Science And Technology Refrigerant composition comprising difluoromethane, 1,1,1-trifluoroethane and 1,1,1,2-tetrafluoroethane
WO2004042009A2 (en) * 2002-10-30 2004-05-21 Genentech, Inc. Inhibition of il-17 production
US20050129108A1 (en) * 2003-01-29 2005-06-16 Everest Vit, Inc. Remote video inspection system
WO2004071517A2 (en) * 2003-02-06 2004-08-26 Schering Corporation Uses of il-23 related reagents
TWI357336B (en) * 2003-03-10 2012-02-01 Schering Corp Uses of il-23 agonists and antagonists; related re
US20050050707A1 (en) * 2003-09-05 2005-03-10 Scott Joshua Lynn Tip tool
EP1694864A2 (en) 2003-11-20 2006-08-30 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US20050162643A1 (en) * 2004-01-22 2005-07-28 Thomas Karpen Automotive fuel tank inspection device
AU2005215527B2 (en) * 2004-02-17 2011-04-07 Merck Sharp & Dohme Corp. Methods of modulating IL-23 activity; related reagents
US20050287593A1 (en) * 2004-05-03 2005-12-29 Schering Corporation Use of cytokine expression to predict skin inflammation; methods of treatment
US20060050983A1 (en) * 2004-09-08 2006-03-09 Everest Vit, Inc. Method and apparatus for enhancing the contrast and clarity of an image captured by a remote viewing device
WO2006068987A2 (en) * 2004-12-20 2006-06-29 Schering Corporation Uses of il-23 antagonists in the treatment of diabetes mellitus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536637A (en) * 1993-04-07 1996-07-16 Genetics Institute, Inc. Method of screening for cDNA encoding novel secreted mammalian proteins in yeast
US20030194797A1 (en) * 1999-01-22 2003-10-16 Young Paul E. Metalloproteinase ADAM 22

Also Published As

Publication number Publication date
US20030166091A1 (en) 2003-09-04
US7323545B2 (en) 2008-01-29
US7285636B2 (en) 2007-10-23
US7357926B2 (en) 2008-04-15
US20030054517A1 (en) 2003-03-20
US20030157630A1 (en) 2003-08-21
US20030199062A1 (en) 2003-10-23
US20030207363A1 (en) 2003-11-06
US20030134362A1 (en) 2003-07-17
US20030166078A1 (en) 2003-09-04
US20030207417A1 (en) 2003-11-06
US20080032332A1 (en) 2008-02-07
US20030129689A1 (en) 2003-07-10
US20030138886A1 (en) 2003-07-24
US20030203462A1 (en) 2003-10-30
US20030207352A1 (en) 2003-11-06
US20030207388A1 (en) 2003-11-06
US20030082764A1 (en) 2003-05-01
US20030175872A1 (en) 2003-09-18
US20030190721A1 (en) 2003-10-09
US20060073568A1 (en) 2006-04-06
US7329730B2 (en) 2008-02-12
US20030180865A1 (en) 2003-09-25
US20030148428A1 (en) 2003-08-07
US20030138883A1 (en) 2003-07-24
US20030134377A1 (en) 2003-07-17
US20030166095A1 (en) 2003-09-04
US20030190722A1 (en) 2003-10-09
US7291702B2 (en) 2007-11-06
US20030203430A1 (en) 2003-10-30
US20050187379A1 (en) 2005-08-25
US7354998B2 (en) 2008-04-08
US20030175877A1 (en) 2003-09-18
US20030203428A1 (en) 2003-10-30
US7329404B2 (en) 2008-02-12
US20030134365A1 (en) 2003-07-17
US20030207349A1 (en) 2003-11-06
US7351793B2 (en) 2008-04-01
US7495082B2 (en) 2009-02-24
US20030068796A1 (en) 2003-04-10
US20030082691A1 (en) 2003-05-01
US20030180874A1 (en) 2003-09-25
US20030190728A1 (en) 2003-10-09
US20030134361A1 (en) 2003-07-17
US20030082710A1 (en) 2003-05-01
US20030129692A1 (en) 2003-07-10
US20030138888A1 (en) 2003-07-24
US20030082712A1 (en) 2003-05-01
US20030129695A1 (en) 2003-07-10
US20030077714A1 (en) 2003-04-24
US20030087355A1 (en) 2003-05-08
US20030194776A1 (en) 2003-10-16
US7285629B2 (en) 2007-10-23
US20030082761A1 (en) 2003-05-01
US20030157616A1 (en) 2003-08-21
US20030207362A1 (en) 2003-11-06
US20030207365A1 (en) 2003-11-06
US20030157602A1 (en) 2003-08-21
US20030073214A1 (en) 2003-04-17
US20030180879A1 (en) 2003-09-25
US7396906B2 (en) 2008-07-08
US20030134384A1 (en) 2003-07-17
US20030073211A1 (en) 2003-04-17
US20030077713A1 (en) 2003-04-24
US20040033559A1 (en) 2004-02-19
US20030211571A1 (en) 2003-11-13
US20030082692A1 (en) 2003-05-01
US20030134354A1 (en) 2003-07-17
US20030077778A1 (en) 2003-04-24
US20030190726A1 (en) 2003-10-09
US20030134791A1 (en) 2003-07-17
US20030073210A1 (en) 2003-04-17
US20030134364A1 (en) 2003-07-17
US20030207378A1 (en) 2003-11-06
US20030148430A1 (en) 2003-08-07
US7304133B2 (en) 2007-12-04
US20030166082A1 (en) 2003-09-04
US20030207429A1 (en) 2003-11-06
US20030100087A1 (en) 2003-05-29
US20030092106A1 (en) 2003-05-15
US20030166100A1 (en) 2003-09-04
US20030190725A1 (en) 2003-10-09
US20030092113A1 (en) 2003-05-15
US20030170790A1 (en) 2003-09-11
US20030157613A1 (en) 2003-08-21
US20030077717A1 (en) 2003-04-24
US20030180878A1 (en) 2003-09-25
US20030166101A1 (en) 2003-09-04
US20030153033A1 (en) 2003-08-14
US20030157604A1 (en) 2003-08-21
US7166703B2 (en) 2007-01-23
US20040038335A1 (en) 2004-02-26
US20030032155A1 (en) 2003-02-13
US20030157603A1 (en) 2003-08-21
US20030186366A1 (en) 2003-10-02
US20030194770A1 (en) 2003-10-16
US8106156B2 (en) 2012-01-31
US20030082690A1 (en) 2003-05-01
US7390877B2 (en) 2008-06-24
US7408033B2 (en) 2008-08-05
US20030036180A1 (en) 2003-02-20
US20030068795A1 (en) 2003-04-10
US20030087357A1 (en) 2003-05-08
US20030148427A1 (en) 2003-08-07
US7317081B2 (en) 2008-01-08
US20030180875A1 (en) 2003-09-25
US20030199031A1 (en) 2003-10-23
US7323550B2 (en) 2008-01-29
US20030148436A1 (en) 2003-08-07
US7282566B2 (en) 2007-10-16
US20030166094A1 (en) 2003-09-04
US20030166074A1 (en) 2003-09-04
US20030166085A1 (en) 2003-09-04
US20030082698A1 (en) 2003-05-01
US20030180866A1 (en) 2003-09-25
US7425609B2 (en) 2008-09-16
US20030207386A1 (en) 2003-11-06
US20030157620A1 (en) 2003-08-21
US7297766B2 (en) 2007-11-20
US20030175865A1 (en) 2003-09-18
US20050153348A1 (en) 2005-07-14
US20030207364A1 (en) 2003-11-06
US20030199026A1 (en) 2003-10-23
US20030148435A1 (en) 2003-08-07
US20030166087A1 (en) 2003-09-04
US20030092147A1 (en) 2003-05-15
US20030068794A1 (en) 2003-04-10
US20030207353A1 (en) 2003-11-06
US20030170789A1 (en) 2003-09-11
US20030190719A1 (en) 2003-10-09
US7432345B2 (en) 2008-10-07
US20030157627A1 (en) 2003-08-21
US7309766B2 (en) 2007-12-18
US7524497B2 (en) 2009-04-28
US20030134369A1 (en) 2003-07-17
US20030190724A1 (en) 2003-10-09
US20030190730A1 (en) 2003-10-09
US20030207376A1 (en) 2003-11-06
US20030138893A1 (en) 2003-07-24
US7291700B2 (en) 2007-11-06
US7276577B2 (en) 2007-10-02
US7396907B2 (en) 2008-07-08
US20030092111A1 (en) 2003-05-15
US20030157624A1 (en) 2003-08-21
US7282558B2 (en) 2007-10-16
US20030073213A1 (en) 2003-04-17
US7297768B2 (en) 2007-11-20
US7385030B2 (en) 2008-06-10
US7326413B2 (en) 2008-02-05
US20030092110A1 (en) 2003-05-15
US7297764B2 (en) 2007-11-20
US20030203431A1 (en) 2003-10-30
US20030190718A1 (en) 2003-10-09
US20030194777A1 (en) 2003-10-16
US7309763B2 (en) 2007-12-18
US20030077791A1 (en) 2003-04-24
US20030092107A1 (en) 2003-05-15
US20030134373A1 (en) 2003-07-17
US20030082765A1 (en) 2003-05-01
US7202345B2 (en) 2007-04-10
US20030207355A1 (en) 2003-11-06
US20030194773A1 (en) 2003-10-16
US7247710B2 (en) 2007-07-24
US20040009548A1 (en) 2004-01-15
US20030207361A1 (en) 2003-11-06
US20030203437A1 (en) 2003-10-30
US20040203125A1 (en) 2004-10-14
US7309765B2 (en) 2007-12-18
US20030199056A1 (en) 2003-10-23
US20030073216A1 (en) 2003-04-17
US20030180923A1 (en) 2003-09-25
US20030087365A1 (en) 2003-05-08
US7319135B2 (en) 2008-01-15
US7309778B2 (en) 2007-12-18
US20040058424A1 (en) 2004-03-25
US20030068798A1 (en) 2003-04-10
US20030148439A1 (en) 2003-08-07
US20030166098A1 (en) 2003-09-04
US20040235092A1 (en) 2004-11-25
US7279552B2 (en) 2007-10-09
US20030207367A1 (en) 2003-11-06
US7335746B2 (en) 2008-02-26
US20030219885A1 (en) 2003-11-27
US20030077781A1 (en) 2003-04-24
US20030148438A1 (en) 2003-08-07
US7189534B2 (en) 2007-03-13
US7285627B2 (en) 2007-10-23
US7105335B2 (en) 2006-09-12
US20050118635A1 (en) 2005-06-02
US7335729B2 (en) 2008-02-26
US20030087350A1 (en) 2003-05-08
US20030082766A1 (en) 2003-05-01
US20030207383A1 (en) 2003-11-06
US20030199030A1 (en) 2003-10-23
US20030077786A1 (en) 2003-04-24
US20030044945A1 (en) 2003-03-06
US20030157631A1 (en) 2003-08-21
US20030194775A1 (en) 2003-10-16
US20030194792A1 (en) 2003-10-16
US20030049817A1 (en) 2003-03-13
US20030134358A1 (en) 2003-07-17
US20030199058A1 (en) 2003-10-23
US20040253666A1 (en) 2004-12-16
US7160993B2 (en) 2007-01-09
US20030203440A1 (en) 2003-10-30
US20030207358A1 (en) 2003-11-06
US7294692B2 (en) 2007-11-13
US20030203439A1 (en) 2003-10-30
US7291716B2 (en) 2007-11-06
US20030207419A1 (en) 2003-11-06
US20030134385A1 (en) 2003-07-17
US20030157607A1 (en) 2003-08-21
US7342097B2 (en) 2008-03-11
US7312314B2 (en) 2007-12-25
US20030203429A1 (en) 2003-10-30
US20030199023A1 (en) 2003-10-23
US20030199061A1 (en) 2003-10-23
US20030157621A1 (en) 2003-08-21
US20030207389A1 (en) 2003-11-06
US7411047B2 (en) 2008-08-12
US20030207415A1 (en) 2003-11-06
US20040214265A1 (en) 2004-10-28
US7311909B2 (en) 2007-12-25
US20030207371A1 (en) 2003-11-06
US20030166096A1 (en) 2003-09-04
US20030194774A1 (en) 2003-10-16
US20030157629A1 (en) 2003-08-21
US20030190717A1 (en) 2003-10-09
US20030207414A1 (en) 2003-11-06
US20030208055A1 (en) 2003-11-06
US20030129690A1 (en) 2003-07-10
US7700736B2 (en) 2010-04-20
US20030087366A1 (en) 2003-05-08
US20030087360A1 (en) 2003-05-08
US20030194772A1 (en) 2003-10-16
US7291329B2 (en) 2007-11-06
US20030077726A1 (en) 2003-04-24
US20030077779A1 (en) 2003-04-24
US20030207423A1 (en) 2003-11-06
US20030207357A1 (en) 2003-11-06
US20030199057A1 (en) 2003-10-23
US20030175873A1 (en) 2003-09-18
US7342098B2 (en) 2008-03-11
US20030157610A1 (en) 2003-08-21
US20040029217A1 (en) 2004-02-12
US20030207370A1 (en) 2003-11-06
US20040253667A1 (en) 2004-12-16
US7408042B2 (en) 2008-08-05
US20060257971A1 (en) 2006-11-16
US20030082687A1 (en) 2003-05-01
US20030032156A1 (en) 2003-02-13
US20030134380A1 (en) 2003-07-17
US7309768B2 (en) 2007-12-18
US7718173B2 (en) 2010-05-18
US20050245730A1 (en) 2005-11-03
US20030153034A1 (en) 2003-08-14
US20030134379A1 (en) 2003-07-17
US7411040B2 (en) 2008-08-12
US20030175874A1 (en) 2003-09-18
US7141652B1 (en) 2006-11-28
US20030077727A1 (en) 2003-04-24
US20040039164A1 (en) 2004-02-26
US20030157605A1 (en) 2003-08-21
US20030207369A1 (en) 2003-11-06
US7306795B2 (en) 2007-12-11
US20030207366A1 (en) 2003-11-06
US20030207427A1 (en) 2003-11-06
US20030175866A1 (en) 2003-09-18
US7390878B2 (en) 2008-06-24
ATE408419T1 (en) 2008-10-15
US20030207377A1 (en) 2003-11-06
US20030207426A1 (en) 2003-11-06
US20030180871A1 (en) 2003-09-25
US7309762B2 (en) 2007-12-18
US7294693B2 (en) 2007-11-13
US20030092115A1 (en) 2003-05-15
US7355006B2 (en) 2008-04-08
US20030059909A1 (en) 2003-03-27
US20030049816A1 (en) 2003-03-13
US20030134383A1 (en) 2003-07-17
US7390883B2 (en) 2008-06-24
US20030082703A1 (en) 2003-05-01
US20030207418A1 (en) 2003-11-06
US20030194768A1 (en) 2003-10-16
US20030068797A1 (en) 2003-04-10
US20030138891A1 (en) 2003-07-24
US20030087358A1 (en) 2003-05-08
US20030157623A1 (en) 2003-08-21
US20030166071A1 (en) 2003-09-04
US20030077712A1 (en) 2003-04-24
US20030199033A1 (en) 2003-10-23
US20040214268A1 (en) 2004-10-28
US7312308B2 (en) 2007-12-25
US20030087346A1 (en) 2003-05-08
US20030087385A1 (en) 2003-05-08
US7521539B2 (en) 2009-04-21
US20030082699A1 (en) 2003-05-01
US20030157618A1 (en) 2003-08-21
US7335740B2 (en) 2008-02-26
US20030194793A1 (en) 2003-10-16
US20030092108A1 (en) 2003-05-15
US20030082759A1 (en) 2003-05-01
US20030077782A1 (en) 2003-04-24
US7408032B2 (en) 2008-08-05
US20030082695A1 (en) 2003-05-01
US20030082701A1 (en) 2003-05-01
US20030134357A1 (en) 2003-07-17
US7074910B2 (en) 2006-07-11
US20030134366A1 (en) 2003-07-17
US20030148431A1 (en) 2003-08-07
US7317088B2 (en) 2008-01-08
US20090209732A1 (en) 2009-08-20
US7319136B2 (en) 2008-01-15
US7291701B2 (en) 2007-11-06
US20030082696A1 (en) 2003-05-01
US20030207356A1 (en) 2003-11-06
US7335745B2 (en) 2008-02-26
US20030087367A1 (en) 2003-05-08
US7297765B2 (en) 2007-11-20
US20030166075A1 (en) 2003-09-04
US20030207390A1 (en) 2003-11-06
US20030082763A1 (en) 2003-05-01
US20030077721A1 (en) 2003-04-24
US20030157606A1 (en) 2003-08-21
US7273926B2 (en) 2007-09-25
US7504484B2 (en) 2009-03-17
US7279551B2 (en) 2007-10-09
US7390887B2 (en) 2008-06-24
US20030087361A1 (en) 2003-05-08
US20030157612A1 (en) 2003-08-21
US7301007B2 (en) 2007-11-27
US7294702B2 (en) 2007-11-13
US20030166102A1 (en) 2003-09-04
US20030087348A1 (en) 2003-05-08
US20030207387A1 (en) 2003-11-06
US20030180873A1 (en) 2003-09-25
US7282559B2 (en) 2007-10-16
US20030068793A1 (en) 2003-04-10
US20030087356A1 (en) 2003-05-08
US20030087359A1 (en) 2003-05-08
US20030134360A1 (en) 2003-07-17
US20030077724A1 (en) 2003-04-24
US20050074837A1 (en) 2005-04-07
US20030082709A1 (en) 2003-05-01
US20030175867A1 (en) 2003-09-18
US20030194767A1 (en) 2003-10-16
US20030082762A1 (en) 2003-05-01
US7417115B2 (en) 2008-08-26
US20030199059A1 (en) 2003-10-23
US7312313B2 (en) 2007-12-25
US7468427B2 (en) 2008-12-23
US20030180868A1 (en) 2003-09-25
US20040126839A1 (en) 2004-07-01
US7351794B2 (en) 2008-04-01
US20030175869A1 (en) 2003-09-18
US7317079B2 (en) 2008-01-08
US20030207373A1 (en) 2003-11-06
US20090142786A1 (en) 2009-06-04
US7417123B2 (en) 2008-08-26
US20030078377A1 (en) 2003-04-24
US7378502B2 (en) 2008-05-27
US20030148429A1 (en) 2003-08-07
US20030148432A1 (en) 2003-08-07
US20030077711A1 (en) 2003-04-24
US20030017563A1 (en) 2003-01-23
US20030166084A1 (en) 2003-09-04
US20030119103A1 (en) 2003-06-26
US20030207359A1 (en) 2003-11-06
US7314920B2 (en) 2008-01-01
US20030166083A1 (en) 2003-09-04
US20050136475A1 (en) 2005-06-23
US20030194794A1 (en) 2003-10-16
US7343721B2 (en) 2008-03-18
US20030134363A1 (en) 2003-07-17
US20030077792A1 (en) 2003-04-24
US20030087347A1 (en) 2003-05-08
US20030207382A1 (en) 2003-11-06
US7189813B2 (en) 2007-03-13
US20030175878A1 (en) 2003-09-18
US20030138885A1 (en) 2003-07-24
US20030134370A1 (en) 2003-07-17
US7309777B2 (en) 2007-12-18
US20030194779A1 (en) 2003-10-16
US20030207416A1 (en) 2003-11-06
US7312307B2 (en) 2007-12-25
US20030207368A1 (en) 2003-11-06
US7220831B2 (en) 2007-05-22
US20030087354A1 (en) 2003-05-08
US20030077718A1 (en) 2003-04-24
US7189806B2 (en) 2007-03-13
US20030073215A1 (en) 2003-04-17
US20030082708A1 (en) 2003-05-01
US7361732B2 (en) 2008-04-22
US7189807B2 (en) 2007-03-13
US20030077719A1 (en) 2003-04-24
US20030134381A1 (en) 2003-07-17
US20040038336A1 (en) 2004-02-26
US20040009547A1 (en) 2004-01-15
US7342096B2 (en) 2008-03-11
US7087428B2 (en) 2006-08-08
US7361337B2 (en) 2008-04-22
US7566774B2 (en) 2009-07-28
US20030082711A1 (en) 2003-05-01
US20030022328A1 (en) 2003-01-30
US20030134382A1 (en) 2003-07-17
US20030077788A1 (en) 2003-04-24
US20030077777A1 (en) 2003-04-24
US7309767B2 (en) 2007-12-18
US20030082686A1 (en) 2003-05-01
US20030207381A1 (en) 2003-11-06
US7098003B2 (en) 2006-08-29
US7291715B2 (en) 2007-11-06
US7439325B2 (en) 2008-10-21
US20030157626A1 (en) 2003-08-21
US20040214269A1 (en) 2004-10-28
US7084258B2 (en) 2006-08-01
US20030175880A1 (en) 2003-09-18
US20030199051A1 (en) 2003-10-23
US20030082702A1 (en) 2003-05-01
US20030186367A1 (en) 2003-10-02
US20030190727A1 (en) 2003-10-09
US7335728B2 (en) 2008-02-26
US20030087363A1 (en) 2003-05-08
US20030180864A1 (en) 2003-09-25
US7285626B2 (en) 2007-10-23
US20030077787A1 (en) 2003-04-24
US20080050758A1 (en) 2008-02-28
US20030194778A1 (en) 2003-10-16
US20030158104A1 (en) 2003-08-21
US20030170788A1 (en) 2003-09-11
US20030087353A1 (en) 2003-05-08
US20030207420A1 (en) 2003-11-06
US20030092103A1 (en) 2003-05-15
US7285644B2 (en) 2007-10-23
US7488586B2 (en) 2009-02-10
US20030077728A1 (en) 2003-04-24
US20030194765A1 (en) 2003-10-16
US20030138889A1 (en) 2003-07-24
US20070224185A1 (en) 2007-09-27
US20030134359A1 (en) 2003-07-17
US20060084139A1 (en) 2006-04-20
US20030157611A1 (en) 2003-08-21
US20030207428A1 (en) 2003-11-06
US20030207351A1 (en) 2003-11-06
US7390486B2 (en) 2008-06-24
US20030087349A1 (en) 2003-05-08
US20030199025A1 (en) 2003-10-23
US20030082697A1 (en) 2003-05-01
US20030194791A1 (en) 2003-10-16
US20030077725A1 (en) 2003-04-24
US20030077790A1 (en) 2003-04-24
US20030148424A1 (en) 2003-08-07
US20040214267A1 (en) 2004-10-28
US7304132B2 (en) 2007-12-04
US20030207384A1 (en) 2003-11-06
US7309764B2 (en) 2007-12-18
US20060194283A1 (en) 2006-08-31
US20030175875A1 (en) 2003-09-18
US20030180870A1 (en) 2003-09-25
US20030077789A1 (en) 2003-04-24
US20030190720A1 (en) 2003-10-09
US7282560B2 (en) 2007-10-16
US20030077783A1 (en) 2003-04-24
US7288626B2 (en) 2007-10-30
US7291717B2 (en) 2007-11-06
US7153941B2 (en) 2006-12-26
US20030129694A1 (en) 2003-07-10
US20030148423A1 (en) 2003-08-07
US20030148425A1 (en) 2003-08-07
US20030077710A1 (en) 2003-04-24
US20030207422A1 (en) 2003-11-06
US7342104B2 (en) 2008-03-11
US20030199027A1 (en) 2003-10-23
US20030077722A1 (en) 2003-04-24
US20070026487A1 (en) 2007-02-01
US7385031B2 (en) 2008-06-10
US20050164279A1 (en) 2005-07-28
US20030134375A1 (en) 2003-07-17
KR20040074090A (en) 2004-08-21
US20030036179A1 (en) 2003-02-20
US20030199063A1 (en) 2003-10-23
US20030175868A1 (en) 2003-09-18
US20040048333A1 (en) 2004-03-11
US20030134368A1 (en) 2003-07-17
US20030087352A1 (en) 2003-05-08
US20030207425A1 (en) 2003-11-06
US20030134378A1 (en) 2003-07-17
US20030082694A1 (en) 2003-05-01
US7375195B2 (en) 2008-05-20
US20030157608A1 (en) 2003-08-21
US20030207424A1 (en) 2003-11-06
US20030143674A1 (en) 2003-07-31
US20030087362A1 (en) 2003-05-08
US20030077784A1 (en) 2003-04-24
US20030077716A1 (en) 2003-04-24
US20030082760A1 (en) 2003-05-01
US20030157622A1 (en) 2003-08-21
US7348414B2 (en) 2008-03-25
US7345146B2 (en) 2008-03-18
US7479545B2 (en) 2009-01-20
US7312312B2 (en) 2007-12-25
US20030077659A1 (en) 2003-04-24
US20030134376A1 (en) 2003-07-17
US20050136515A1 (en) 2005-06-23
US20030082689A1 (en) 2003-05-01
US20030166081A1 (en) 2003-09-04
US20030157601A1 (en) 2003-08-21
US7109305B2 (en) 2006-09-19
US20030022331A1 (en) 2003-01-30
US20050170396A1 (en) 2005-08-04
US20030157628A1 (en) 2003-08-21
US20030082700A1 (en) 2003-05-01
US7285628B2 (en) 2007-10-23
US20030207354A1 (en) 2003-11-06
US20030207379A1 (en) 2003-11-06
US20030134355A1 (en) 2003-07-17
US20030180876A1 (en) 2003-09-25
US7323544B2 (en) 2008-01-29
US20030166090A1 (en) 2003-09-04
US20030190729A1 (en) 2003-10-09
US7193049B2 (en) 2007-03-20
US7288625B2 (en) 2007-10-30
US20030054516A1 (en) 2003-03-20
US20030134367A1 (en) 2003-07-17
US20030199032A1 (en) 2003-10-23
US20030092105A1 (en) 2003-05-15
US20030157619A1 (en) 2003-08-21
US20030166092A1 (en) 2003-09-04
US20030199064A1 (en) 2003-10-23
US20030087345A1 (en) 2003-05-08
US7704496B2 (en) 2010-04-27
US20030073212A1 (en) 2003-04-17
US7371824B2 (en) 2008-05-13
US20030134356A1 (en) 2003-07-17
US20030157617A1 (en) 2003-08-21
US7304131B2 (en) 2007-12-04
US20030199028A1 (en) 2003-10-23
US20030199052A1 (en) 2003-10-23
US20030175879A1 (en) 2003-09-18
US20040077064A1 (en) 2004-04-22
US20030175871A1 (en) 2003-09-18
US20020137890A1 (en) 2002-09-26
US20030199055A1 (en) 2003-10-23
US7635478B2 (en) 2009-12-22
US20030194769A1 (en) 2003-10-16
DE60228997D1 (en) 2008-10-30
US20030166088A1 (en) 2003-09-04
US20030166093A1 (en) 2003-09-04
US7294705B2 (en) 2007-11-13
US7307151B2 (en) 2007-12-11
US20030092104A1 (en) 2003-05-15
US7371827B2 (en) 2008-05-13
US20070264686A1 (en) 2007-11-15
US20060040351A1 (en) 2006-02-23
US20030157609A1 (en) 2003-08-21
US20030207385A1 (en) 2003-11-06
US20030087351A1 (en) 2003-05-08
US20030138887A1 (en) 2003-07-24
US20050153396A1 (en) 2005-07-14
US20030082693A1 (en) 2003-05-01
US20030194771A1 (en) 2003-10-16
US20060084138A1 (en) 2006-04-20
US20030199054A1 (en) 2003-10-23
US20030166077A1 (en) 2003-09-04
US20050019823A1 (en) 2005-01-27
US7317074B2 (en) 2008-01-08
US7318922B2 (en) 2008-01-15
US20030082706A1 (en) 2003-05-01
US7396916B2 (en) 2008-07-08
US20030203432A1 (en) 2003-10-30
US20030207350A1 (en) 2003-11-06
US20030082704A1 (en) 2003-05-01
US7425621B2 (en) 2008-09-16
US20030077723A1 (en) 2003-04-24
US20030157614A1 (en) 2003-08-21
US20030207374A1 (en) 2003-11-06
US7288627B2 (en) 2007-10-30
US7294495B2 (en) 2007-11-13
US20030157632A1 (en) 2003-08-21
US20030096386A1 (en) 2003-05-22
US7304140B2 (en) 2007-12-04
US20030082705A1 (en) 2003-05-01
US20030077715A1 (en) 2003-04-24
US20030148437A1 (en) 2003-08-07
US20030148433A1 (en) 2003-08-07
US20030166089A1 (en) 2003-09-04
US20030207421A1 (en) 2003-11-06
US20040033558A1 (en) 2004-02-19
US7317080B2 (en) 2008-01-08
US20030194766A1 (en) 2003-10-16
US20030134372A1 (en) 2003-07-17
US20030166086A1 (en) 2003-09-04
US20030087364A1 (en) 2003-05-08
US20030077780A1 (en) 2003-04-24
US20030199060A1 (en) 2003-10-23
US20030129693A1 (en) 2003-07-10
US20030199029A1 (en) 2003-10-23
US20030207360A1 (en) 2003-11-06
US20030148434A1 (en) 2003-08-07
US7220568B2 (en) 2007-05-22
US7449554B2 (en) 2008-11-11
US20050214846A1 (en) 2005-09-29
US20030138892A1 (en) 2003-07-24
US20030077785A1 (en) 2003-04-24
US20030166076A1 (en) 2003-09-04
US20030166080A1 (en) 2003-09-04
US20030199053A1 (en) 2003-10-23
US20030077720A1 (en) 2003-04-24
US20030077776A1 (en) 2003-04-24
US20030175870A1 (en) 2003-09-18
US20030190723A1 (en) 2003-10-09
US20060084144A1 (en) 2006-04-20
ZA200404849B (en) 2006-05-31
US7355007B2 (en) 2008-04-08
US20030190731A1 (en) 2003-10-09
US20030134374A1 (en) 2003-07-17
US7429640B2 (en) 2008-09-30
US7294703B2 (en) 2007-11-13
US20030148426A1 (en) 2003-08-07

Similar Documents

Publication Publication Date Title
US7470774B2 (en) Anti-pro1477 antibodies
US20050202496A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP2228446A1 (en) Secreted and transmembrane polypeptieds and nucleic acids encoding the same
US20060003370A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20050202497A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7414112B2 (en) Antibodies to PRO1550 polypeptides
US20050266490A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20050221352A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP1637541B1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20050202475A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20060088854A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20060040289A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20060188914A1 (en) Secreted and transmembrane polypeptides and nucleic acids encoding the same

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION