WO2000073452A2 - Compositions and methods for the treatment of immune related diseases - Google Patents

Compositions and methods for the treatment of immune related diseases Download PDF

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Publication number
WO2000073452A2
WO2000073452A2 PCT/US2000/015264 US0015264W WO0073452A2 WO 2000073452 A2 WO2000073452 A2 WO 2000073452A2 US 0015264 W US0015264 W US 0015264W WO 0073452 A2 WO0073452 A2 WO 0073452A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antι
polypeptide
prol
pro306
Prior art date
Application number
PCT/US2000/015264
Other languages
French (fr)
Other versions
WO2000073452A3 (en
Inventor
Avi J. Ashkenazi
Kevin P. Baker
Betty Chan
Audrey Goddard
Paul J. Godowski
Austin L. Gurney
Caroline Hebert
William Henzel
Rhona C. Kabakoff
David L. Shelton
Daniel Tumas
Colin K. Watanabe
William I. Wood
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
Priority claimed from PCT/US1999/012252 external-priority patent/WO1999063088A2/en
Priority claimed from PCT/US1999/020111 external-priority patent/WO2000012708A2/en
Priority claimed from PCT/US1999/021090 external-priority patent/WO2000015796A2/en
Priority claimed from PCT/US1999/021547 external-priority patent/WO2000015797A2/en
Priority claimed from PCT/US1999/028313 external-priority patent/WO2000032221A2/en
Priority claimed from PCT/US1999/028634 external-priority patent/WO2000036102A2/en
Priority claimed from PCT/US1999/028565 external-priority patent/WO2000037638A2/en
Priority claimed from PCT/US1999/028551 external-priority patent/WO2000053750A1/en
Priority claimed from PCT/US1999/030911 external-priority patent/WO2000075316A1/en
Priority claimed from PCT/US2000/000219 external-priority patent/WO2000053753A2/en
Priority claimed from PCT/US2000/000376 external-priority patent/WO2000053755A2/en
Priority claimed from PCT/US2000/003565 external-priority patent/WO2001053486A1/en
Priority claimed from PCT/US2000/004341 external-priority patent/WO2000053756A2/en
Priority claimed from PCT/US2000/004342 external-priority patent/WO2000078961A1/en
Priority claimed from PCT/US2000/004414 external-priority patent/WO2001004311A1/en
Priority claimed from PCT/US2000/005004 external-priority patent/WO2000053757A2/en
Priority claimed from PCT/US2000/004914 external-priority patent/WO2000075327A1/en
Priority claimed from PCT/US2000/005601 external-priority patent/WO2000056889A2/en
Priority claimed from PCT/US2000/005841 external-priority patent/WO2000053758A2/en
Priority claimed from PCT/US2000/006884 external-priority patent/WO2001005972A1/en
Priority claimed from PCT/US2000/007377 external-priority patent/WO2001019991A1/en
Priority claimed from PCT/US2000/007532 external-priority patent/WO2000070050A1/en
Priority claimed from PCT/US2000/008439 external-priority patent/WO2000073454A1/en
Priority claimed from PCT/US2000/013705 external-priority patent/WO2000073445A2/en
Priority claimed from PCT/US2000/014042 external-priority patent/WO2000077037A2/en
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU54601/00A priority Critical patent/AU5460100A/en
Priority to AU6802801A priority patent/AU6802801A/en
Priority to EP06000589A priority patent/EP1661997A1/en
Priority to CA002492070A priority patent/CA2492070A1/en
Priority to CA002490909A priority patent/CA2490909A1/en
Priority to EP06000582A priority patent/EP1666495A1/en
Priority to CA002491258A priority patent/CA2491258A1/en
Priority to CA002391455A priority patent/CA2391455A1/en
Priority to CA002492049A priority patent/CA2492049A1/en
Priority to EP05025102A priority patent/EP1672070A3/en
Priority to CA002494705A priority patent/CA2494705A1/en
Priority to EP00983846A priority patent/EP1250426A2/en
Priority to CA002491610A priority patent/CA2491610A1/en
Priority to EP06000585A priority patent/EP1661996A1/en
Priority to AU20554/01A priority patent/AU2055401A/en
Priority to CA002491433A priority patent/CA2491433A1/en
Priority to CA002496312A priority patent/CA2496312A1/en
Priority to EP06000584A priority patent/EP1669371A3/en
Priority to EP06000583A priority patent/EP1686134A3/en
Priority to EP06000587A priority patent/EP1690872A3/en
Priority to PCT/US2000/032678 priority patent/WO2001040466A2/en
Priority to EP10005292A priority patent/EP2228446A1/en
Priority to EP06000588A priority patent/EP1690873A3/en
Priority to JP2001542531A priority patent/JP2004522404A/en
Priority to CA002490853A priority patent/CA2490853A1/en
Priority to EP06000581A priority patent/EP1666494A1/en
Priority to CA2709291A priority patent/CA2709291A1/en
Priority to EP06000586A priority patent/EP1688497A1/en
Publication of WO2000073452A2 publication Critical patent/WO2000073452A2/en
Priority to EP07016900A priority patent/EP1897944B1/en
Priority to EP10009490A priority patent/EP2290081A3/en
Priority to AU25909/01A priority patent/AU2590901A/en
Priority to DK07016903.2T priority patent/DK1897947T3/en
Priority to ES07016901T priority patent/ES2380958T3/en
Priority to AT07016899T priority patent/ATE537258T1/en
Priority to AT07016903T priority patent/ATE541931T1/en
Priority to DK07016901.6T priority patent/DK1897945T3/en
Priority to AT07016901T priority patent/ATE541930T1/en
Priority to EP00989401A priority patent/EP1240325B1/en
Priority to JP2001546918A priority patent/JP2003527104A/en
Priority to ES10009491.1T priority patent/ES2458349T3/en
Priority to DE60043069T priority patent/DE60043069D1/en
Priority to AT00989401T priority patent/ATE444361T1/en
Priority to AT07016900T priority patent/ATE519847T1/en
Priority to EP07016901A priority patent/EP1897945B1/en
Priority to US09/747,259 priority patent/US6569645B2/en
Priority to ES07016903T priority patent/ES2380812T3/en
Priority to PT00989401T priority patent/PT1240325E/en
Priority to ES07016899T priority patent/ES2379101T3/en
Priority to ES09009972.2T priority patent/ES2506665T3/en
Priority to EP07016902A priority patent/EP1897946B1/en
Priority to PT07016903T priority patent/PT1897947E/en
Priority to CA002391374A priority patent/CA2391374A1/en
Priority to EP07016903A priority patent/EP1897947B1/en
Priority to DK00989401.5T priority patent/DK1240325T3/en
Priority to EP09009972.2A priority patent/EP2163625B1/en
Priority to PT07016900T priority patent/PT1897944E/en
Priority to EP07016899A priority patent/EP1897943B1/en
Priority to PT07016901T priority patent/PT1897945E/en
Priority to PCT/US2000/034956 priority patent/WO2001046420A2/en
Priority to EP10009491.1A priority patent/EP2258848B1/en
Priority to ES07016902T priority patent/ES2388748T3/en
Priority to DK07016900.8T priority patent/DK1897944T3/en
Priority to ES00989401T priority patent/ES2333772T3/en
Priority to CA002401448A priority patent/CA2401448A1/en
Priority to CA002534391A priority patent/CA2534391A1/en
Priority to CA002533831A priority patent/CA2533831A1/en
Priority to PCT/US2001/006520 priority patent/WO2001068848A2/en
Priority to CA002534030A priority patent/CA2534030A1/en
Priority to AU2001268028A priority patent/AU2001268028A1/en
Priority to CA002539214A priority patent/CA2539214A1/en
Priority to CA002534186A priority patent/CA2534186A1/en
Priority to JP2001567332A priority patent/JP2004508805A/en
Priority to EP01945919A priority patent/EP1259614A2/en
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Priority to CA002533903A priority patent/CA2533903A1/en
Priority to CA002533991A priority patent/CA2533991A1/en
Priority to CA002531917A priority patent/CA2531917A1/en
Priority to US09/816,744 priority patent/US6579520B2/en
Priority to PCT/US2001/017092 priority patent/WO2001092331A2/en
Priority to AU2001265019A priority patent/AU2001265019A1/en
Priority to US09/866,034 priority patent/US20030170864A1/en
Priority to AU6531101A priority patent/AU6531101A/en
Priority to EP04005726A priority patent/EP1489095A1/en
Priority to EP05024038A priority patent/EP1702928A3/en
Priority to JP2002501551A priority patent/JP2004510409A/en
Priority to EP01939834A priority patent/EP1286749A1/en
Priority to CA002591656A priority patent/CA2591656A1/en
Priority to CA002591590A priority patent/CA2591590A1/en
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Priority to CA002591583A priority patent/CA2591583A1/en
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Priority to EP05024032A priority patent/EP1659177A3/en
Priority to AU2001265311A priority patent/AU2001265311A1/en
Priority to CA002591841A priority patent/CA2591841A1/en
Priority to CA002591930A priority patent/CA2591930A1/en
Priority to EP05024028A priority patent/EP1686174A1/en
Priority to EP05024033A priority patent/EP1666493A1/en
Priority to PCT/US2001/017800 priority patent/WO2001093983A1/en
Priority to EP05024031A priority patent/EP1666492A1/en
Priority to CA002585822A priority patent/CA2585822A1/en
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Priority to CA002591929A priority patent/CA2591929A1/en
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Priority to CA002594659A priority patent/CA2594659A1/en
Priority to EP05024030A priority patent/EP1666491A1/en
Priority to US09/874,503 priority patent/US20020177188A1/en
Publication of WO2000073452A3 publication Critical patent/WO2000073452A3/en
Priority to US09/902,713 priority patent/US20030082541A1/en
Priority to US09/902,615 priority patent/US20030092002A1/en
Priority to US09/902,692 priority patent/US20030054400A1/en
Priority to US09/902,759 priority patent/US20030077654A1/en
Priority to US09/902,634 priority patent/US20030082540A1/en
Priority to US09/902,853 priority patent/US20020192659A1/en
Priority to US09/902,736 priority patent/US20030049676A1/en
Priority to US09/902,979 priority patent/US20030113718A1/en
Priority to US09/902,903 priority patent/US20030044839A1/en
Priority to US09/902,572 priority patent/US20030108983A1/en
Priority to US09/903,603 priority patent/US20030148419A1/en
Priority to US09/903,806 priority patent/US20030130489A1/en
Priority to US09/903,943 priority patent/US20030054349A1/en
Priority to US09/903,562 priority patent/US6965015B2/en
Priority to US09/903,640 priority patent/US7208308B2/en
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Priority to US10/173,694 priority patent/US20030166107A1/en
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Priority to US10/174,569 priority patent/US20030166111A1/en
Priority to US10/174,581 priority patent/US7153939B2/en
Priority to US10/174,576 priority patent/US7125962B2/en
Priority to US10/174,591 priority patent/US20030166115A1/en
Priority to US10/174,586 priority patent/US20030032106A1/en
Priority to US10/174,585 priority patent/US20030032105A1/en
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Priority to US10/174,590 priority patent/US20030008352A1/en
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Priority to US10/174,583 priority patent/US7211645B2/en
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Priority to US10/175,739 priority patent/US20030027267A1/en
Priority to US10/175,754 priority patent/US20030166123A1/en
Priority to US10/175,752 priority patent/US20030022295A1/en
Priority to US10/175,747 priority patent/US20030032107A1/en
Priority to US10/175,741 priority patent/US20030073171A1/en
Priority to US10/175,736 priority patent/US20030166117A1/en
Priority to US10/175,751 priority patent/US20030166122A1/en
Priority to US10/175,742 priority patent/US20030166118A1/en
Priority to US10/175,735 priority patent/US20030082715A1/en
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Priority to US10/176,989 priority patent/US20030170803A1/en
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Priority to US10/176,759 priority patent/US20030166128A1/en
Priority to US10/176,917 priority patent/US20030044918A1/en
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Priority to US10/176,991 priority patent/US20030027324A1/en
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Priority to US10/176,982 priority patent/US20030044919A1/en
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Priority to US10/176,978 priority patent/US20030032116A1/en
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Priority to US10/176,752 priority patent/US20030170799A1/en
Priority to US10/176,489 priority patent/US20030166125A1/en
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Priority to US10/179,520 priority patent/US20030096353A1/en
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Priority to US10/179,510 priority patent/US20030032117A1/en
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Priority to US10/180,559 priority patent/US20030032124A1/en
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Abstract

The present invention relates to a composition containing novel proteins and methods for the diagnosis and treatment of immune related diseases.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE RELATED DISEASES
Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of immune related diseases
Background of the Invention Immune related and inflammatory diseases are the manifestation or consequence of fairly complex, often multiple interconnected biological pathways which in normal physiology are critical to respond to insult or injury, initiate repair from insult or injury, and mount innate and acquired defense against foreign organisms Disease or pathology occurs w hen these normal physiological pathways cause additional msult or injury either as directly related to the intensity of the response, as a consequence of abnormal regulation or excessive stimulation, as a reaction to self or as a combination of these Though the genesis of these diseases often mvolves multistep pathways and often multiple different biological
Figure imgf000003_0001
s intervention at critical points in one or more of these pathways can have an ameliorative or therapeutic effect Therapeutic intervention can occur by either antagonism of a detrimental process/pathway or stimulation of a beneficial process/pathway
Many immune related diseases are known and have been extensively studied Such diseases include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases neoplasm, etc
T lymphocytes (T cells) are an important component of a mammalian immune response T cells recognize antigens which are associated with a self-molecule encoded by genes within the major histocompatibility complex (MHC) The antigen may be displayed together with MHC molecules on the surface of antigen presentmg cells, virus infected cells, cancer cells, grafts, etc The T cell system eliminates these altered cells which pose a health threat to the host mammal T cells include helper T cells and cytotoxic T cells Helper T cells proliferate extensively following recognition of an antigen -MHC complex on an antigen presenting cell. Helper T cel also secrete a variety of cytokines, i e , lymphokines, which play a central role m the activation of B cells, cytotoxic T cells and a variety of other cells which participate m the immune response A central event m both humoral and cell mediated immune responses is the activation and clonal expansion of helper T cells Helper T cell activation is initiated by the interaction of the T cell receptor (TCR) - CD3 complex with an antigen-MHC on the surface of an antigen presenting cell This interaction mediates a cascade of biochemical events that induce the resting helper T cell to enter a cell cycle (the GO to Gl transition) and results in the expression of a high affinity receptor for IL-2 and sometimes IL-4 The activated T cell progresses through the cycle proliferating and differentiating into memory cells or effector cells
In addition to the si nals mediated through the TCR, activation of T cells involves additional costimulation mduced by cvtokines released by the antigen presenting cell or through interactions with membrane bound molecule- en the antigen presenting cell and the T cell The cytokines IL-1 and IL-6 have been shown to provide a cosumulatory signal Also, the interaction between the B7 molecule expressed on the surface of an antigen presentmg cell and CD28 and CTLA-4 molecules expressed on the T cell surface effect T cell activation Activated T cells express an increased number of cellular adhesion molecules, such as ICAM-1, integrins, VLA-4, LFA-1, CD56 etc
T-cell proliferation in a mixed lymphocyte culture or mixed lymphocvte reaction (MLR) is an established indication of the ability of a compound to stimulate the immune system In many immune responses, inflammatory cells infiltrate the site of injury or infection The migrating cells may be neutrophihc, eosinophihc, monocytic or hmphocytic as can be determined by histologic examination of the affected tissues Current Protocols in Immunology ed John E Cohgan, 1994, John Wiley & Sons, Ine T-cell activity is also positively affected by stimulation with antι-CD3 and antι-CD28 antibodies Thus, the ability of a compound to inhibit the costimulation or alternatively replace antι-CD28 is indicative of the inhibitory or stimulatory effect, respectively, on the immune svstem Immune related diseases can be treated by suppressing the immune response Using neutralizing antibodies that inhibit molecules having immune stimulatory activity would be beneficial in the treatment of immune-mediated and inflammatory diseases Molecules which inhibit the immune response can be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease Summary of the Invention
A Embodiments
The present invention concerns compositions and methods for the diagnosis and treatment of immune related disease in mammals including humans The present invention is based on the identification of proteins (including agonist and antagonist antibodies) which either stimulate or inhibit the immune response in mammals Immune related diseases can be treated by suppressing or enhancing the immune response Molecules that enhance the immune response stimulate or potentiate the immune response to an antigen Molecules which stimulate the immune response can be used therapeutically where enhancement of the immune response would be beneficial Alternatively, molecules that suppress the immune response attenuate or reduce the immune response to an antigen (e g , neutralizing antibodies) can be used therapeutically where attenuation of the immune response would be beneficial (e g , mflammation) Accordmgly, the PRO polypeptides, agonists and antagonists thereof are also useful to prepare medicines and medicaments for the treatment of immune- related and inflammatory diseases In a specific aspect, such medicines and medicaments comprise admixmg a therapeutically effective amount of a PRO polypeptide, agonists or antagonist thereof with a pharmaceutically acceptable carrier Preferabh the admixture is stenle In a further embodiment, the mvention concerns a method of identifying agonists or antagonists to a
PRO polypeptide which comprises contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated b\ said PRO polypeptide Preferably, the PRO polypeptide is a native sequence PRO polypeptide In a specific aspect, the PRO agonists or antagonist is an anti-PRO antibody
In another embodiment, the mvention concerns a composition of matter containing PRO polypeptide or an agonist or antagonist antibodv which bmds the polypeptide m admixture with a earner or excipient In one aspect, the composition contains a therapeutically effective amount of the peptide or antibody In another aspect, when the composition contams an immune stimulating molecule, the composition is useful for (a) mcreasmg infiltration of inflammatory cells mto a tissue of a mammal in need thereof, (b) stimulatmg or enhancing an immune response m a mammal in need thereof, (c) mcreasmg the proliferation of T-lymphocytes in a mammal in need thereoi in response to an antigen, (d) stimulatmg the activity of T-lymphocytes or (e) increasing the vasular permeability In a further aspect, when the composition contains an immune inhibiting molecule, the composition is useful for (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, (c) decreasing the activity of T-lymphocytes or (d) decreasing the proliferation of T-lymphocytes in a mammal m need thereof in response to an antigen In another aspect, the composition contains a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent Preferably, the composition is stenle
In another embodiment, the ιn\ ention concerns a method of treating an immune related disorder in a mammal in need thereof, compnsmg admimstenng to the mammal an effective amount of a PRO polypeptide, an agonist thereof, or an antagonist thereto In a preferred aspect, the immune related disorder is selected form the group consisting of s\ stemιc lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic aπhπtis, spondyloarthropathies systemic sclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome, systemic vascu tis, sarcoidosis. autoimmune hemolytic anemia, autoimmune thrombocytopema, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelmating diseases of the central and peripheral nervous systems such as multiple sclero is, idiopathic demyelmating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory dem\ elrnatmg polyneuropathy, hepatobihary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, mflanunatory bowel disease gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune- mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psonasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, lmmunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumomtis transplantation associated diseases including graft rejection and graft -versus-host- disease.
In another embodiment, the mvention provides an antibody which specifically bmds to any of the above or below descnbed polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody In one aspect, the present invention concerns an isolated antibody which bmds a PRO polypeptide In another aspect, the antibody mimics the activity of a PRO polypeptide (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of a PRO polypeptide (an antagonist antibody) In another aspect, the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region
(FR) residues. The antibody may be labeled and may be immobilized on a solid support In a further aspect, the antibody is an antibody fragment, a monoclonal antibody, a single-chain antibody, or an anti-idiotypic antibody
In yet another emboctiment, the present invention provides a composition comprising an anti-PRO antibody in admixture with a pharmaceutically acceptable earner In one aspect, the composition compnses a therapeutically effective amount of the antibody Preferably, the composition is stenle The composition may be administered in the form of a liquid pharmaceutical formulation, which may be preserved to achieve extended storage stability Alternatively, the antibody is a monoclonal antibody, an antibody fragment, a humanized antibody, or a singie-chain antibody
In a further embodiment, the invention concerns an article of manufacture, compnsmg. (a) a composiuen of matter compnsmg a PRO polypeptide or agonist or antagonist thereof; (b) a container containing said composition, and
(c) an instruction affixed to said container, or a package insert included in said container referring to the use of said PRO polypeptide or agonist or antagonist thereof in the treatment of an immune related disease The composition ma\ comprise a therapeutically effective amount of the PRO polypeptide or the agonist or antagonist thereof
In yet another embodiment, the present invention concerns a method of diagnosing an immune related disease in a mammal, compnsmg detecting the level of expression of a gene encodmg a PRO polypeptide (a) m a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample as compared to the control sample indicates the presence of immune related disease in the mammal from which the test tissue cells were obtained
In another embodiment the present invention concerns a method of diagnosing an immune disease in a mammal, compπsing (a) contacting an anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the respective PRO polypeptide, respectively, in the test sample, wherein the formation of said complex is indicative of the presence or absence of said disease The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type A larger quantity of complexes formed in the test sample indicates the presence or absence of an immune disease in the mammal from which the test tissue cells were obtained The antibody preferably carnes a detectable label Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluonmetry, or other techniques known in the art The test sample is usually obtained from an individual suspected of havmg a deficiency or abnormality of the immune system
In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a sample compnsmg exposmg a test samples of cells suspected of containing the PRO polypeptide to an anti-PRO antibody and determining the bmding of said antibody to a component of said sell sample In a specific aspect me sample compnses a cell suspected of containing the PRO polypeptide and the antibody binds to the cell Tne antibody is preferably detectably labeled and/or bound to a solid support
In another embodiment, the present invention concerns an immune-related disease diagnostic kit, compnsmg an anti-PRO antiDody and a earner m suitable packaging The kit preferably contains instructions for using the antibody to detect the presence of the PRO polypeptide Preferably the earner is pharmaceutically acceptable
In another embodiment, the present invention concerns a diagnostic kit, containing an anti-PRO m suitable packagmg The kit preferably contams instructions for using the antibody to detect the PRO polypeptide In another embodiment, the invention provides a method of diagnosing an immune-related disease in a mammal which compnses detecting the presence or absence or a PRO polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of a PRO polypeptide in said test sample is indicative of the presence of a immune-related disease in said mammal
In another embodiment, the present invention concerns a method for identifying an agonist of a PRO polypeptide compnsmg (a) contacting cells and a test compound to be screened under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide, and
(b) determining the induction of said cellular response to determine if the test compound is an effective agonist, herein the induction of said cellular response is indicative of said test compound being an effective agonist
In another embodiment the present invention provides a method for identifying an agonist of a PRO polypeptide comprising
(a) contacting cells and a test compound to be screened under conditions suitable for the stimulation of cell proliferation by a PRO pohpeptide, and (b) measuring the proliferation of said cells to determine if the test compound is an effective agonist, wherein the stimulation of cell proliferation is indicative of said test compound being an effective agonist
In another embodiment the invention concerns a method for identifying a compound capable of inhibiting the expression and or activity of a PRO polypeptide by contacting a candidate compound with a PRO polypeptide under condition and for a time sufficient to allow these two components to interact and determining whether the actι\ ιt\ of the PRO polypeptide is inhibited In a specific aspect, either the candidate compound or the PRO pohpeptide is immobilized on a solid support In another aspect, the non-immobilized component carries a detectable label In a preferred aspect, this method comprises the steps of
(a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the mduction of a cellular response normally induced by a PRO polypeptide, and (b) determining the mduction of said cellular response to determine if the test compound is an effective antagonist In another preferred aspect, the method compnses the step of
(a) contacting cells and a test compound to be screened in the presence of a PRO polypeptide under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide under conditions suitable for the stimulation of cell proliferation by a PRO polypeptide, and (b) measuring the proliferation of the cells to determine if the test compound is an effective antagonist
In another embodiment the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that normally express the polypeptide, wherein the method comprises contacting the cells with a test compound and determining whether the expression of the PRO polypeptide is inhibited In a preferred aspect- this method compnses the steps of (a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PRO polypeptide, and
(b) determining the inhibition of expression of said polypeptide
In yet another emboαiment, the present invention concerns a method for treating an immune-related disorder in a mammal that suffers therefrom compnsmg admimstenng to the mammal a nucleic acid molecule that codes for either (a) a PRO polypeptide, (b) and agonist of a PRO polypeptide or (c) an antagonist of a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO antibody In a prefered embodiment, the mammal is human In anotne^ prefened embodiment, the nucleic acid is administered via ex vivo gene therapy
In a further preferred embodiment, the nucleic acid is compnsed within a vector, more preferably an adenoviral, adeno-associated viral, lentmral or retroviral vector In yet another aspect- the invention provides a recombinant retroviral particle compnsmg a retroviral vector consisting essentiallv of a promoter nucleic acid encoding (a) a PRO polvpeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) an antagonist polypeptide of a PRO polypeptide and a signal sequence for cellular secretion of the pohpeptide, wherein the retroviral vector is in association with retroviral structural proteins Preferably the signal sequence is from a mammal, such as from a native PRO polypeptide In a still further embodiment, the invention concerns an ex \ ιx o producer cell comprising a nucleic acid construct that expresses retro\ iral structural proteins and also comprises a retroviral \ ector consisting essentially of a promoter, nucleic acid encoding (a) a PRO polypeptide (b) an agonist polypeptide or a PRO polypeptide or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association \\ ith the structural protems to produce recombinant retro\ iral particles
In a still further embodiment, the invention provides a method for mcreasmg the infiltration of inflammatory cells from the \ asculature into a tissue of a mammal comprising administering a therapeutically effective amount of (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the infiltration of inflammatory cells from the vasculature in the mammal is increased In a still further embodiment, the invention provides a method for decreasing the infiltration of inflammatory cells from the \ asculature into a tissue of a mammal comprising administering a therapeutically effective amount of (a) a PRO polypeptide (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherem the infiltration of inflammatory cells from the vasculature in the mammal is decreased
In a still further embodiment, the invention provides for a method of increasing the activity of T- lymphocytes in a mammal compnsmg administering a therapeutically effective amount of (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the activity of T-lymphocytes m the mammal is increased
In a still further embodiment, the invention provides for a method of decreasing the activity of T- lymphocytes in a mammal compnsmg administenng a therapeutically effective amount of (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherein the activity of T-lymphocytes in the mammal is decreased
In a still further embodiment, the invention provides for a method of mcreasmg the proliferation of T- lymphocytes in a mammal compnsmg administenng a therapeutically effective amount of (a) a PRO polypeptide, (b) an agonist ol a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherem the proliferation of T-lymphocvtes m the mammal is increased
In a still further embodiment, the invention provides for a method of decreasing the proliferation of T- lymphocytes in a mammal compnsmg administenng a therapeutically effective amount of (a) a PRO polypeptide, (b) an agonist ot a PRO polypeptide, or (c) an antagonist of a PRO polypeptide, wherem the proliferation of T-lymphocvtes in the mammal is decreased In a still further embodiemnt, the invention provides for a method of affectmg the proliferation of T- cells compnsmg contacting PBMC cells with an effective amount of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526 PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO106^ PRO1031, PR01157, PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR019I7, PR05723, PRO4405, PRO4302, PRO9940, PRO6006 polypeptide and measunng the change in prohieration from control levels In a still further embodiment the invention provides for of stimulating the activity of T-cells comprising contacting CD4- cells or PBMC cells with an effective amount of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333 PR0526, PR0381, PR0364, PR0356, PR0719, PRO86 I, PR0769, PR0788, PR0826, PR0982, PR0779 PRO1068, PRO1031, PROH57, PROH59 PR01475. PR01271, PR01343, PR01375, PR01418, PR014'74 PR01917. PR05723, PRO4405, PRO4302, PRO9940, PRO6006 polypeptide in combination with an effecm e amount of antι-CD3 antibody and measuring the change in activity from control levels
In a still further embodiment, the invention provides for a method of inhibiting the activity of T-cells compnsmg contacting CD4- cells which have been previously stimulated by treatment with antι-CD3 and anti- CD28 antibodies, with an effective amount of PRO 184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364 PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH5- PROH59, PR01475, PR01271 , PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405 PRO4302, PRO9940, PRO6006 polypeptide and measunng the change in activity from control levels
In a still further embodiment, the mvention provides for a method of stimulating the proliferation of T- lymphocytes in a mammal compnsmg administering a therapeutically effective amount of a PR0861, PR0788, PR01159, PR01646, PR014^5, PR01917, PRO9940, PR05723 or PRO6006 polypeptide, wherem the proliferation of T-lymphocytes m the mammal is stimulated In a still further embodiment, the mvention provides for a method of decreasing the proliferation of T- lymphocytes in a mammal compnsmg administenng a therapeutically effective amount of a PR0184, PRO306, PR0779, PR01271, PR013~5 or PR01474 polypeptide, wherem the proliferation of T-lymphocytes is decreased
In a still further embodiment, the invention provides for a method of stimulatmg the activity of T- lyphocytes comprising admmistenng a therapeutically effective amount of a PR0245, PR0266, PRO306, PR0333, PR0356, PR0364 PR0381, PR0526, PR0719, PR0769, PR0826, PRO1031, PRO1069, PR01343, PR01375 or PR01418 polypeptide, wherem the activity of T-lymphocytes is increased
In a still further embodiment, the invention provides for a method of decreasing the activity of T- lymphocytes compnsmg administering a therapeutically effective amount of a PR0184, PR0212, PRO306, PR0333, PR0364, PR0381 PR0982, PRO1068, PR01157, PR01343, PRO4302 or PRO4405 polypeptide, wherem the activity of T-lymphocytes is decreased
B Additional Embodiments
In other embodiments of the present mvention, the invention provides vectors compnsmg DNA encoding any of the herein descnbed 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 descnbed polypeptides is further provided and compnses cultunng host cells under conditions suitable for expression of the desired pohpeptide and recovenng the desired polypeptide from the cell culture
In other embodiments, the mvention provides chimenc molecules compnsmg any of the herein descnbed polypeptides fused to a heterologous polypeptide or ammo acid sequence Example of such chimeric molecules compnse any of the herein descnbed polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
In another embodiment, the invention provides an antibody which specifically bmds to any of the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or smgle-cham antibody
In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
In other embodiments, 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, altematnely 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, alternativeh 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, alternate ely at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encodmg a PRO polypeptide havmg a full- length ammo acid sequence as disclosed herem, an ammo acid sequence lackmg 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 specificalh defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the D\ A 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° o nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternativeh 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, altemam ely 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, alternatπ eh 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 99c o nucleic acid sequence identity to (a) a DNA molecule compnsmg the codmg sequence of a full-length PRO polypeptide cDN A 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 ammo acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)
In a further aspect, the
Figure imgf000011_0001
concerns an isolated nucleic acid molecule comprising a nucleotide sequence havmg at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternativeh 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, alternate ely 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, alternativeh at least about 94% nucleic acid sequence identity, alternatively at least about
95% nucleic acid sequence identitv 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
Figure imgf000011_0002
any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the D\A molecule of (a)
Another aspect the
Figure imgf000011_0003
provides an isolated nucleic acid molecule compnsmg a nucleotide sequence encodmg a PRO pohpeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherem the transmembrane domaιn(s) of such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein descnbed PRO polypeptides are contemplated
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that ma\ find use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionalh encode a polypeptide compnsmg a binding site for an anti-PRO antibody or as antisense oligonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides m length, alternatively at least about 50 nucleotides m length, alternatively at least about 60 nucleotides in length, alternatively at least about 7υ nucleotides m length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides m length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides m length, alternatively at least about 120 nucleotides m length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 15υ nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 17υ nucleotides in length, alternatively at least about 180 nucleotides m length, alternatively at least about 19υ nucleotides in length, alternatively at least about 200 nucleotides m length, alternatively at least about 25u nucleotides in length, alternatively at least about 300 nucleotides m length, alternatively at least about 35υ nucleotides m length, alternatively at least about 400 nucleotides m length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides m length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides m length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein m this context the term "about" means the referenced nucleotide sequence length plus or mmus 10% of that referenced length It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined m 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, preferabh those PRO polypeptide fragments that compπse a bindmg site for an anti-PRO antibody
In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences heremabove identified In a certain aspect, the invention concerns an isolated PRO polypeptide, compnsmg an amino acid sequence havmg at least about 80% ammo acid sequence identity, alternatively at least about 81% ammo acid sequence identity, alternativeh at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence ldentitj . alternatively at least about 84% ammo 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% ammo acid sequence identity, alternatively at least about 88% ammo acid sequence identity, alternativeh at least about 89% ammo acid sequence identity, alternatively at least about 90% ammo acid sequence identity, alternatively at least about 91% ammo 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° o amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternate eh at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence ldent . alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% ammo 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 here , an extracellular domain of a transmembrane protem. v> ith or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length ammo acid sequence as disclosed herem
In a further aspect, the mvention concerns an isolated PRO polypeptide comprising an ammo acid sequence havmg at least about 80% amino acid sequence identity, alternatively at least about 81% ammo acid sequence identity, alternate eh at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence ident . alternatively at least about 84% ammo acid sequence identity, alternatively at least about 85% ammo acid seσuence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 8"% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternate eh at least about 89% amino acid sequence identity, alternatively at least about 90% ammo acid sequence ldentits . alternatively at least about 91% ammo acid sequence identity, alternatively at least about 92% ammo acid sequence identity, alternatively at least about 93% ammo acid sequence identity, alternatively at least about 94° o ammo acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatπ eh at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identitv alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% ammo 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 further aspect the m\ ention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81% positives, alternatively at least about 82% positives, alternatn eh at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives, alternatively at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives alternatively at least about 91% positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98° o positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO polypeptide having a full-length ammo acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herem, 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 specific aspect the mvention 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 ammo acid sequence as herembefore descnbed Processes for producing the same are also herem descnbed, wherem those processes comprise cultuπng a host cell compnsmg a vector which comprises the appropriate encodmg nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
Another aspect the
Figure imgf000013_0001
ention provides an isolated PRO polypeptide which is either transmembrane domam-deleted or transmembrane domain-inactivated Processes for producing the same are also herem descnbed, wherem those processes compnse cultuπng a host cell comprising a vector which compnses 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 mvention concerns agonists and antagonists of a native PRO polypeptide as defined herem 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 compnse contactmg 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 compnsmg a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, m combmation with a earner Optionally, the earner is a pharmaceutically acceptable earner
Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore descnbed, 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
Brief Description of the Drawings
Figure 1 shows DNA28500 (SEQ ID NO' l) Figure 2 shows the native sequence PRO 184 polypeptide (SEQ ID NO 2)
Figure 3 shows DNA30942-1134 (SEQ ID NO:3)
Figure 4 shows the native sequence PR0212 polypeptide (SEQ ID NO 4)
Figure 5 shows DNA35638-1141 (SEQ ID NO.8)
Figure 6 shows the native sequence PR0245 polypeptide (SEQ ID NO.9) Figure 7 shows DNA37150-1178 (SEQ ID NO- 13)
Figure 8 shows the native sequence PR0266 polypeptide (SEQ ID NO.14)
Figure 9 shows DNA39984- 1221 (SEQ ID NO.18).
Figure 10 shows the native sequence PRO306 polypeptide (SEQ ID NO:19).
Figure 11 shows DNA41374-1312 (SEQ ID NO:26). Figure 12 shows the native sequence PR0333 polypeptide (SEQ ID NO:27)
Figure 13 shows DN A44184- 1319 (SEQ ID NO:28).
Figure 14 shows the native sequence PR0526 polypeptide (SEQ ID NO.29)
Figure 15 shows DNA44194-1317 (SEQ ID NO:33)
Figure 16 shows the native sequence PR0381 polypeptide (SEQ ID NO:34). Figure 17 shows DNA47365- 1206 (SEQ ID NO:38).
Figure 18 shows the native sequence PR0364 polypeptide (SEQ ID NO:39).
Figure 19 shows DNA47470-1130 (SEQ ID NO:48).
Figure 20 shows the native sequence PR0356 polypeptide (SEQ ID NO:49).
Figure 21 shows DNA49646-1327 (SEQ ID NO:53). Figure 22 shows the native sequence PR0719 polypeptide (SEQ ID N0 54)
Figure 23 shows DNA50798 (SEQ ID NO:58).
Figure 24 shows the native sequence PR0861 polypeptide (SEQ ID NO:59).
Figure 25 shows DN .54231-1366 (SEQ ID NO:60).
Figure 26 shows the native sequence PR0769 polypeptide (SEQ ID NO:61). Figure 27 shows DNA56405-1357 (SEQ ID NO:66).
Figure 28 shows the native sequence PR0788 polypeptide (SEQ ID N0.67).
Figure 29 shows DNA57694-1341 (SEQ ID NO:68)
Figure 30 shows the native sequence PR0826 polypeptide (SEQ ID NO:69).
Figure 31 shows DNA57700-1408 (SEQ ID NO:70). Figure 32 shows the native sequence PR0982 polypeptide (SEQ ID NO:71).
Figure 33 shows DNA58801-1052 (SEQ ID NO:72).
Figure 34 shows the native sequence PR0779 polypeptide (SEQ ID NO:73).
Figure 35 shows DNA59214-1449 (SEQ ID NO:76).
Figure 36 shows the native sequence PRO1068 polypeptide (SEQ ID NO;77). Figure 37 shows DNA59294-1381 (SEQ ID NO:78). Figure 38 shows the natι\ e sequence PRO 1031 polypeptide (SEQ ID NO 79)
Figure 39 shows DNA60292-1506 (SEQ ID NO 80)
Figure 40 shows the nam e sequence PRO 1157 polypeptide (SEQ ID NO 81)
Figure 41 shows DNA60627-1508 (SEQ ID NO 82) Figure 42 shows the native sequence PRO 1 159 polypeptide (SEQ ID NO 83)
Figure 43 shows DNA61 185-1646 (SEQ ID NO 84)
Figure 44 shows the native sequence PR01475 polypeptide (SEQ ID NO 85)
Figure 45 shows DNA66309-1538-1 (SEQ ID NO 91)
Figure 46 shows the nativ e sequence PRO 1271 polypeptide (SEQ ID NO 92) Figure 47 shows DNA66675-1587 (SEQ ID NO 93)
Figure 48 shows the native sequence PRO 1343 polypeptide (SEQ ID NO 94)
Figure 49 shows DNA67004-1614 (SEQ ID NO 98)
Figure 50 shows the native sequence PR01375 polypeptide (SEQ ID NO 99)
Figure 51 shows DNA68864-1629 (SEQ ID NO 100) Figure 52 shows the nativ e sequence PRO 1418 polypeptide (SEQ ID NO 101)
Figure 53 shows DN A"3739-1645 (SEQ ID NO 102)
Figure 54 shows the native sequence PR01474 polypeptide (SEQ ID NO 103)
Figure 55 shows DNA76400-2528 (SEQ ID NO 104)
Figure 56 shows the native sequence PR01917 polypeptide (SEQ ID NO 105) Figure 57 shows DN A82361 (SEQ ID NO 106)
Figure 58 shows the native sequence PR05723 polypeptide (SEQ ID NO 107)
Figure 59 shows DN A84920-2614 (SEQ ID NO 108)
Figure 60 shows the native sequence PRO4405 polypeptide (SEQ ID NO 109)
Figure 61 shows DN 4.92218-2554 (SEQ ID NO 113) Figure 62 shows the native sequence PRO4302 polypeptide (SEQ ID NO 114)
Figure 63 shows DN 4.92282 (SEQ ID NO 115)
Figure 64 shows the native sequence PRO9940 polypeptide (SEQ ID NO 116)
Figure 65 shows DN 4.105782-2693 (SEQ ID NO 117)
Figure 66 shows the native sequence PRO6006 polypeptide (SEQ ID NO 118)
Detailed Descπption of the Prefened Embodiments I Definitions
The term "immune related disease" means a disease in which a component of the immune system of a mammal causes, mediates or otherwise contnbutes to a morbidity in the mammal Also mcluded are diseases m which stimulation or rnterv ention of the immune response has an ameliorative effect on progression of the disease Included withm tms term are immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases immunodeficiency diseases, neoplasm, etc
The term "T cell mediated" disease means a disease in which T cells directly or indirectly mediate or otherwise contribute to a morbidity in a mammal The T cell mediated disease may be associated with cell mediated effects, lymphokme mediated effects, etc , and even effects associated with B cells if the B cells are stimulated, for example, bv the lvmphokmes secreted by T cells
Examples of immune-related and inflammatory diseases, some of which are immune or T cell mediated, which can be treated according to the invention mclude systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vascuhtis, sarcoidosis, autoimmune hemolvtic anemia (immune pancytopema, paroxysmal nocturnal hemoglobinuπa), autoimmune thrombocytopema (idiopathic thrombocytopemc purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephπtis, tubulointerstitial nephritis), demyelmating diseases of the central and peπpheral nervous systems such as multiple sclerosis, idiopathic demyelmating polyneuropathv or Guillain-Barre syndrome, and chronic inflammatory demyelmating polyneuropathy, hepatobiharv diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non- hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skm diseases including bullous skm diseases, erythema multifoπne and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, lmmunologic diseases of the lung such as eosinophi c pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft -versus-host-disease Infectious diseases including viral diseases such as AIDS (HI\ mfection), hepatitis A, B, C, D, and E, herpes, etc , bactenal infections, fungal mfections, protozoal infections and parasitic mfections
"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoprotems having the same general structural charactenstics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibodv -like molecules which lack antigen specificity Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas The term "antibody" is used m the broadest sense and specifically covers, without limitation, intact monoclonal antibodies (including agonist, antagonist and neutralizing antibodies), polyclonal antibodies, multispecific antibodies (e g bispecifϊc antibodies) formed rrom at least two intact antibodies, single chain antibodies bindmg the epitopes specific to the PRO polypepude and antibody fragments so long as they exhibit the desired biological activity An anti-PRO antibody is an antibody which rmmunologically binds to a PRO polypeptide The antibody may bmd to any domam of the PRO polypeptide which may be contacted by the antibody For example, the antibody may bmd to any extracellular domam of the polypeptide and when the entire polypeptide is secreted, to any domam on the polypeptide w ich is available to the antibody for bmdmg "Native antibodies and "native immunoglobulins" are usually heterotetrameπc glycoproteins of about
150,000 daltons, composed ot two identical light (L) chains and two identical heavy (H) chains Each light cham is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light cham also has regularly spaced uitrachain disulfide bndges Each heavy chain has at one end a vanable domain (VH) followed by a number of constant domains Each light chain has a vanable domam at one end (V ) and a constant domam at its other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain ot the heavy chain Particular amino acid residues are believed to form an interface between the light- and heaw-cham vanable doma s
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the bmding and specificity of each particular antibody for its particular antigen Howe er the variability is not evenly distributed throughout the variable domams of antibodies It is concentrated three or four segments called "complementarity-determining regions" (CDRs) or "hypervaπable regions" in both in the light-chain and the heavy-chain variable domains The more highly conserved portions of vanable domains are called the framework (FR) The variable domains of native heavy and light chains each compπse four or five FR regions, largely adopting a β-sheet configuration, connected by the CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other cham, contnbute to the formation of the antigen-binding site of antibodies (see Kabat et al , NIH Publ No 91-3242, Vol I, pages 647-669 (1991)) There are at least two techniques for determining the extent of the CDRs (1) An approach based on the extent of cross-species sequence vanabi ty (i e Kabat et al , Sequences of Pioteins of Immunological Intei est (National Institute of Health, Bethesda, MD), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia, C et al , (1989), Nature 342 877) Moreover, CDR's can also be defined using a hybrid approach incorporating the residues identified by both of the previous techniques The constant domams are not involved directly in binding an antibody to an antigen, but exhibit vanous effector functions, such as participation of the antibody in antibody-dependent cellular toxicity
Depending on the ammo 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 mav be further divided into subclasses (isotypes), e g , IgGl, IgG2, IgG3, IgG4, IgA, and IgA2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, / e , the individual antibodies comprising the population are identical except for possible naturally occumng mutations that may be present in minor amounts Monoclonal antibodies are highly specific, bemg directed against a single antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificitv . the monoclonal antibodies are advantageous in that they are synthesized by the hybndoma culture, uncontam nated by other immunoglobulins The modifier "monoclonal" indicates the character of the antibody as bemg obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybndoma method first descnbed by Kohler et al , Nature, 256 495 [1975], or may be made by recombinant DNA methods (see, eg , U S Patent No 4,816,567) The "monoclonal antibodies" may also be isolated from phage antibody libraries usmg the techniques described in Clackson et al , Natw e, 352 624-628 [1991] and Marks et al , J Mol Biol . 222 581-597 (1991). for example See also U S Patent Nos 5,750,373, 5,571,698, 5,403,484 and 5,223,409 which describe the preparation of antibodies using phagemid and phage v ectors
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavv and or light chain is identical with or homologous to conesponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chaιn(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4,816,567, Morrison et al , Pi oc Natl Acad Sci USA, 81 6851-6855 [1984]) "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) vv hich contain minimal sequence derived from non-human immunoglobulin For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibodv ) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity In some instances Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may compnse residues which are found neither in the recipient antibody nor m the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will compπse substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions coπespond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence The humanized antibody optimally also will compnse at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin For further details, see Jones et al Nature, 321 522-525 (1986), Reichmann et al , Nature, 332 323-329 [1988], and Presta, Curr Op Struct Biol 2 593-596 (1992) The humanized antibody includes a "pπmatιzed"antιbody where the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest Antibodies contaming residues from Old World monkeys are also possible within the invention See, for example, U S Patent Nos 5,658,570, 5,693,780, 5,681,722, 5,750,105, and 5,756,096 Antibodies and fragments thereof m this invention also include "affinity matured" antibodies in which an antibody is altered to change the amino acid sequence of one or more of the CDR regions and/or the framework regions to alter tne affinity of the antibody or fragment thereof for the antigen to which it bmds Affinity maturation may result m an mcrease or in a decrease in the affinity of the matured antibody for the antigen relative to the starting antibody Typically, the starting antibody will be a humanized, human, chimeric or murine antibody and the affinity matured antibody will have a higher affinity than the starting antibody Dunng the maturation process one or more of the amino acid residues in the CDRs or in the framework regions are changed to a different residue using any standard method Suitable methods include point mutations using well known cassette mutagenesis methods (Wells et al , 1985, Gene 34 315) or oligonucleotide mediated mutagenesis methods (Zoller et al , 1987, Nucleic Acids Res 10 6487-6504) Affinity maturation may also be performed using known selection methods in which many mutations are produced and mutants having the desired affinity are selected from a pool or library of mutants based on improv ed affinity for the antigen or ligand Known phage displav techniques can be conveniently used in this approach See, for example, U S 5,750,373, U S 5,223,409. etc
Human antibodies are also with in the scope of the antibodies of the invention Human antibodies can 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 58 (1991)] The techniques of Cole et al and Boerner et al are also av ailable for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancel Thetapy , Alan R Liss, p 77 (1985), Boerner et al , J Immunol , 147 (1) 86- 95 (1991), U S 5,750, 373] Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgemc animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completeh mactivated Upon challenge, human antibody production is observed, which closely resembles that seen m humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example, in U S Patent 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), Lonbeτg et al Natw e 368 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al , Nature Biotechnology J4 845-51 (1996), Neuberger, Natwe Biotechnology J4, 826 (1996), Lonberg and Huszar, Intel n Rev Immunol J_3 65-93 (1995)
The term "effecti e amount" is at least the minimum concentration or amount of a PRO polypeptide and/or agonist/antagonist which causes, mduces or results in either a detectable improvement m a component of the immune response m mammals as measured in an in vitro assay For example, an increase or decrease in the proliferation of T-cells and/or v ascular permeability as measured in Examples provided herem Furthermore, a "therapeutically effective amount" is the minimum concentration or amount of a PRO polypeptide and/or agonist/antagonist which would be effective in at least attenuating a pathology (mcreasmg or decreasing as the case may be) a component of the immune response in mammals, the results of which effects a treatment as defined in the previous paragraph
The "pathology" of an immune related disease includes all phenomena that compromise the well-being of the patient This mcludes v\ ithout limitation, abnormal or uncontrollable cell growth, antibody production, auto-antibody production, complement production and activation, mterference with the normal functioning of neighboring cells, release of cytokmes or other secretory products at abnormal levels, suppression or aggravation of any inflammatory or rmmunological response, infiltration of inflammatory cells (neutrophi c, eosinophihc, monocytic, lymphocytic) mto tissue spaces, etc
The term "cytotoxic agent" as used herem refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is mtended to include radioactive isotopes (e g , I131, 1125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bactenal, fungal, plant or animal ongm, or fragments thereof
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents mclude adnamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabmoside (" Ara-C"), cyclophosphamide thiotepa. busulfan, cytoxin, taxoids, e g , pachtaxel (Taxol, Bristol-Myers Squibb Oncology, Pnnceton, NJ) and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, France), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycm C, mitoxantrone, vincristine, vmorelbme carboplatin, temposide, daunomycin carmmomycm, aminopterm, dactinomycin, mitomycins, esperamicms (see U S Pat No 4 675,187), melphalan and other related nitrogen mustards Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapπstone A "growth inhibitors agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase) such as agents that induce Gl anest and M-phase aπest Classical M-phase blockers mclude the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunombicin, etoposide, and bleomycin Those agents that arrest Gl also spill over mto S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin methotrexate, 5-fluorouracil, and ara-C Further information can be found in The Molecular Basis of Cancel Mendelsohn and Israel, eds , Chapter 1 , entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" bv Murakami et al (WB Saunders Philadelphia, 1995), especially p 13
The term "cytokine is a genenc term for proteins released by one cell population which act on another cell as intercellular mediators Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones Included among the cytokmes are growth hormone such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, promsulm, relaxm, prorelaxm glycoprotem hormones such as follicle stimulating hormone (FSH), thyroid stimulatmg hormone (TSH) and luteimzing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen tumor necrosis factor-α and -β, mulleπan-inhibitmg substance, mouse gonadotropm-associated peptide inhibin, activm, vascular endothelial growth factor, integnn, thrombopoietm (TPO), nerve growth factors such as NGF-β, platelet-growth factor, transf ormmg growth factors (TGFs) such as TGF-α and TGF- β, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, mterferons such as interferon- α -β and -γ, colony stimulating factors (CSFs) such as macrophage-CSF (M- CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-lα, IL-2, IL-3, IL-4, IL-5 IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, a tumor necrosis factor such as TNF-α or TNF-β, and other polypeptide factors including LIF and kit ligand (KL) As used herem, the term cytokine mcludes proteins from natural sources or from recombmant cell culture and biologically active equivalents of the native sequence cytokmes
"Active" or "activitv ' the context of variants of the PRO polypeptide refers to form(s) of proteins of the mvention which retain the biologic and/or the ability to induce the production of an antibody against an antigenic epitope possessed bv the PRO polypeptide More specifically, "biological activity" refers to a biological function (either inhibitory or stimulatory) caused by a native sequence or naturally-occurring PRO polypeptide Even more specifically, "biological activity" m the context of an antibody or another molecule that can be identified by the screening assays disclosed herein (e g , an organic or inorganic small molecule, peptide, etc ) can be the ability of such molecules to mduce or inhibit infiltration of inflammatory cells mto a tissue, to stimulate or inhibit T-cell proliferation or activation, to stimulate or inhibit cytokine release by cells or to increase or decrease vascular permeability Another specific biological activity is the increased vascular permeability or the inhibition thereof
II Additional Definitions
The terms "PRO pohpeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to v arious polypeptides wherein the complete designation (; e , PRO/number) refers to specific polypeptide sequences as described herem 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 po peptides and polypeptide ariants (which are further defined herem) The PRO polypeptides described herem 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 herem All disclosures in this specification which refer to the "PRO polypeptide" refer to each of the polypeptides individually as well as jointly For example, descnptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions contaming, 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 pohpeptide deπved 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-occurnng truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occumng variant forms (e g , alternatively spliced forms) and naturalh -occumng allelic variants of the polypeptide In vaπous embodiments of the mvention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides compnsmg the full-length ammo 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 m the accompanymg figures are shown to begin with methionine residues designated herein as ammo acid position 1 m the figures it is conceivable and possible that other methionme residues located either upstream or downstream from the amino acid position 1 the figures may be employed as the starting ammo acid residue for the PRO pohpeptides The PRO polypeptide extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domams 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 domams It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to cntena routmely 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 ammo acids at either end of the domain as initially identified herem 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, how ever, that the C-terminal boundary of a signal peptide mav 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 herem wherem the C-terminal boundary of the signal peptide may be identified pursuant to cnteπa routmely employed in the art for identifying that type of amino acid sequence element (e g Nielsen et al , Pi ot Eng 10 1-6 (1997) and von Heinje et al , Nucl Acids Res 14 4683-4690 (1986)) Moreov er 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 mv ention
"PRO polypeptide v aπant" 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 pohpeptide 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 vanants include, for mstance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-termmus of the full-length native amino acid sequence Ordinanly, a PRO polypeptide variant will have at least about 80% ammo acid sequence identitv . alternatively at least about 81% ammo 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% ammo acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternativ eh at least about 86% ammo acid sequence identity, alternatively at least about 87% amino acid sequence identitv alternatively at least about 88% ammo 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% ammo acid sequence identity, alternatively at least about 92% ammo acid sequence identity, alternativ eh at least about 93% amino acid sequence identity, alternatively at least about 94% ammo acid sequence identitv alternatively at least about 95% ammo acid sequence identity, alternatively at least about 96% ammo acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% ammo acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herem, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domam of a PRO polypeptide, with or without tne signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herem Ordinarily, PRO variant polypeptides are at least about 10 amino acids in lengU alternatively at least about 20 ammo acids in length, alternatively at least about 30 ammo acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 ammo acids m length, alternativ ely at least about 60 amino acids in length, alternatively at least about 70 ammo acids in length, alternativeh at least about 80 ammo acids m length, alternatively at least about 90 amino acids m length, alternatively at least about 100 ammo acids m length, alternatively at least about 150 amino acids m length, alternatively at least about 200 ammo acids m length, alternatively at least about 300 ammo acids in length, or more "Percent (%) ammo acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of ammo 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 achiev e 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 achiev ed m various ways that are withm the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megahgn (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, % ammo acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherem the complete source code for the ALIGN-2 program is provided in Table 1 below The ALIGN-2 sequence compaπson computer program was authored by Genentech, Ine 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 Copyπght Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech. Ine South San Francisco, California or may be compiled from the source code provided m 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 compansons, the % amino acid sequence identity of a given ammo acid sequence A to, with, or against a given ammo acid sequence B (which can alternatively be phrased as a given ammo acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or agamst a given amino acid sequence B) is calculated as follows
100 tunes the fraction X/Y
where X is the number of ammo 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 ammo acid sequence A is not equal to the length of ammo acid sequence B, the % ammo 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 % ammo acid sequence identity of the ammo acid sequence designated "Comparison Protein" to the ammo acid sequence designated "PRO", wherem "PRO" represents the ammo acid sequence of a hypothetical PRO polypeptide of mterest, "Comparison Protein" represents the ammo acid sequence of a polypeptide agamst which the "PRO" polypeptide of interest is being compared, and "X, " Y" and "Z" each represent different hypothetical amino acid residues
Unless specificalh stated otherwise, all % am o acid sequence identity values used herem are obtained as descnbed in me immediately preceding paragraph using the ALIGN-2 computer program However, % amino acid sequence identity values may also be obtained as descnbed 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, / 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 ammo acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B". the ammo 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 downloaded from http //www ncbi nlm mh gov or otherwise 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 ammo acid sequence comparisons, the % ammo acid sequence identity of a giv en ammo acid sequence A to, with, or against a given ammo acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or compnses a certain % amino acid sequence identity to, w ith. or agamst a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of ammo 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 m B It will be appreciated that where the length of ammo acid sequence A is not equal to the length of amino acid sequence B, the % ammo acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A "PRO vanant 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 herem. 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 Ordmanly, a PRO vanant 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, alternativ eh 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, alternativ ely 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, alternativ eh 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 domam of a PRO polypeptide, with or without the signal sequence, as disclosed herem or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence
Ordinarily, PRO v anant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides m 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, alternativ ely at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternati ely at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternativ ely at least about 900 nucleotides in length, or more "Percent (%) nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequences identified herem 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 identitv can be achieved in various ways that are withm the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software For purposes herem however, % nucleic acid sequence identity values are generated using the sequence companson computer program ALIGN-2, wherem the complete source code for the ALIGN-2 program is provided m Table 1 below The ALIGN-2 sequence comparison computer program was authored by Genentech, Ine and the source code shown m Table 1 below has been filed with user documentation in the U S Copynght 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, Ine , South San Francisco, California 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 operatmg system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary In situations where 4LIGN-2 is employed for nucleic acid sequence compansons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or agamst a given nucleic acid sequence D (which can alternatively be phrased a a given nucleic acid sequence C that has or compnses 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 vv ill 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 identitv of the nucleic acid sequence designated "Companson 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-DN 4 nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent different hypothetical nucleotides
Unless specificalh 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 ( ltschul et al Methods in Enzymolos 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 sconng matrix = BLOSUM62 When WU-BLAST-2 is employed, a % nucleic acid sequence identity value is determined by dividing (a) the number of matchmg identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encodmg nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encodmg nucleic acid and the companson nucleic acid molecule of interest (l e , the sequence against w hich the PRO polypeptide-encodmg nucleic acid molecule of interest is bemg compared which may be a vanant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encodmg nucleic acid molecule of mterest For example, in the statement "an isolated nucleic acid molecule compnsmg 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 companson nucleic acid molecule of mterest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encodmg nucleic acid molecule of mterest
Percent nucleic acid sequence identity may also be determined using the sequence companson program NCBI-BLAST2 (Altschul et al Nucleic Acids Res 25 3389-3402 (1997)) The NCBI-BLAST2 sequence companson program may be downloaded from http //www ncbi nlm n gov or otherwise obtamed 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 lo .v complexity length = 15/5, multi-pass e-value = 0 01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scormg matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for sequence compansons, 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 compnses a certain % nucleic acid sequence identity to, with, or agamst 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 m 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 hybndization 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
The term "positives' in the context of sequence comparison performed as described above, includes residues in the sequences compared that are not identical but have similar properties (e g as a result of conservative substitutions, see Table 6 below) For purposes herein, the % value of positives is determined by dividing (a) the number of ammo acid residues scoring a positive value between the PRO polypeptide amino acid sequence of interest hav mg a sequence derived from the native PRO polypeptide sequence and the comparison amino acid sequence of mterest (i e , the ammo acid sequence agamst which the PRO polypeptide sequence is being compared) as determined m the BLOSUM62 matrix of WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest Unless specifically stated otherwise, the % value of positives is calculated as described m the immediately precedmg paragraph However, m the context of the amino acid sequence identity comparisons performed as described for ALIGN-2 and NCBI-BLAST-2 above, includes ammo acid residues m the sequences compared that are not only identical, but also those that have similar properties Amino acid residues that score a positive value to an amino acid residue of mterest are those that are either identical to the amino acid residue of interest or are a prefened substimtion (as defined in Table 6 below) of the ammo acid residue of mterest
For amino acid sequence compansons usmg ALIGN-2 or NCBI-BLAST2, the % value of positives of a given amino acid sequence A to with or against a given ammo acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given ammo acid sequence B) is calculated as follows
100 tunes the fraction X Y
where X is the number of ammo acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues m B It will be appreciated that where the length of ammo acid sequence A is not equal to the length of a mo acid sequence B, the % positives of A to B will not equal the % positives of
B to A
"Isolated," when used to descnbe the various polypeptides disclosed herem, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural env ironment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide and mav include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes In prefened embodiments, the polypeptide will be purified (1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal ammo acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducmg or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide mcludes polypeptide in situ withm recombinant cells, since at least one component of the PRO polvpeptide natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared bv at least one purification step
An "isolated" PRO polypeptide-encodmg nucleic acid or other polypeptide-encodmg 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-encodmg nucleic acid An isolated polypeptide-encoding nucleic acid molecule is other than m 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-encodmg nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is m 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 nbosome binding site Eukaryotic cells are known to utilize promoters polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed mto 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 preprotem that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a codmg sequence if it affects the transcription of the sequence, or a ribosome bmdmg site is operably linked to a codmg sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences bemg linked are contiguous, and, m the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by gation at conv enient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used m accordance with conventional practice The term "antibody is used m the broadest sense and specifically covers, for example, single anti-PRO monoclonal antibodies (mcludmg 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 obtamed from a population of substantially homogeneous antibodies, I e , the individual antibodies compnsmg the population are identical except for possible naturally-occumng mutations that may be present in minor amounts
"Stringency" of hvbndization 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 Hybndization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperamre The higher the degree of desired homology between the probe and hybπdizable sequence the higher the relative temperature which can be used As a result it follow s 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 Cut tent Piotocols 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 cιtrate/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% bovme serum albumιn 0 1% Fιcoll/0 1% polyvιnylpyrrolιdone/50mM 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 x SSC (0 75 M NaCl, 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8 ) 0 1% sodium pyrophosphate, 5 x 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 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0 1 x SSC containing EDTA at 55 C
"Moderately stnngent conditions" may be identified as described by Sambrook et al Molecular Cloning A Laboratory Manual New York Cold Sprmg Harbor Press, 1989, and include the use of washmg solution and hybndization conditions (e g , temperamre, ionic strength and %SDS) less strmgent that those described above An example of moderately stnngent conditions is overnight mcubation at 37°C in a solution compnsmg 20% formamide 5 x SSC (150 mM NaCl, 15 mM tnsodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters m 1 x 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 herem refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag pohpeptide" The tag polypeptide has enough residues to provide an epitope agamst 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 ith other epitopes Suitable tag polypeptides generally have at least six amino acid residues and usually bet een 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 bmdmg specificity of a heterologous protem (an "adhesin") with the effector functions of immunoglobulin constant domains Structuralh the lmmunoadhesrns compnse a fusion of an amino acid sequence with the desired bmdmg specificity hich is other than the antigen recognition and binding site of an antibody (t e , is "heterologous"), and an immunoglobulin constant domain sequence The adhesin part of an immunoadhesin molecule typically is a contiguous ammo acid sequence compnsmg 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-occumng PRO, wherem "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occumng PRO other than the ability to induce the production of an antibody agamst an antigemc epitope possessed by a native or naturally-occumng PRO and an "immunological" activity refers to the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally-occumng 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 ammo acid sequence variants of native PRO polypeptides, peptides. antisense oligonucleotides, small organic molecules, etc Methods for identifying agonists or antagomsts 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, wherem 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 peπod of time "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic m 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 combmation with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutiv e administration in any order
"Carners" as used herem include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal bemg exposed thereto at the dosages and concentrations employed Often the physiologically acceptable earner is an aqueous pH buffered solution Examples of physiologically acceptable carners mclude buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, protems, such as serum albumin, gelatin, or immunoglobulins. hydrophilic polymers such as polyvinylpyno done, amino acids such as glycme, glutamme, asparagine, arginine or lysme, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins chelatmg agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt- forming countenons such as sodium, and or nomonic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™
"Antibody fragments' compnse a portion of an mtact antibody, preferably the antigen bmding or vanable region of the mtact 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-bindmg 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-combinmg sites and is still capable of cross-linking antigen
"Fv" is the minimum antibodv fragment which contains a complete antigen-recognition and -binding site This region consists of a dimer of one heavy- and one light-chain variable domam m tight, non-covalent association It is m 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-bindmg specificity to the antibody However, even a single vanable 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 cham and the first constant domain
(CHI) 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 CHI domain including one or more cysteines from the antibody hmge region Fab'-SH is the designation herem for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab')2 antibody fragments onginally were produced as pairs of Fab' fragments which have hmge 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 distmct types, called kappa and lambda, based on the amino acid sequences of their constant domams
Dependmg on the ammo 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 mav be further divided mto subclasses (isotypes), e g , IgGl, IgG2, IgG3, IgG4, IgA, and IgA2
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domams of antibody, wherem these domains are present in a smgle polypeptide chain Preferably, the Fv polypeptide further compπses 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 m The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds , Spnnger-Verlag, New York, pp 269-315 (1 94)
The term "diabodies' refers to small antibody fragments with two antigen-bmdmg sites, which fragments compπse a heaw-chain vanable domam (VH) connected to a light-chain vanable domain (VL) m the same polypeptide cham (VH-\ _) By usmg a linker that is too short to allow pairing between the two domains on the same cham, the domains are forced to pair with the complementary domains of another chain and create two antigen-bmdmg sites Diabodies are descnbed more fully in, for example, EP 404,097, WO 93/11161, and Hollmger 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 env ironment 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 nonproiemaceous solutes In preferred embodiments, the antibody will be punfied (1) to greater than 95% by weight of antibodv 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-termmal or internal ammo acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducmg conditions using Coomassie blue or, preferably, silver stam 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, how ev er isolated antibody will be prepared by at least one purification step
The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or lndirecth 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), polysacchaπdes (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 Patent No 4,275,149
A "liposome" is a small vesicle composed of various types of lipids, phosphohpids 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
Table 1
/*
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0 */
#define _M -8 /* value of a match with a stop */ int day [26] [26] = {
/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A*/ 2, 0,-2, 0, 0.-4, l.-l.-l, 0.-1,-2,-1, 0,_M, 1, 0,-2, 1.1.0.0,-6, 0,-3, 0}, /*B */ 0, 3,-4, 3, 2,-5, 0.1.-2, 0, 0.-3,-2, 2,_M,-1, 1, 0, 0.0.0,-2,-5.0,-3.1}, l*C */ -2,-4,15,-5,-5,-4.-3.-3.-2.0,-5.-6,-5.-4._M,-3,-5,-4, 0.-2.0,-2,-8, 0, 0,-5}, /*D*/ 0, 3.-5, 4, 3,-6.1.1.-2, 0.0.-4,-3, 2,_M,-1, 2,-1, 0, 0.0.-2,-7, 0,-4.2}, l*E*l 0, 2,-5, 3, 4,-5, 0.1.-2.0, 0.-3,-2, 1,_M.-1, 2,-1, 0.0.0,-2,-7, 0,-4.3}, /* p */ -4,-5,-4,-6,-5, 9,-5.-2.1, 0.-5, 2.0,-4,_M,-5,-5,-4,-3.-3.0,-1, 0.0, 7,-5},
/*G*/ 1, 0,-3, 1, 0,-5, 5.-2.-3.0,-2.-4,-3, 0,_M,-l,-l,-3, 1.0.0,-1,-7.0,-5, 0},
/*H*/ -1, 1,-3, 1, 1,-2,-2.6.-2.0.0.-2.-2, 2,_M, 0, 3, 2.-1.-1.0.-2,-3, 0, 0.2},
1*1*1 [-1,-2,-2,-2,-2, 1.-3.-2.5.0.-2.2.2.-2,_M,-2,-2,-2,-l.0.0, 4,-5.0,-1,-2},
1*1*1 0, 0, 0, 0, 0, 0, 0.0.0, 0, 0.0, 0, 0,_M, 0, 0, 0, 0.0.0, 0, 0, 0, 0, 0},
/*K*/ -1, 0,-5, 0, 0,-5,-2.0.-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0.0.0,-2,-3, 0,-4, 0},
/*L*/ [-2,-3,-6,-4,-3, 2,-4.-2.2.0,-3.6.4,-3._M,-3,-2,-3,-3.-l.0, 2,-2.0,-1,-2},
/*M*/ -1.-2,-5,-3,-2, 0.-3.-2.2.0, 0, 4, 6,-2,_M,-2,-l, 0.-2.-1.0.2,-4, 0.-2,-1},
/*N*/ 0, 2,-4, 2, 1,-4.0.2.-2, 0, 1.-3,-2, 2,_M,-1, 1, 0, 1.0, 0,-2,-4, 0,-2, 1},
/*0 */ _M,_M,_M,_M,_M._M._M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M, M,_M,_M,_M,_M,_M,_M},
/* p */ 1,-1,-3,-1,-1,-5.-1.0.-2, 0,-1, -3,-2,-1, _M, 6 ,0,0, 1,0.0,-1,-6,0,-5,0},
/*Q*/ 0, 1,-5, 2, 2.-5.-1.3.-2.0, 1.-2,-1, 1,_M, 0, 4, 1,-1,-1,0,-2.-5,0,-4, 3}, /*R*/ ■2, 0,-4,-1,-1,-4.-3.2.-2, 0, 3,-3, 0, 0,_M, 0 1,6,0,-1,0,-2,2,0,-4,0}, /*S*/ 1, 0, 0, 0, 0,-3, l.-l.-l, 0.0,-3,-2, 1,_M, 1, -1,0,2, 1,0,-1,-2,0.-3,0}, /* T */ 1, 0,-2, 0, 0,-3, 0.-1, 0, 0, 0,-1,-1, 0,_M, 0, -1,-1, 1,3,0,0,-5.0,-3,0},
1*11*1 0, 0, 0, 0, 0, 0, 0.0.0, 0, 0.0, 0, 0. M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, I* v */ 0,-2,-2,-2,-2,-1.-1.-2.4, 0.-2, 2, 2,-2,_M,-l ,-2,-2,-1, 0, 0, 4,-6, 0,-2,-2},
/* w*/ ■6,-5,-8,-7,-7, 0.-7.-3.-5, 0,-3,-2,-4,-4,_M,-6 ,-5,2,-2.-5.0,-6,17, 0.0,-6}, ι*x*ι 0, 0, 0, 0, 0, 0.0.0.0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0.0.0, 0, 0, 0, 0, 0},
I* Y */ •3,-3, 0,-4,-4, 7.-5.0,-1, 0,-4,-1, -2,-2,_M,-5 ,-4,-4,-3.-3.0,-2,0,0,10,-4}, ι*z*ι 0, 1,-5, 2, 3,-5.0.2.-2.0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0.0,-2,-6, 0,-4, 4} };
Page 1 of day. h Table 1 (conf)
/* */
^include < stdio.h> ^include < ctype.h >
#define MAXJMP 16 /* max jumps in a diag */
#define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#defϊne JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp */
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /*■ penalty for mismatched bases */
#defϊne DINS0 8 /* penalty for a gap */
#define DINS1 1 /* penalty per base */
#define PINS0 8 /* penalty for a gap */
#defιne PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]. /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]: /* base no. of jmp in seq x */
}; /* limits seq to 2"16 -l */ struct diag { int score /* score at last jmp * '■•// long offset: /* offset of prev block */ short ij∞p: /* current jmp index */ struct jmp jp: / * list of jmps
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]: /* loc of jmp (last elem before gap) */
}; char *ofile; /* output file name */ char *namex[2]: /* seq names: getseqs() */ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqsQ */ int dmax; /* best diag: nw() */ int dmaxO; /* final diag */ int dna; /* set if dna: main() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy: /* total gaps in seqs */ int lenO, leπl; /* seq lens */ int ngapx, ngapy: /* total size of gaps */ int smax; /* max score: nw() */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds path for seqs */ char *calloc(). *malloc(). *mdex(), *strcpy(); char *getseq(). *g_calloc();
Page 1 of nw.h Table 1 (conf)
/* Needleman-Wunsch alignment program usage progs filel file2 where filel and file2 are two dna or two protein sequences The sequences can be in upper- or lower-case an may contain ambiguity
; Any lines beginning with ' ' ' > ' or ' < ' are ignored Max file length is 65535 (limited by unsigned short x in the jmp struct)
: A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
; Output is in the file "align out'
* The program may create a tmp file in /tmp to hold mfo about traceback
* Original version developed under BSD 43 on a vax 8650 */
^include "nw.h" #include "day h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0.0.12,0.3,15.0,0.0,5,6,8,8,7,9,0,10,0
static _pbval[26] = {
1,2|(1<<('D'-'A'))|(1<<('N'-'A')), 4, 8.16, 32,64, 128, 256, OxFFFFFFF.1< < 10.1< < 11, 1< < 12, 1< < 13.1< < 14. 1<<15, 1<<16.1<<17, 1<<18, 1<<19, 1< <20, 1<<21, 1< <22, 1<<23, 1<<24.1<<25|(1<<('E'-'A'))|(1<<('Q'-'A'))
};
Figure imgf000035_0001
char *av[]; prog = av[0], if(ac != 3){ fpπntf(stderτ. ' usage %s filel file2\n", prog), fpπntf(stderr.' where fuel and file2 are two dna or two protein sequences \n"), fpπntf(stderr."The sequences can be in upper- or lower-case\n"), fprιntf(stderr. ' Any lines beginning with ',' or ' < ' are ιgnored\n"), fpπntf(stdeπ." Output is xn the file V'ahgn out\"\n"), exιt(l),
} namex[0] = av[l], namex[l] = av[2], seqx[0] = getseq(namex[0]. &len0), seqx[l] = getseq(namex[l], &lenl), xbm = (dna)9 dbval • ibval, endgaps = 0; /* 1 to penalize endgaps */ ofile = "align. out" /* output file */ nw(); /* fill in the matrix, get the possible jmps */ readjmρs(); /* get the actual jmps */ pπntO; /* prmt stats, alignment */ cleanup(O); /* unlink any tmp files */
Page 1 of nw.c Table 1 (conf)
/* do the alignment return best score maιn() * dna values in Fitch and Smith PNAS 80 1382- 1386 1983
* pro PAM 250 values
* When scores are equal we preter mismatches to any gap preler
* a new gap to extending an ongoing gap and prefer a gap m seqx
* to a gap in seq y
*/ nw() n
{ char *px *py /* seqs and ptrs */ int *ndelv *dely /* keep tiack of dely */ int ndelx delx /* keep track of delx */ int *tmp /* for swapping rowO rowl */ iinntt mmiiss /* score for each type */ int insO msl /* insertion penalties */ register id /* diagonal index */ register 1J /* jmp index */ register *col0 *coll /* score for curr, last row */ rreeggiisstteerr Xxλx M y\ /* index into seqs */ dx = (struct diag *)g_calloc( to et diags ', lenO+lenl + 1 sizeof (struct diag)), ndely = (int *)g_calloc( to get ndely lenl + 1 , sιzeof(int)) dely = (int *)g_calloc( to get dely lenl + 1 sizeof(int)) colO = (int *)g_calloc( to get coIO lenl + 1 sιzeof(int)) coll = (int *)g_calloc("to get coll , lenl + l sizeof(int)), insO = (dna)9 DINS0 PINSO, msl = (dna)9 DINS1 PINS1 smax = -10000, if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1, yy < = lenl, yy+ +) { col0[yy] = delypyy] = col0[yy-l] - msl, ndely [yy] = yy,
} col0[0] = 0 /* Waterman Bull Math Biol 84 */
} else for (yy = 1 y> < = lenl , yy+ +)
Figure imgf000036_0001
/* fill m match matrix */ for (px = seqx[0], xx = 1 xx < = lenO, px+ + , xx+ +) {
/* initialize first entry m col */ if (endgaps) { if ( = = 1) coll[0] = delx = -(msO+insl), else coll[0] = delx = col0[0] - msl , ndelx = xx
} } else { coll[0] = 0, deh = -insO, ndelx = 0,
Page 2 of nw c Table 1 (conf) for (py = seq\[l ] yy = 1 yy < = lenl , py+ + , yy + +) {
Figure imgf000037_0001
mis + = (xbm[*px-'A'l&xbm[*py-'A ])"> DMAT DMIS else mis + = _day[*px-'A'][:,py-'A ]
10 /* update penalty for del in x seq,
* fav or new del over ongong del
* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndely[yy] < MAXGAP) { 15 if (col0[yy] - insO > = dely[yy]) { dely[yy] = col0[yy] - (insO + insI) ndely [yy] = 1 ,
Figure imgf000037_0002
20 ndely[yy] + + ,
}
} else { if (col0[yy] - (ιns0+ ιnsl) > = dely[yy]) { dely[yy] = col0[yy] - (msO+ iπsl ), 25 ndely[yy] = 1 ,
} else ndely[yy] + + ,
}
30 /* update penalty for del m y seq,
* fav or new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll [yy-l] - msO > = delx) { 35 delx = coll[yy-l] - (insO+insI), ndelx = 1 , } else { delx -= msl , ndelx + + , 40 }
} else { if (coll[yy-l] - (insO + insI) > = delx) { delx = coll[yy-l] - (insO + insI), ndelx = 1 , 45 } else ndelx + + ,
}
/* pick the maximum score, we're favoring 50 * mis over any del and delx over dely
*/
55
60
Page 3 of nw c Table 1 (conf) id = λ\ yy + lenl - 1 if (mis > = delx && mis > = dely[yy])
Figure imgf000038_0001
else if (delx > = dely[yy]) { coll[yy] = delx, lj = dx[ιd] rjmp, if (dxfid] jp n[0] && ('dna ] | (ndelx > = MAXJMP && xx > dx[ιd] jp x[ιj] + MX) | | mis > dx[ιd] score+DINSO)) { dx[ιd] ιjmp+ + , if ( + +ιj > = MAXJMP) { wπtejmps(ιd), rj = dx[ιd] ljmp = 0, dx[ιd] offset = offset, offset + = sizeof(struct jmp) + sizeof(offset),
}
} dxfid] jp n[ιj] = ndel dx[ιd] jp x[ιj] = xx, dx[ιd] score = delx,
} else { coll[yy] = dely[yy], IJ = dx[ιd] rjmp, if (dxfid] jp n[0] && Cdna ] | (ndely [yy] > = MAXJMP
&& xx > dxfid] jp x[y] + MX) | | mis > dxfid] score+DINSO)) { dxfid] rjmp + + , if ( + +ij > = MAXJMP) { wπtejmps(ιd), ij = dxfid] ljmp = 0, dxfid] offset = offset, offset + = sizeof(struct jmp) + sizeof(offset), }
} dxfid] jp n[ιj] = -ndely [yy], dxfid] jp x[ιj] = xx, dxfid] score = delyfyy],
} if (xx = = lenO && yy < lenl) { /* last col
*/ if (endgaps) collfyy] -= ιnsO+ιnsl *(lenl-yy) if (coll[yy] > smax) { smax = collfyy], dmax = id, } } } if (endgaps && xx < lenO) coll[yy-l] -= ms0+insl*(len0-xx), if (collfyy- 1] > smax) { smax = coll[yy-l], dmax = id, } tmp = colO, colO = coll , coll = tmp,
}
(void) free((char *)ndel) ), (void) free((char *)dely) (void) free((char *)col0)
(void) ffee((char *)coll)
Page 4 of nw c Table 1 (conf)
/* *
* pπnt() - only routine visible outside this module *
* static
* getmat() - trace back best path count matches pπnt()
* pr_ahgn() - print alignment of described in array p[] pπnt()
* dumpblockO — dump a block of lines ith numbers stars pr_ahgn() * nums() - put out a number line dumpblockO
* putlineO — put out a line (name [num] seq [num]) dumpblockO
* stars() - put a line of stars dumpblockO
* stripnameO - strip any path and prefix from a seqname */
^include nw h'
#define SPC 3
#define P LINE 256 /* maximum ouψut line */ #define P_SPC 3 /* space between name or num and seq */ extern day [26] [26], int olen, /* set output line length */
FILE *fx, I" ouφut file */ prιnt() print
{ int lx ly, firstgap lastgap, /* overlap */ if ((fx = fopen(ofile " ")) = = 0) { fpπntf(stdeιτ " %s can t write %s\n", prog, ofile), cleanup(l)
} fpπntf(fx, ' < first sequence %s (length = %d)\n' , namexfO], lenO), fpπntf(fx, < second sequence %s (length = %d)\n' namexfl], lenl) olen = 60, lx = lenO, ly = lenl , firstgap = lastgap = 0 if (dmax < lenl - 1) { /* leading gap in x */ ppfO] spc = firstgap = lenl - dmax - 1 , ly - = pp[0] spc
} else if (dmax > lenl - 1) { /* leading gap in y */ PPfl] spc = firstgap = dmax - (lenl - 1), lx - = pp[l] spc,
} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = ienO - dmaxO -1 , lx - = lastεap
} else if (dmaxO > IenO - 1) { /* trailmg gap in y */ lastgap = dmaxO - (IenO - 1), ly -= lastgap } getmat(lx, ly, firstgap lastgap), pr_ahgn(),
Page 1 of nwprint c Table 1 (conf)
/*
* trace back the best path, count matches */ static getmat(lx, ly, firstgap. lastgap) getmat int lx. ly; /* "core" (minus endgaps) */ int firstgap. lastεap: /* leading trailing overlap */
{ int nm. iO. il . sizO, sizl char outx[32]: double pet: register nO. nl : register char *p0. *pl :
/* get total matches, score
*/ iO = il = sizO = sizl = 0: pO = seqx[0] + pp[l].spc: pi = seqx[l] + pp[0].spc: nO = pp[l].spc + 1 : nl = pp[0].spc + 1 : nm = 0; while ( *p0 && *pl ) { if (siz0) { pl + + ; nl + + : sizO-:
} else if (sizl) { p0+ + ; n0+ + ; sizl-:
} else { if (xbm[*p0-'A']&xbmf*pl-'A']) nm+ + ; if (nO+ + = = pp[0].x[i0]) sizO = pp[0].n[i0+ +]; if (nl + + = = pp[l].x[il]) sizl = pp[l].n[il + +]; p0+ + : pl + + :
}
/* pet homology:
* if penalizing endgaps, base is the shorter seq * else, knock off overhangs and take shorter core
*/ if (endgaps) lx = (IenO < lenl)? IenO : lenl; else lx = (lx < ly)? lx : ly; pet = 100.*(doublejnm/(double)lx; fprintf(fx, "\n"); fprintf(fx, " < %d match %s in an overlap of %d: % .2f percent similarity \n" , nm, (nm = = 1)? " " : "es" . lx, pet);
Page 2 of nwprint.c Table 1 (conf) fprintf(fx. " < gaps in first sequence: %d" , gapx); ...getmat if (gapx) {
(void) sprintfioutx. " ( %d %s%s)" , ngapx. (dna)? "base": "residue" , (ngapx = = 1)? " ": "s"): fprintf(fx. " % s" . outx): fprintf(fx, ". gaps in second sequence: %d", gapy); if (gapy) {
(void) sprintf(outx, " ( %d %s%s)", ngapy. (dna)? "base" : "residue" , (ngapy = = 1)? " " : "s"); fprintf(fx, " % s" . outx):
} if (dna) fprintf(fx,
"\n < score: %d (match = %d, mismatch %d, gap penalty = %d + %d per base)\n" , smax, DMAT. DMIS. DINS0, DINS1); else fprintf(fx,
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n" smax, PINS0. PINS1): if (endgaps) fprintf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap: %d %s%s\n" . firstgap, (dna)? "base" : "residue" , (firstgap = = 1)? " " : "s" , lastgap, (dna)? "base" : "residue", (lastgap = = 1)? " " : "s"); else fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static nip]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next ouφut char slot */ static char out[2][P LINE] /* ouφut line */ static char star[P LINE]: /* set by stars() */
* print alignment of described in struct path ppf] */ static pr align() pr align
{ int nn: /* char count */ int more; register i: for (i = 0, lmax = 0: i < 2; i+ +) { nn = stripname(namexfi]); if (nn > lmax) lmax = nn: ncfi] = 1: nifi] = 1: sizfi] = ijfi] = 0; psfi] = seqxfi]: pofi] = outfi];
Page 3 of nwprint.c Table 1 (conf) for (nn = nm = 0 more = 1 more, ) { ...pr align for (l = more = 0 ι < 2 ι + + ) { /*
* do we have more of this sequence ' */
Figure imgf000042_0001
continue more+ + , if (pp[ι] spc) { /* leading space */ *po[ι] + + = ' ', pp[ι] spc- ,
} else if (sizfi]) { /* in a gap */
*po[ι]+ + = '-', sizfi]- ,
} else { /* we're putting a seq element
*/ *po[ι] = *ps[ι], if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]), po[ι] + + , ps[ι]+ + ,
/*
* are we at next gap for this seq9
*/ if (nι[ι] = = ppfi] xfyfi]]) { /*
* we need to merge all gaps
* at this location */ sizfi] = pp[ι] n[ιj[ι] + +], while (nι[ι] = = ppfi] x[ιj[ι]])
Figure imgf000042_0002
for (i = 0 l < 2, ι+ +) po[ι] = out[ι], nn = 0, }
/*
* dump a block of lines, including numbers, stars pr_ahgn() */ static dumpblockO dumpblock
{ register 1, for (ι = 0, l < 2, ι+ +) *po[ι]- = "\0 ,
Page 4 of nwprint c Table 1 (conf)
.dumpblock
(void) putc( \n', fx) for(ι = 0 l < 2 ι + +) { if (*out[ι] && (*out[ι] ' = *(po[ι])'= ' ')){ if (i = = 0) nums(ι), if(ι == 0&& *out[l]) stars() puthne(ι) if (l == 0&&*out[l]) fpπntf(fx, star),
Figure imgf000043_0001
nums(ι),
* put out a number line dumpblockO
*/ static nums(ιx) iiuins int ix, /* index in out|
{ char nlιne[P_LINE] register i J register char *pn *px, *py, for(ρn = nline, l = 0 I < Imax + P SPC, ι+ + ,pn++)
*ρn = ' ', for (l = ncfix], py = outfix], *py, py+ + , pn+ +) { if(*py == j I *py == '-') *pn = ' ' else { if (ι%10 == 0 I I (l == 1 && ncfix] ' = D){ j = (l < 0)9 -l l, for (px = pn, j, j /= 10, px— )
*px =j%10 + '0 , if (l < 0)
} else
*pn = ι+ + .
*ρn = '\0', ncfix] = = 1, for (pn = nline, *ρn pn++)
(void) putc(*pn fx).
(void) putc('\n', fx).
}
/*
* put Ol it a line (name, [num] seq [num]) dumpblockO
*/ static putlined x) putline
{ Page 5 of nwpπnt c Table 1 (eonf)
...putline int I: register char *px: for (px = namex[ix]. i = 0: *px && *px ! = px+ + . i++)
(void) ρutc(*px. fx): for (: i < lmax + P SPC: i++)
(void)putc(' '. fx):
/* these count from 1:
* ni[] is cunent element (from 1)
* nef] is number at start of current line */ for (px = out[ix]; *px: px+ +)
(void) putc(*px&0x7F. fx); (void) putc('\n', fx):
/*
* put a line of stars (seqs always in outfO]. out[l]): dumpblockO
*/ static stars() stars { int register char *p0. *pl, ex. *px; if('.*out[0] || (*out[0] &&*(po[0]) == ") I !*out[l] I I (*out[l] &&*(po[l])== ' ')) return; px = star; for (i = lmax + P_SPC: i: i~)
*px++ = ' ': for (pO = outfO], pi = outfi]: *p0 && *pl; p0+ + , pl + +) { if (isalpha(*p0) && isalpha(*pl)) { if (xbm[*pO-'A']&xbmf*pl-'A']) { ex = '*'; nm++;
} elseif(!dna&&_day[*pO-'A'][*ρl-'A'] > 0) ex = '.'; else ex = ' ';
} else ex = ' ':
*px++ = ex;
}
*px++ = '\n'; *px = '\0';
Page 6 of nwprint.c Table 1 (conV)
/*
* strip path or prefix from pn. return len: pr_align()
*/ static stripname(pn) stripname char *pn; /* file name (may be path) */
{ register char *px. *py:
PY = 0; for (px = pn; *px; px+ +) if (*pχ = = ' /') py = px + 1 ; if (py)
(void) strcpyφn, py): return(strlen(pn)) ;
Page 7 of nwprint.c Table 1 (conf)
/*
* cleanupO - cleanup any tmp file
* getseqO — read in seq set dna len maxlen * g_calloc() calloc() with error checkin
* readjmpsO ~ et the good jmps from tmp file it necessary
* writejmpsO — write a filled arrav of jmps to a tmp file nw() */
^include 'nw h ^include < sy s/file h > char *jname = /tmp/homgXXXXXX /* tmp file for jmps */ FILE *fj, int cleanupO, /* cleanup tmp file */ long lseek(),
/*
* remove any tmp file if we blow
*/ cleanup(ι) cleanup int 1,
{ if (fj)
(void) unlιnk(jname) exιt(ι), }
/*
* read, remrn ptr to seq, set dna len, maxlen
* skip lines starting with ' , ' < or ' > '
* seq in upper or lower case
*/ char * getseq(file, len) getseq char *file, /* file name */ int *len, /* seq len */ char lιne[1024] *pseq, register char *px *py, int natac tlen,
FILE *fp~ if ((fp = fopen(file "r")) = = 0) { fpπntf(stderr "%s can t read %s\n' , prog, file), exιt(l),
} tlen = natgc = 0, while (fgets(hne 1024 fp)) { if (*lιne = = | | *luιe = = ' < | | *hne = = ' > ') continue, for (px = line. *px ' = \n' , px+ +) if (ιsupper(*px) 1 1 ιslower(*px)) tlen+ +,
} if ((pseq = malloc((unsigned)(den+6))) = = 0) { fpπntf(stdeπ," %s mallocO failed to get %d bytes for %s\n' , prog, tlen+6, file), exιt(l),
} pseqfO] = pseqfl] = pseq[2] = pseqf3] = '\0' ,
Page 1 of nwsubr c Table 1 (conf)
...getseq py = pseq + 4. *len = tlen; rew d(fp), while (fgets(hne.1024. fp)) { if (*hπe == '.' 11 *hne == '<' | | *hne == '>') continue, for (px = lme: *px != '\n'; px++) { if (ιsupper(*px))
*py++ = *px; else if dslower(*px))
*py+ + = toupper(*px); if (ιndex( " ATGCU " , *(py- 1 ))) natgc + + ; } }
*py++ = '\0';
*py = '\0';
(void) fclose(fp); dna = natgc > (tlen3). return(pseq+4);
} char * g_calloc(msg, nx, sz) g calloc char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */
{ char "px. *calloc(); if ((px = calloc((unsigned)nx. (unsigned)sz)) = = 0) { if (*msg) { fpπntf(stderr, "%s: g_calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz); exit(l), } } return(ρx);
}
/*
* get final jmps from dxf] or tmp file, set ppf], reset dmax: main()
*/ readjmpsO readjmps { int fd = -1:
Figure imgf000047_0001
register i, j, xx; if(fj){
(void) fclose(fi): if ((fd = opentjname. O RDONLY, 0)) < 0) { fprιntf(stderr, "%s: can't open() %s\n", prog, jname); cleanup(l); } } for (I = iO = il = 0. dmaxO = dmax, xx = IenO; ; i+ +) { while (1) { for (j = dxfdmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j-)
Page 2 of nwsubr.c Table 1 (conQ
...readjmps if (j < 0 && dx[dmax] offset && fj) {
(void) lseek(fd dx[dmax] offset 0) (void) read(fd (char *)&dx[dmax] jp sizeof(struct jmp))
(void) read(fd (char *)&dx[dmax] offset sιzeof(dx[dmax] offset)), dxfdmax] ljmp = MAXJMP- 1
} else break
} if (ι > = JMPS) { fpπntf(stderr %s too many gaps in ahgnmenΛn prog), cleanup(l) } if
Figure imgf000048_0001
xx = dxfdmax] jp x[j]
Figure imgf000048_0002
if (siz < 0) { /* gap in second seq */
Figure imgf000048_0003
/* id = xx - yy + lenl - 1 */ pp[l] x[ιl] = xx dmax + lenl 1 , gapy + + , ngapy -= siz /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP 1 1 endgaps)9 siz MAXGAP, ιl + + ,
} else if (siz > 0) { /* gap in first seq */
Figure imgf000048_0004
gapx+ + , ngapx + = siz, /* ignore MAXGAP when domg endgaps */ siz = (siz < MAXGAP | | endgaps)9 siz MAXGAP, ι0+ + , }
} else break
} /* reverse the order of jmps
*/ for 0 = 0, iO- , j < iO j + + ι0~) { i = ppfO] nfj] pp[0] nfj] = ppfO] nfiO], ppfO] nfiO] = i, i = ppfO] xlj] ppfO] xfj] = ppfO] x[ι0], pp[0] x[ι0] = i, } for d = 0, ιl-, j < il j + + , ιl-) { i = pp[l] nfj], pp[l] nfj] = pp[l] n[ιl], ppfi] nfil] = i, i = PP[1] ϋl PP[1] x )] = PPtl] xlil], PPtl] fil] = i. } if (fd > = 0)
(void) close(fd) if (fj) {
(void) unlιnk(jname), fj = 0 offset = 0
}
} Page 3 of nwsubr c Table 1 (conf)
/*
* write a filled jmp struct offset of the prev one (if any) nw()
*/ wπtejmps(ιx) writejmps
Figure imgf000049_0001
char *mktemp(). if (!fj) { if (mktemp(jname) < 0) { fpπntf(stderr. " %s can't mktempO %s\n", prog, j name), cleanupO ). } if ((fj = fopendname. "w")) = = 0) { fpπntf(stderr, " %s can't write %s\n", prog, jname), exιt(l),
} }
(void) fwπte((char *)&dx[ιx] jp. sizeof(struct jmp), 1, fj), (void) fwπte((char *)&dx[ιx] offset, sizeof(dx[ιx]. offset), 1 , fj),
Page 4 of nwsubr c Table 2
PRO XXXXXXXXXXXXXXX (Length = 15 am o acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% ammo 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 Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% 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 DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% 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 DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matchmg nucleotides between the two nucleic acid sequences as determmed by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =
divided by 12 33 3% III Compositions and Methods of the Invention
A Full-Length PRO polypeptides
The present invention prov ides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides In particular, cDNAs encoding vaπous PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced m separate expression rounds may be given different PRO numbers but the UNQ number is unique for any giv en 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 refened 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 m the art The predicted amino acid sequence can be determined from the nucleotide sequence usmg routine skill For the PRO polypeptides and encoding nucleic acids described herem, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time
B PRO Polvpeptide Vanants
In addition to the full-length native sequence PRO polypeptides described herem, it is contemplated that PRO vanants can be prepared PRO vanants can be prepared by introducing appropnate nucleotide changes into the PRO DNA and/or by synthesis of the desired PRO polypeptide Those skilled 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 anchonng characteristics
Vanations in the nativ e full-length sequence PRO or in various domains of the PRO described herein, can be made, for example us g any of the techniques and guidelines for conservative and non-conservative mutations set forth, for mstance, m U S Patent No 5,364,934 Vanations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change m 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 ammo acid in one or more of the domains of the PRO Guidance in determining which amino acid residue mav 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 ammo acid sequence changes made in regions of high homology Ammo acid substitutions can be the result of replacing one amino acid with another ammo acid having similar structural and or chemical properties such as the replacement of a leucme with a senne, t e , conservative ammo acid replacements Insertions or deletions may optionally be m the range of about 1 to 5 ammo acids The vanation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids m the sequence and testmg the resultmg 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 ammo 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 chemicallv svnthesized 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 mterest are shown in Table 6 under the heading of prefened substitutions If such substitutions result in a change m biological activity, then more substantial changes, denommated exemplary substitutions in Table 6, or as further descnbed below in reference to amino acid classes, are introduced and the products screened
Table 6
Original Exemplary Prefened Residue Substitutions Substitutions
Figure imgf000054_0001
Arg (R) lvs gin asn lys Asn (N) gin his, lys, arg gin Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro ala ala His (H) asn gin, lys, arg arg He (I) leu val, met, ala, phe, norleucme leu
Leu (L) norleucme, lie, val,
Figure imgf000054_0002
Lys (K) arg, gin, asn arg
Figure imgf000054_0003
Phe (F) leu val, lie, ala, tyr leu Pro (P) ala ala
Figure imgf000054_0004
Thr (T) ser ser Trp (W) tyr. phe tyr Tyr (Y) tip phe, thr, ser phe Val (V) lie leu met, phe, ala norleucme leu
Substantial modifications in function or immunological identity of the PRO polypeptide are accomplished by selectmg substitutions that differ significantly in their effect on maintaining (a) the stmcture 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 mto groups based on common side-chain properties
(1) hydrophobic norleucme, met, ala, val, leu, lie, (2) neutral hydrophilic cys. ser, thr,
(3) acidic asp, glu,
(4) basic asn, gin, his, lys, arg,
(5) residues that influence cham onentation gly, pro, and
(6) aromatic tip, 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 conservativ e substitution sites or more preferably, into the remainmg (non-conserved) sites
The variations can be made using methods known in the art such as ohgonucleotide-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 , K) 6487 (1987)], cassette mutagenesis [Wells et al Gene, 34 315 (1985)], restnction selection mutagenesis [Wells et al Ph o 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 prefened scanning amino acids are relatively small, neutral amino acids Such amino acids include alanine, glycme, serine, and cysteine Alanine is typically a prefened scanning ammo 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 vanant [Cunningham and Wells. Science, 244 1081-1085 (1989)] Alanme is also typically prefened because it is the most common amino acid Further, it is frequently found m both buried and exposed positions [Creighton The Proteins, (W H Freeman & Co , N Y ), Chothia, J Mol Biol , 150 1 (1976)] If alanme substitution does not yield adequate amounts of variant, an lsoteπc 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 derivatizmg agent that is capable of reactmg with selected side chams or the N- or C- terminal residues of the PRO Denvatization with bifunctional agents is useful, for mstance, for crosslinkmg PRO to a water-msoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa Commonly used crosslinkmg agents include, e g , 1 , 1 -bιs(dιazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azιdosalιcyhc acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dιthιobιs(succmιmιdylpropιonate), bifunctional maleimides such as bιs-N-maleιmιdo-1,8- octane and agents such as methvI-3-[(p-azιdoρhenyl)dιthιo]propιoιmιdate
Other modifications mclude deamidation of glutaminyl and asparagmyl residues to the coπesponding glutamyl and aspartyl residues, respectively, hydroxylation of prohne and lysme, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginme, 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 amme, and amidation of any C-termmal carboxyl group
Another type of covalent modification of the PRO polypeptide included withm the scope of this mvention compnses 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 m native sequence PRO (either by removing the underlymg 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 m the native sequence PRO In addition the phrase includes qualitative changes in the glycosylation of the native protems, involving a change m the nature and proportions of the various carbohydrate moieties present Addition of glycosvlation sites to the PRO polypeptide may be accomplished by altering the ammo acid sequence The alteration mav be made, for example by the addition of, or substitution by, one or more serine or threonine residues to the nativ e sequence PRO (for O-hnked 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 couplmg of glvcosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 11 September 1987, and in Aplin and Wπston, 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 ammo acid residues that serve as targets for glycosylation Chemical deglvcosylation techniques are known in the art and described, for instance, by Hakimuddin, et a! , 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 descnbed 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 vanety of nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent 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 compnsmg PRO fused to another heterologous polypeptide or ammo acid sequence
In one embodiment, such a chimenc 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 ammo- or carboxyl- termmus of the PRO The presence of such epitope-tagged forms of the PRO can be detected usmg an antibody against the tag polypeptide Also, provision of the epitope tag enables the
PRO to be readily punfied bv affinity punfication usmg an anti-tag antibody or another type of affinity matrix that bmds to the epitope tag Vaπous 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 vims glycoprotem 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 ,
BwTechnology, 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-Freyerrnuth et al , Proc Natl Acad Set USA, 87 6393-6397 (1990)]
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO with an immunoglobulm 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 mclude the substitution of a soluble (transmembrane domam deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule In a particularly prefened embodiment, the immunoglobulin fusion mcludes the hmge, CH2 and CH3. or the hmge, CHI, CH2 and CH3 regions of an IgGl molecule For the production of immunoglobulin fusions see also US Patent No 5.428.130 issued June 27, 1995 D Preparation of PRO
The description below relates primarily to production of PRO by cultuπng 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, e 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, CA (1969), Mernfield, 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, CA) using manufacturer's instructions Various portions of the PRO may be chemically synthesized separately and combmed 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 protem encoded by it Screenmg the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as descnbed in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spπng 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 Sprmg Harbor Laboratory Press, 1995)]
The Examples below descπbe 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 labelmg are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stnngency and high stringency, are provided Sambrook et al , supra
Sequences identified m such library screenmg methods can be compared and aligned to other known sequences deposited and available m public databases such as GenBank or other pπvate sequence databases Sequence identity (at either the ammo acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determmed usmg methods known in the art and as descnbed herem
Nucleic acid havmg protem codmg sequence may be obtained by screening selected cDNA or genomic branes usmg the deduced ammo acid sequence disclosed herein for the first time, and, if necessary, usmg conventional pnmer extension procedures as descnbed 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, selectmg 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 m 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. CaP04, liposome-mediated and electroporation Dependmg on the host cell used, transformation is performed usmg standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as descnbed in Sambrook et al , supra, or electroporation is generally used for prokaryotes Infection with Agr obacterium tumefaciens is used for transformation of certain plant cells, as descnbed by Shaw et al , Gene, 23 315 (1983) and WO 89/05859 published 29 June 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 descnbed in U S Patent No 4.399.216 Transformations into yeast are typically earned out according to the method of Van Sol gen et al , J Bact , 130 946 (1977) and Hsiao et al , Proc Natl Acad Sci (USA), 76 3829 (1979) However, other methods for mtroducmg DNA into cells, such as by nuclear micro injection, electroporation, bactenal protoplast fusion with mtact cells, or polycations, e g , polybrene, polyornithine, may also be used For vaπous 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 clonmg or expressing the DNA m the vectors herein mclude prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes mclude but are not limited to eubactena, such as Gram-negative or Gram-positive organisms, for example, Enterobactenaceae such as E coli Vanous E coli strams 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 Enterobactenaceae such as Escherichia, e g , E coli, Enterobacter, Erwinia, Klebstella, Proteus, Salmonella, e.g., Salmonella typhtmurium, Serratta, e g., Serratia marcescans, and Shtgella, as well as Bacilli such as B subtihs and B hcheniformis (e g , B hcheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aerugtnosa, and Streptomyces These examples are illustrative rather than limiting Strain W3110 is one particularly prefened host or parent host because it is a common host strain for recombmant 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 protems endogenous to the host, with examples of such hosts including .-? coli W3110 strain 1A2, which has the complete genotype ton A , 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 ; E coh W3110 stram 37D6. which has the complete genotype tonA ptr3 phoA El 5 (argF-lac) 169 degP ompT rbs7 ilvG /can' ', E coli W31 10 stram 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E coli strain havmg mutant penplasmic protease disclosed m U.S Patent No 4,946,783 issued 7 August 1990 Alternatively in vitr o methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable
In addition to prokarvotes eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encodmg vectors Sacchar omyces cer evisiae is a commonly used lower eukaryotic host microorganism Others include Schizo acchar omyces pombe (Beach and Nurse, Natur e, 290 140 [1981], EP 139,383 published 2 Mav 1985), Kluvver omyces hosts (U S Patent 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 fiagihs (ATCC 12,424) K bulgancus (ATCC 16,045), K wickeramu (ATCC 24,178), K waltu (ATCC 56,500), K ώ osoph arum (ATCC 36,906, Van den Berg et al , Bio/Technology, 8 135 (1990)) K thermotoleians, and K marxianus varrowia (EP 402,226), Pichia pastoris (EP 183,070, Sreeknshna 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 Schwanniomvces occidentals (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g , Neurospora, Pemcilhum Tohpocladium (WO 91/00357 published 10 January 1991), and Aspergύlus hosts such as A nidulans (Ballance et al Biochem Biophys Res Commun , 1 12 284-289 [1983], Tilburn et al , Gene, 26 205-221 [1983], Yelton et al , Proc Natl Acad Sci USA, SΛ 1470-1474 [1984]) and A niger (Kelly and Hynes, EMBO J , 4 475-479 [1985]) Methylotropic yeasts are suitable herem and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomvces 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 mclude 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 lme (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 Chasm, Proc Natl Acad Set 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 appropnate host cell is deemed to be within the skill in the art 3 Selection and Use of a Rephcable Vector
The nucleic acid (e g . cDNA or genomic DNA) encoding PRO may be inserted mto a rephcable 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 appropnate nucleic acid sequence may be inserted mto the vector by a variety of procedures In general, DNA is inserted into an appropnate restnction endonuclease sιte(s) usmg techniques known in the art Vector components generally mclude, but are not limited to one or more of a signal sequence, an oπgm of replication, one or more marker genes, an enhancer element, a promoter, and a transcnption termination sequence Construction of suitable vectors containing one or more of these components employs standard gation 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-termmus of the mature protem or polypeptide In general, the signal sequence may be a component of the vector, or it may be a part of the PRO-encodmg 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, penicilhnase, lpp, or heat-stable enterotoxm II leaders For yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccha omxces and Kluvveromx ces α-factor leaders, the latter described in U S Patent No 5.010,182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362,179 published 4 Apπl 1990), or the signal described in WO 90/13646 published 15 November 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 ongin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and vanous viral origins (SV40, polyoma, adenovims, VSV or BPV) are useful for cloning vectors m mammalian cells
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker Typical selection genes encode protems that (a) confer resistance to antibiotics or other toxins, e g , ampicillm, neomycin, methotrexate, or tetracychne, (b) complement auxotrophic deficiencies, or (c) supply critical nutnents not available from complex media, e g , the gene encoding D-alamne 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-encodmg nucleic acid, such as DHFR or thymidine kmase An appropnate host cell when w ild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as descnbed by Urlaub et al , Proc Natl Acad Sci USA, 77 4216 (1980) A suitable selection gene for use in yeast is the trp\ 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 trp\ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow m 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 vanety 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 (tip) promoter system [Goeddel, Nucleic Acids Res , 8 4057 (1980), EP 36,776], and hybnd promoters such as the tac promoter [deBoer et al , Proc Natl Acad Sci USA, 80 21-25 (1983)] Promoters for use m bactenal systems also will contam 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, hexokmase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase 3-phosphoglycerate mutase, pymvate kinase. tπosephosphate 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, metallotfuonein, 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 vims, fowlpox vims (UK 2,211,504 published 5 July 1989), adenovims (such as Adenovims 2), bovine papilloma vims, avian sarcoma vims, cytomegalovirus, a retrovims, hepatitis-B vims and Simian Vims 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 (globm, elastase, albumin α-fetoprotem. and insulin) Typically, however, one will use an enhancer from a eukaryotic cell vims Examples include the SV40 enhancer on the late side of the replication oπgm (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication ongm, 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, anrmal, 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 contam 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 descnbed in Gethmg 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, P/oc Natl Acad Sci USA, 77 5201-5205 (1980)], dot blottmg (DΝA analysis), or in situ hybridization, using an appropnately labeled probe, based on the sequences provided herem Alternatively, antibodies may be employed that can recognize specific duplexes, mcludmg DΝA duplexes, RΝA duplexes, and DΝA-RΝA hybrid duplexes or DΝA-protem duplexes The antibodies in turn may be labeled and the assay may be earned 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 stammg of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful tor 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 agamst 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 recov ered 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 Tπton-X* 100) or by enzymatic cleavage Cells employed m expression of PRO can be dismpted by various physical or chemical means, such as freeze-thaw cycling, sonication mechanical disruption, or cell lysing agents
It may be desired to punfv PRO from recombinant cell protems 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, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bmd epitope-tagged forms of the PRO Vanous methods of protem purification may be employed and such methods are known in the art and descnbed for example in Deutscher, Methods in Enzvmology, 182 (1990), Scopes, Protein Pur ification Principles and Practice, Spnnger-Verlag, New York (1982) The puπfication step(s) selected will depend, for example, on the nature of the production process used and the particular PRO produced
E Anti-PRO Antibodies
The present invention further provides anti-PRO antibodies Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies 1 Polvclonal Antibodies The anti-PRO antibodies may compnse 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 mjected m 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 protem known to be immunogemc in the mammal being immunized Examples of such immunogemc protems mclude but are not limited to keyhole limpet hemocyanm, semm albumin, bovine thyroglobulm, 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 usmg hybndoma methods, such as those descnbed by Kohler and Milstein, Nature, 256 495 (1975) In a hybndoma 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 bmd to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro The immunizing agent w ill typically include the PRO polypeptide or a fusion prote thereof Generally, either peripheral blood lvmphocytes ("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 hybndoma cell [Godmg, Monoclonal Antibodies Pr inciples 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 hybndoma 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 phosphonbosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminoptenn and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells
Prefened immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibodv -producing cells, and are sensitive to a medium such as HAT medium More prefened immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center San Diego, California and the American Type Culture Collection, Manassas, Virginia 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, Ine , New York, (1987) pp 51-63] The culture medium in which the hybndoma cells are cultured can then be assayed for the presence of monoclonal antibodies duected agamst PRO Preferably, the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by lmmunoprecipitation or by an in vitro bindmg assay, such as radioimmunoassay (RIA) or enzyme-linked lmmunoabsorbent assay (ELISA) Such techniques and assays are known in the art The bmdmg 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 hvbπdoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Godmg, supra] Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal The monoclonal antibodies secreted by the subclones may be isolated or punfied from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described m U S Patent No 4,816,567 DNA encodmg the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by usmg oligonucleotide probes that are capable of binding specifically to genes encodmg the heavy and light chains of murine antibodies) The hybndoma cells of the mvention serve as a prefened source of such DNA Once isolated, the DNA may be placed mto 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 protem, to obtain the synthesis of monoclonal antibodies in the recombmant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heaw and light chain constant domains in place of the homologous murine sequences [U S Patent No 4 816 567 Momson et al supi a] or bv covalently joining to the immunoglobulin coding sequence all or part of the codmg sequence for a non-immunoglobuhn polypeptide Such a non- lmmunoglobuhn polypeptide can be substituted for the constant domains of an antibodv 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 mav 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 heaw chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinkmg Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prev ent crosslinkmg
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-bindmg subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies mclude 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 havmg the desired specificity, affinity and capacity In some instances, Fv framework residues of the human immunoglobulin are replaced by conesponding non- human residues Humanized antibodies may also compnse residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will compnse substantially all of at least one and typically two, vanable domains, m which all or substantially all of the CDR regions conespond 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 rmmunoglobulm 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 ammo acid residues introduced into it from a source which is non-human These non- human amino acid residues are often refened to as "import" residues, which are typically taken from an "import" vanable domam Humamzation can be essentially performed following the method of Winter and co- workers [Jones et al , Nature 321 522-525 (1986), Riechmann et a! , Nature, 332 323-327 (1988), Verhoeyen et al , Science, 239 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the conesponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeπc antibodies (U S Patent No 4,816,567), wherein substantially less than an mtact human vanable domam 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 Boemer 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 Boemer et al, J. Immunol, 147(l):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 reaπangement. assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent 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); Moπison, 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. Prefened 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 conect bispecific stmcture. The purification of the conect 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, EMBOJ., 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 prefened to have the first heavy-chain constant region (CHI) 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 96/2701 1, 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 prefened 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 vv ith larger side chains (e g tyrosine or tryptophan) Compensatory "cavities" of identical or similar size to the large side chaιn(s) are created on the interface of the second antibody molecule by replacing large ammo acid side chains with smaller ones (e g alanme or threonine) This provides a mechanism for increasing the v leld of the heterodimer over other unwanted end-products such as homodimers
Bispecific antibodies can be prepared as full length antibodies or antibodv 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 complexmg agent sodium arsemte to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thio trobenzoate (TNB) deπvatives One of the Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercap toethylamine 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 coh and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bmd to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes agamst human breast tumor targets Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been descnbed For example, bispecific antibodies have been produced usmg leucine zippers Kostelny et al , J Immunol 148(5) 1547-1553 (1992) The leucme 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 descnbed by Hollinger et al , Proc Natl Acad Set USA 90 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments compnse a heavy-chain vanable domain (VH) connected to a light-chain vanable domain (VL) by a linker which is too short to allow pairing between the two domams on the same cham Accordingly, the VH and V domams of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby formmg 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, Graber et al , J Immunol 152 5368 (1994)
Antibodies with more than two \ alencies are contemplated For example, tnspecific 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 polvpeptide ami 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-bmding arm and an arm which bmds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA Another bispecific antibody of interest bmds the PRO polypeptide and further binds tissue factor (TF)
5 Heterocomugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently jomed antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent 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 crosslinkmg agents For example, lmmunotoxins may be constmcted using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methyl-4-mercaptobutyπmιdate and those disclosed, for example, m U S Patent No 4,676,980
6 Effector Function Engineenng
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e , the effectiveness of the antibody in treatmg cancer For example, cysteine resιdue(s) may be introduced mto the Fc region, thereby allowing interchain disulfide bond formation in this region The homodrmenc 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 , J 6 1191-1195 (1992) and Shopes, J Immunol , L48 2918-2922 (1992) Homodrmenc 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 Immunocomugates The mvention also pertains to immunoconjugates compnsmg 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 ongm, or fragments thereof), or a radioactive isotope (i e , a radioconjugate)
Chemotherapeutic agents useful in the generation of such immunoconjugates have been descnbed above Enzymatically active toxms and fragments thereof that can be used include diphtheria A cham, nonbindmg active fragments of diphthena toxin, exotoxm A chain (from Pseudomonas aeruginosa), πcin A chain, abnn A chain, modeccin A chain, alpha-sarcin, Aleuntes fordu proteins, dianthm proteins, Phytolaca americana proteins (PAPI. PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonana officina s inhibitor, gelon . mitogelhn, restπctocm, phenomycin, enomycin, and the tncothecenes A vanety of radionuchdes are available for the production of radioconjugated antibodies Examples include 212Bι, I31I, In, 9Υ, and 186Re Conjugates of the antibody and cytotoxic agent are made usmg a vanety of bifunctional protein-coupling agents such as N-succιnιmιdyl-3-(2-pyπdyldιthιol) propionate (SPDP), lminothiolane (IT) bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL) active esters (such as disuccinimidyl suberate), aldehvdes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamme) bis-diazonium derivatives (such as bιs-(p-dιazonmmbenzoyl)-ethylenedιamιne), dnsocyanates (such as tolyene 2 6-dιιsocyanate) and bis-active fluorine compounds (such as l,5-dιfluoro-2,4- dinitrobenzene) For example a ncin lmmunotoxin can be prepared as described m Vitetta et al , Science, 238 1098 (1987) Carbon- 14-labeled 1-ιsothιocyanatobenzyl 3-methyldιethylene tπaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See W094/1 1026
In another embodiment the antibody may be conjugated to a "receptor' (such streptavidin) for utilization m tumor pretargetmg 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 avidm) that is conjugated to a cytotoxic agent (e g a radionucleotide) 8 Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes Liposomes containmg 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 w lth enhanced circulation time are disclosed in U S Patent No 5,013,556
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition compnsmg phosphatidylcho ne, cholesterol, and PEG-denvatized phosphatidylethanolamine (PEG-PE) Liposomes are extmded 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 descnbed 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 withm 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 screenmg assavs disclosed hereinbefore, can be administered for the treatment of vanous disorders in the form of pharmaceutical compositions
If the PRO polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are prefened However, hpofections or liposomes can also be used to deliver the antibody, or an antibody fragment, mto cells Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the bmdmg domam of the target protem is preferred For example, based upon the vaπable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protem sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology See, e g , Marasco et al , Proc Natl Acad Sci L/SΛ, 90 7889-7893 (1993) The formulation herem 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 m 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 mav also be entrapped in microcapsules prepared for example, by coacervation techniques or by interfacial polv meπzation, for example hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal dmg 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
Figure imgf000069_0001
administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
Sustained-release preparations may be prepared Suitable examples of sustamed-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 matnces include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or ρoly(vmvlalcohol)), polylactides (U S Pat No 3,773,919) copolvmers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene- vmyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprohde acetate), and poly-D-(-)-3- hydroxybutyric acid While polvmers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 davs certain hydrogels release proteins for shorter time penods When encapsulated antibodies remain m the bodv 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 mtermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropnate additives, and developing specific polymer matrix compositions F Tissue Distnbution
The location of tissues expressing the PRO can be identified by determining mRNA expression in vanous human tissues The location of such genes provides information about which tissues are most likely to be affected by the stimulatmg and inhibiting activities of the PRO polypeptides The location of a gene in a specific tissue also provides sample tissue for the activity blocking assays discussed below
As noted before, gene expression in various tissues may be measured by conventional Southern blottmg, Northern blotting to quantitate the transcnption of mRNA (Thomas, Proc Natl Acad Sci USA, 77 5201-5205 [1980]), dot blottmg (DNA analysis), or in situ hybndization, usmg an appropnately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DΝA-RΝA hybrid duplexes or DΝA-protein duplexes
Gene expression m vanous tissues, alternatively, may be measured by immunological methods, such as immunohistochemical stammg of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for immunohistochemical stammg 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 agamst a native sequence of a PRO polypeptide or against a synthetic peptide based on the DΝA sequences encodmg the PRO polypeptide or against an exogenous sequence fused to a DΝA encodmg a PRO polypeptide and encodmg a specific antibody epitope General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided below G Antibodv Binding Studies
The activity of the PRO polypeptides can be further verified by antibody bmding studies, in which the ability of an anti-PRO antibodv to inhibit the effect of the respective PRO polypeptide on tissue cells is tested Exemplary antibodies include polyclonal. monoclonal humanized, bispecific, and heteroconjugate antibodies, the preparation of which will be described herembelow
Antibody binding studies may be earned out in any known assay method, such as competitive binding assays, direct and indirect sandw ich assays, and immunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques, pp 147- 158 (CRC Press, Ine , 1987) Competitive bmdmg assays rely on the ability of a labeled standard to compete with the test sample analyte for bindmg with a limited amount of antibody The amount of target protem in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies preferably are msolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound
Sandwich assays mv oh e the use of two antibodies, each capable of binding to a different immunogemc portion, or epitope, of the protem to be detected In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody bmds to the analyte, thus formmg an insoluble three-part complex See, e , US Pat No 4,376,110 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobuhn antibody that is labeled with a detectable moiety (mdirect sandwich assay) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme
For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example H Cell-Based Assays
Cell-based assays and animal models for immune related diseases can be used to further understand the relationship between the genes and polypeptides identified herem and the development and pathogenesis of immune related disease
In a different approach, cells of a cell type known to be involved in a particular immune related disease are transfected with the cDNAs descnbed herem, and the ability of these cDNAs to stimulate or inhibit immune function is analyzed Suitable cells can be transfected with the desired gene, and monitored for immune function activity Such transfected cell lmes can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit or stimulate immune function, for example to modulate T-cell proliferation or inflammatory cell infiltration Cells transfected with the codmg sequences of the genes identified herem can further be used to identify drag candidates for the treatment of immune related diseases
In addition, primary cultures denved from transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are prefened Techniques to deπve contmuous cell lines from transgenic animals are w ell known in the art (see, e g , Small et al , Mol Cell Biol 5 642-648 [1985])
One suitable cell based assay is the mixed lymphocyte reaction (MLR) Current Protocols in Immunology, unit 3 12; edited by J E Coligan, A M Kmisbeek, D H Marghes, E M Shevach, W Strober, National Institutes of Health. Published by John Wiley & Sons, Inc. In this assay, the ability of a test compound to stimulate or inhibit the proliferation of activated T cells is assayed. A suspension of responder T cells is cultured with allogeneic stimulator cells and the proliferation of T cells is measured by uptake of tritiated thymidine. This assay is a general measure of T cell reactivity. Since the majority of T cells respond to and produce IL-2 upon activation, differences in responsiveness in this assay in part reflect differences in IL-2 production by the responding cells. The MLR results can be verified by a standard lymphokine (IL-2) detection assay. Current Protocols in Immunology, above, 3.15, 6.3.
A proliferative T cell response in an MLR assay may be due to direct mitogenic properties of an assayed molecule or to external antigen induced activation. Additional verification of the T cell stimulatory activity of the PRO polypeptides can be obtained by a costimulation assay. T cell activation requires an antigen specific signal mediated through the T-cell receptor (TCR) and a costimulatory signal mediated through a second ligand binding interaction, for example, the B7 (CD80, CD86)/CD28 binding interaction. CD28 crosslinking increases lymphokine secretion by activated T cells. T cell activation has both negative and positive controls through the binding of ligands which have a negative or positive effect. CD28 and CTLA-4 are related glycoproteins in the Ig superfamily which bind to B7. CD28 binding to B7 has a positive costimulation effect of T cell activation; conversely, CTLA-4 binding to B7 has a negative T cell deactivating effect. Chambers, C. A. and Allison, J. P., Curr. Opin. Immunol. (1997) 9:396. Schwartz, R. H., Cell (1992) 71:1065; Linsey, P. S. and Ledbetter, J. A., Annu. Rev. Immunol. (1993) 11 :191; June, C. H. et al, Immunol. Today (1994) 15:321; Jenkins, M. K., Immunity (1994) 1:405. In a costimulation assay, the PRO polypeptides are assayed for T cell costimulatory or inhibitory activity.
PRO polypeptides, as well as other compounds of the invention, which are stimulators (costimulators) of T cell proliferation and agonists, e.g., agonist antibodies, thereto as determined by MLR and costimulation assays, for example, are useful in treating immune related diseases characterized by poor, suboptimal or inadequate immune function. These diseases are treated by stimulating the proliferation and activation of T cells (and T cell mediated immunity) and enhancing the immune response in a mammal through administration of a stimulatory compound, such as the stimulating PRO polypeptides. The stimulating polypeptide may, for example, be a PROl 84, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PR01157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940, PRO6006 polypeptide or an agonist antibody thereof.
Direct use of a stimulating compound as in the invention has been validated in experiments with 4- IBB glycoprotein, a member of the tumor necrosis factor receptor family, which binds to a ligand (4-1BBL) expressed on primed T cells and signals T cell activation and growth. Alderson, M. E. et al, J. Immunol. (1994) 24:2219. The use of an agonist stimulating compound has also been validated experimentally. Activation of 4-
1BB by treatment with an agonist anti-4-lBB antibody enhances eradication of tumors. Hellstrom, I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998) 18:1. Immunoadjuvant therapy for treatment of tumors, described in more detail below, is another example of the use of the stimulating compounds of the invention.
An immune stimulating or enhancing effect can also be achieved by antagonizing or blocking the activity of a PRO which has been found to be inhibiting in the MLR assay. Negating the inhibitory activity of the compound produces a net stimulatory effect Suitable antagonists/blocking compounds are antibodies or fragments thereof which recognize and bind to the inhibitory protein thereby blocking the effective interaction of the protein with its receptor and inhibiting signaling through the receptor This effect has been validated in experiments using anti-CTL A.-4 antibodies which enhance T cell proliferation, presumably by removal of the inhibitory signal caused by CTLA-4 bmding Walunas, T L et l Immunity (1994) 1 405
Alternatively, an immune stimulating or enhancing effect can also be achieved by administration of a PRO which has vascular permeability enhancing properties Enhanced vacuolar permeability would be beneficial to disorders which can be attenuated by local infiltration of immune cells (e g . monocytes, eosinophils, PMNs) and inflammation On the other hand. PRO polypeptides, as well as other compounds of the mvention, which are direct inhibitors of T cell proliferation activation, lymphokine secretion, and/or vascular permeability can be directly used to suppress the immune response These compounds are useful to reduce the degree of the immune response and to treat immune related diseases characterized by a hyperactive, superoptimal, or autoimmune response This use of the compounds of the invention has been validated by the experiments described above in which CTLA-4 bindmg to receptor B7 deactivates T cells The direct inhibitory compounds of the invention function m an analogous manner The use of compound which suppress vascular permeability would be expected to reduce mflammation Such uses would be beneficial in treating conditions associated with excessive inflammation
Alternatively, compounds, e g , antibodies, which bind to stimulating PRO polypeptides and block the stimulating effect of these molecules produce a net inhibitory effect and can be used to suppress the T cell mediated immune response by inhibiting T cell proliferation activation and/or lymphokine secretion Blocking the stimulating effect of the polypeptides suppresses the immune response of the mammal This use has been validated m expenments using an antι-IL2 antibody In these expenments, the antibody binds to IL2 and blocks bmding of IL2 to its receptor thereby achievmg a T cell inhibitory effect I Animal Models
The results of the cell based in vitro assays can be further verified usmg in vivo animal models and assays for T-cell function A vanety of well known animal models can be used to further understand the role of the genes identified herem m the development and pathogenesis of immune related disease, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides, mcludmg small molecule antagonists The in vivo nature of such models makes them predictive of responses in human patients Animal models of immune related diseases include both non-recombinant and recombmant (transgenic) animals Non-recombinant animal models mclude, for example, rodent, e g , murine models Such models can be generated by introducing cells into syngeneic mice using standard techniques, e g , subcutaneous injection, tail vem injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, etc
Graft-versus-host disease occurs when immunocompetent cells are transplanted mto lmmunosuppressed or tolerant patients The donor cells recognize and respond to host antigens The response can vary from life threatening severe inflammation to mild cases of dianhea and weight loss Graft-versus-host disease models provide a means of assessmg T cell reactivity against MHC antigens and minor transplant antigens A suitable procedure is described in detail m Cunent Protocols in Immunology, above, unit 4 3 An animal model for skm allograft rejection is a means of testing the ability of T cells to mediate in vivo tissue destmction and a measure of their role in transplant rejection The most common and accepted models use murine tail-skm grafts Repeated experiments have shown that skm allograft rejection is mediated by T cells, helper T cells and killer-effector T cells, and not antibodies Auchincloss, H Jr and Sachs, D H , Fundamental Immunology 2nd ed , W E Paul ed , Raven Press, NY, 1989, 889-992 A suitable procedure is described in detail in Current Pr otocols in Immunology, above, unit 4 4 Other transplant rejection models which can be used to test the compounds of the invention are the allogeneic heart transplant models described by Tanabe, M et al, Transplantation (1994) 58 23 and Tinubu, S A et al, J Immunol (1994) 4330-4338
Animal models for delayed type hypersensitivity provides an assay of cell mediated immune function as well Delayed type hypersensitivity reactions are a T cell mediated in vivo immune response characterized by inflammation which does not reach a peak until after a period of time has elapsed after challenge with an antigen These reactions also occur in tissue specific autoimmune diseases such as multiple sclerosis (MS) and expenmental autoimmune encephalomyelitis (EAE, a model for MS) A suitable procedure is described m detail in Current Protocols in Immunology, above, unit 4 5 EAE is a T cell mediated autoimmune disease charactenzed by T cell and mononuclear cell inflammation and subsequent demyehnation of axons in the central nervous system EAE is generally considered to be a relevant animal model for MS in humans Bolton, C , Multiple Sclerosis (1995) 1 143 Both acute and relapsing-remitting models have been developed The compounds of the mvention can be tested for T cell stimulatory or inhibitory activity against immune mediated demyelmating disease using the protocol descnbed in Current Protocols in Immunology, above, units 15 1 and 15 2 See also the models for myelin disease in which oligodendrocytes or Schwann cells are grafted into the central nervous system as described in Duncan, I D et al, Molec Med Today (1997) 554-561
Contact hypersensitivity is a simple delayed type hypersensitivity in \ ιvo assay of cell mediated immune function In this procedure, cutaneous exposure to exogenous haptens which gives nse to a delayed type hypersensitivity reaction which is measured and quantitated Contact sensitivity involves an initial sensitizing phase followed by an ehcitation phase The ehcitation phase occurs when the T lymphocytes encounter an antigen to which they have had previous contact Swelling and inflammation occur, making this an excellent model of human allergic contact dermatitis A suitable procedure is descnbed in detail m Current Protocols in Immunology , Eds J E Cologan, A M Kmisbeek, D H Marguhes, E M Shevach and W Strober, John Wiley & Sons, Ine , 1994, unit 4 2 See also Grabbe, S and Schwarz, T, lmmun Today 19 (1) 37-44 (1998)
An animal model for arthntis is collagen-induced arthritis This model shares clinical, histological and immunological charactenstics of human autoimmune rheumatoid arthntis and is an acceptable model for human autoimmune arthntis Mouse and rat models are characteπzed by synovitis, erosion of cartilage and subchondral bone The compounds of the invention can be tested for activity agamst autoimmune arthritis using the protocols descnbed m Current Protocols in Immunology, above, units 15 5 See also the model using a monoclonal antibody to CD18 and VLA-4 mtegnns descnbed in Issekutz, A C et al , Immunology (1996) 88'569
A model of asthma has been descnbed m which antigen-induced airway hyper-reactivity, pulmonary eosrnophilia and inflammation are induced by sensitizing an animal with ovalbumin and then challenging the animal with the same protein delivered by aerosol Several animal models (guinea pig, rat, non-human primate) show symptoms similar to atopic asthma in humans upon challenge with aerosol antigens Murine models have many of the features of human asthma Suitable procedures to test the compounds of the invention for activity and effectiveness in the treatment of asthma are described by Woly ec, W W et al Am J Respir Cell Mol Biol (1998) 18 777 and the references cited therein
Additionally, the compounds of the mvention can be tested on animal models for psoriasis like diseases Evidence suggests a T cell pathogenesis for psoriasis The compounds of the invention can be tested in the scid scid mouse model described by Schon, M P et al, Nat Med (1997) 3 183, m which the mice demonstrate histopathologic skm lesions resembling psoriasis Another suitable model is the human skin/scid mouse chimera prepared as described by Nickoloff, B J et al, Am J Path (1995) 146 580
Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein mto the genome of animals of interest, using standard techniques for producing transgenic animals Animals that can serve as a target for transgenic manipulation mclude, without limitation, mice, rats, rabbits, guinea pigs sheep, goats, pigs, and non-human primates, e g baboons, chimpanzees and monkeys Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U S Patent No 4,873,191), retrovirus-mediated gene transfer mto germ lines (e g , Van der Putten et al Proc Natl Acad Sci USA 82, 6148-615 [1985]), gene targeting m embryonic stem cells (Thompson et al Cell 56, 313-321 [1989]), electroporation of embryos (Lo, Mol Cel Biol 3, 1803- 1814 [1983]), sperm-mediated gene transfer (Lavitrano et al , Cell 57, 717-73 [1989]) For review, see, for example, U S Patent No 4.736,866
For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA 89, 6232-636 (1992)
The expression of the transgene in transgenic animals can be monitored by standard techniques For example, Southern blot analvsis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as in situ hybndization, Northern blot analysis, PCR, or immunocytochemistry The animals may be further examined for signs of immune disease pathology, for example by histological examination to determine infiltration of immune cells into specific tissues Blockmg expenments can also be performed in which the transgenic animals are treated with the compounds of the invention to determine the extent of the T cell proliferation stimulation or inhibition of the compounds In these expenments, blockmg antibodies which bind to the PRO polypeptide, prepared as descnbed above, are administered to the animal and the effect on immune function is determined
Alternatively, "knock out" animals can be constmcted which have a defective or altered gene encoding a polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced mto an embryonic cell of the animal For example, cDNA encoding a particular polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques A portion of the genomic DNA encodmg a particular polvpeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor mtegration 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 m 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 eg, Bradley, in Teratocarcinomas and Embn onic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 113-152] A chimenc 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 then 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 polypeptide J ImmunoAdiuvant Therapy
In one embodiment the lmmunostimulatmg compounds of the invention can be used m lmmunoadjuvant therapy for the treatment of tumors (cancer) It is now well established that T cells recognize human tumor specific antigens One group of tumor antigens, encoded by the MAGE, BAGE and GAGE families of genes, are silent m all adult normal tissues , but are expressed in significant amounts in tumors, such as melanomas, lung tumors head and neck tumors, and bladder carcinomas DeSmet, C et al , (1996) Proc Natl Acad Sci USA, 93 7149 It has been shown that costimulation of T cells induces tumor regression and an antitumor response both in x itio and in vivo Melero, I et al , Nature Medicine (1997) 3 682, Kwon, E D et al , Proc Natl Acad Sci ISA (1997) 94 8099, Lynch, D H et al, Natwe Medicine (1997) 3 625, Finn, O J and Lotze, M T , J Immunol (1998) 21 114 The stimulatory compounds of the invention can be administered as adjuvants, alone or together with a growth regulating agent, cytotoxic agent or chemotherapeutic agent, to stimulate T cell proliferation/ activation and an antitumor response to tumor antigens The growth regulating, cytotoxic, or chemotherapeutic agent may be administered m conventional amounts using known administration regimes lmmunostimulatmg activity by the compounds of the invention allows reduced amounts of the growth regulatmg, cytotoxic, or chemotherapeutic agents thereby potentially lowering the toxicity to the patient K Screening Assays for Dmg Candidates
Screening assays for dmg candidates are designed to identify compounds that bind to or complex with the polypeptides encoded by the genes identified herein or a biologically active fragment thereof, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assays will include assays amenable to high-throughput screenmg of chemical libraries, making them particularly suitable for identifying small molecule dmg candidates Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptιde-ιmmunoglobulιn fusions, and, m particular antibodies mcludmg, without limitation, poly- and monoclonal antibodies and antibody fragments, smgle-cham antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments The assays can be performed in a vanety of formats, mcludmg protein-protein b dmg assays, biochemical screenmg assays, immunoassays and cell based assays which are well characterized in the art
All assays are common in that they call for contacting the drag candidate w ith a 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 polypeptide encoded by the gene identified herein or the dmg 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 polypeptide and drying Alternatively, an immobilized antibody, e g a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface The assay is performed by adding the non-immobilized component hich 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 cames a detectable label, the detection of label immobilized on the surface indicates that complexmg occuned Where the originally non-immobilized component does not carry a label, complexmg can be detected, for example, by using a labelled antibody specifically binding the immobilized complex
If the candidate compound interacts with but does not bind to a particular protein encoded by a gene identified herem, its interaction with that protein can be assayed by methods well known for detecting protein- protem interactions Such assays include traditional approaches, such as, cross-linking, co- unmunoprecipitation, and co-punfication 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 transcnptional activators, such as yeast GAL4, consist of two physically discrete modular domams, one acting as the DNA-binding domam while the other one functioning as the transcnption activation domain The yeast expression system descnbed in the foregomg publications (generally refened to as the "two-hybrid system") takes advantage of this property, and employs two hybnd proteins, one in which the target protein is fused to the DNA-bmding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain The expression of a GALl -lacL reporter gene under control of a GAL4- activated promoter depends on reconstitution of G AL4 activ lty via protein-protein interaction Colonies containmg mteractmg 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 This system can also be extended to map protein domams mvolved in specific protein interactions as well as to pinpoint ammo acid residues that are crucial for these interactions
In order to find compounds that interfere with the interaction of a gene identified herem and other mtra- or extracellular components can be tested, a reaction mixture is usually prepared containmg the product of the gene and the mtra- or extracellular component under conditions and for a time allowing for the interaction and bindmg of the two products To test the ability of a test compound to inhibit bindmg, the reaction is ran m 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 mtra- or extracellular component present in the mixture is monitored as described abov e The formation of a complex in the control reactιon(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 L Compositions and Methods for the Treatment of Immune Related Diseases
The compositions useful in the treatment of immune related diseases include, without limitation, proteins, antibodies, small organic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc that inhibit or stimulate immune function, for example, T cell proliferation/activation, lymphokine release, or immune cell infiltration For example, antisense RNA and RΝA molecules act to directly block the translation of mRΝA by hybridizing to targeted mRΝA and preventing protem translation When antisense DΝA is used, oligodeoxyπbonucleotides den ed from the translation initiation site, e g between about -10 and +10 positions of the target gene nucleotide sequence, are prefened
Ribozymes are enzymatic RΝA molecules capable of catalyzmg the specific cleavage of RΝA Ribozymes act by sequence-specific hybridization to the complementary target RΝA, followed by endonucleolytic cleavage Specific nbozyme cleavage sites within a potential RΝA 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 September 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 tπple helix formation via Hoogsteen base painng 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 molecules can be identified by any or any combination of the screenmg assays discussed above and/or by any other screening techniques well known for those skilled in the art M Pharmaceutical Compositions
The active PRO molecules of the invention (e g , PRO polypeptides, anti-PRO antibodies, and/or vanants of each) as well as other molecules identified by the screenmg assays disclosed above, can be administered for the treatment of immune related diseases, in the form of pharmaceutical compositions Therapeutic formulations of the active PRO molecule, preferably a polypeptide or antibody of the mvention, are prepared for storage by mixing the active molecule having the desired degree of purity with optional pharmaceutically acceptable earners, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed [1980]), in the form of lyophilized formulations or aqueous solutions Acceptable carners, 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 and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chlonde, benzalkonium chloride, benzethomum chlonde, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcmol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, protems, such as semm albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamme, asparagme, histidine, argmine, or lysme, monosacchaπdes, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA sugars such as sucrose, mannitol, trehalose or sorbitol, salt- forming counter- ions such as sodium, metal complexes (e g Zn-protem complexes), and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) Compounds identified by the screenmg assays disclosed herein can be formulated in an analogous manner, usmg standard techniques well known in the art
Lipofections or liposomes can also be used to deliver the PRO molecule into cells Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the b dmg domain of the target protem is preferred For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which 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 herem 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 a cytotoxic agent, cytokine or growth inhibitory agent Such molecules are suitably present in combination m amounts that are effective for the purpose intended
The active PRO molecules may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by lnterfacial polymenzation, for example, hydroxymethylcellulose or gelatin- microcapsules and ρoly-(methy Imethacylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumm microspheres, microemulsions, nano-particles and nanocapsules) or m macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980)
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 or the PRO molecules may be prepared Suitable examples of sustained-release preparations mclude semipermeable matnces of solid hydrophobic polymers contaming the antibody, which matnces are m the form of shaped articles, e g , films, or microcapsules Examples of sustained-release matnces mclude polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vιnylalcohol)), 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™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydroxybutync acid While polymers such as ethylene-vinyl acetate and lactic acid- glyco c acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time penods 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, resultmg in a loss of biological activity and possible changes in lmmunogemcity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be mtermolecular S-S bond formation through thio- disulfϊde mterchange, stabilization may be achieved by modifymg sulfhydryl residues, lyophi zing from acidic solutions, controlling moisture content, using appropnate additives, and developing specific polymer matrix compositions
N Methods of Treatment
It is contemplated that the polypeptides antibodies and other active compounds of the present invention may be used to treat v aπous immune related diseases and conditions such as T cell mediated diseases, including those characterized by infiltration of inflammatory cells into a tissue, stimulation of T-cell proliferation, inhibition of T-cell proliferation, increased or decreased vascular permeability or the inhibition thereof
Exemplary conditions or disorders to be treated with the polypeptides, antibodies and other compounds of the mvention include, but are not limited to systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic mflammatory myopafhies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vascu tis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia paroxysmal nocturnal hemoglobinuπa), autoimmune thrombocytopenia (idiopathic thrombocytopemc purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephπtis, tubulointerstitial nephntis), demyelmating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelmating polyneuropathy or Guillam-Bane syndrome, and chronic inflammatory demyelmating polyneuropathy, hepatobi ary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non- hepatotropic vimses), autoimmune chronic active hepatitis, primary biliary cinhosis, granulomatous hepatitis, and sclerosmg cholangitis, mflammatory bowel disease (ulcerative colitis Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skm diseases, erythema multiforme and contact dermatitis, psonasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, rmmunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases mcludmg graft rejection and graft -versus-host-disease
In systemic lupus erythematosus, the central mediator of disease is the production of auto-reactive antibodies to self proteins/tissues and the subsequent generation of immune-mediated inflammation antibodies either directly or indirectly mediate tissue injury Though T lymphocytes have not been shown to be directly involved in tissue damage, T lymphocytes are required for the development of auto-reactive antibodies The genesis of the disease is thus T lymphocyte dependent Multiple organs and systems are affected clinically mcludmg kidney, lung, musculoskeletal system, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood
Rheumatoid arthntis (RA) is a chronic systemic autoimmune inflammatory disease that mainly mvolves the synovial membrane of multiple joints with resultant injury to the articular cartilage The pathogenesis is T lymphocyte dependent and is associated with the production of rheumatoid factors, auto- antibodies directed against self IgG, with the resultant formation of immune complexes that attam high levels in joint fluid and blood These complexes the jomt may induce the marked infiltrate of lymphocytes and monocytes into the synovium and subsequent marked synovial changes, the joint space/fluid if infiltrated by similar cells with the addition of numerous neutrophils Tissues affected are pnmaπly the joints, often in symmetncal pattern However, extra- articular disease also occurs in two major forms One form is the development of extra-articular lesions w ith ongoing progressive joint disease and typical lesions of pulmonary fibrosis, vascuhtis and cutaneous ulcers The second form of extra-articular disease is the so called Felty's syndrome which occurs late in the RA disease course sometimes after joint disease has become quiescent, and involves the presence of neutropema thrombocytopenia and splenomegaly This can be accompanied by vascuhtis in multiple organs vv ith formations of infarcts, skm ulcers and gangrene Patients often also develop rheumatoid nodules in the subcutis tissue overlying affected jomts the nodules late stage have necrotic centers sunounded by a mixed inflammatory cell infiltrate Other manifestations which can occur in RA include pencarditis, pleuπtis coronarv arteπtis, intestinal pneumomtis with pulmonary fibrosis, keratoconjunctivitis sicca, and rhematoid nodules Juvenile chronic arthritis is a chronic idiopathic inflammatory disease which begins often at less than
16 years of age Its phenotvpe has some similarities to RA, some patients which are rhematoid factor positive are classified as juvenile rheumatoid arthritis The disease is sub-classified into three major categories pauciarticular, polyarticular and systemic The arthritis can be severe and is typically destructive and leads to joint ankylosis and retarded growth Other manifestations can include chronic anterior uveitis and systemic amyloidosis
Spondyloarthropathies are a group of disorders with some common clinical features and the common association with the expression of HLA-B27 gene product The disorders include ankylosing spony tis, Reiter's syndrome (reactive arthntis), arthntis associated with inflammatory bowel disease, spondylitis associated with psonasis, juvenile onset spondyloarthropathy and undifferentiated spondyloarthropathy Distmguishing features mclude sacroileitis with or without spondylitis, inflammatory asymmetric arthritis, association with HLA-B27 (a serologically defined allele of the HLA-B locus of class I MHC), ocular mflammation, and absence of autoantibodies associated with other rheumatoid disease The cell most implicated as key to mduction of the disease is the CD8+ T lymphocyte, a cell which targets antigen presented by class I MHC molecules CD8+ T cells may react against the class I MHC allele HLA-B27 as if it were a foreign peptide expressed bv MHC class I molecules It has been hypothesized that an epitope of HLA-B27 may mimic a bactenal or other microbial antigemc epitope and thus induce a CD8+ T cells response
Systemic sclerosis (scleroderma) has an unknown etiology A hallmark of the disease is induration of the skm, likely this is induced by an active inflammatory process Scleroderma can be localized or systemic, vascular lesions are common and endothelial cell injury in the microvasculature is an early and important event m the development of systemic sclerosis, the vascular injury may be immune mediated An immunologic basis is implied by the presence of mononuclear cell infiltrates m the cutaneous lesions and the presence of anti- nuclear antibodies m many patients ICAM-1 is often upregulated on the cell surface of fibroblasts in skm lesions suggestmg that T cell interaction with these cells may have a role in the pathogenesis of the disease Other organs involved mclude the gastrointestinal tract smooth muscle atrophy and fibrosis resulting in abnormal penstalsis/motilitv kidney concentnc subendothelial intimal proliferation affectmg small arcuate and interlobular artenes with resultant reduced renal cortical blood flow, results in proteinuna, azotemia and hypertension, skeletal muscle atrophy, interstitial fibrosis, mflammation, lung interstitial pneumomtis and interstitial fibrosis, and heart contraction band necrosis, scarnng/fibrosis
Idiopathic inflammatory myopathies mcludmg dermatomyositis, polymyositis and others are disorders of chronic muscle mflammation of unknown etiology resulting in muscle weakness Muscle injury/inflammation is often svmmetnc and progressive Autoantibodies are associated with most forms These myositis-specific autoantibodies are directed against and inhibit the function of components, proteins and RNAs, involved in protein synthesis
Sjogren's syndrome is due to immune mediated inflammation and subsequent functional destruction of the tear glands and sa varv glands The disease can be associated with or accompanied by inflammatory connective tissue diseases The disease is associated with autoantibody production against Ro and La antigens, both of which are small RN A-protem complexes Lesions result in keratoconjunctivitis sicca, xerostomia, with other manifestations or associations including bilary cirrhosis, peripheral or sensory neuropathy, and palpable purpura Systemic vascuhtis are diseases in hich the primary lesion is inflammation and subsequent damage to blood vessels which results in ischemia/necrosis/degeneration to tissues supplied by the affected vessels and eventual end-organ dysfunction in some cases Vascuhtides can also occur as a secondary lesion or sequelae to other immune-inflammatory mediated diseases such as rheumatoid arthritis, systemic sclerosis, etc , particularly in diseases also associated with the formation of immune complexes Diseases in the primary systemic vascuhtis group include systemic necrotizmg vascuhtis polyarteritis nodosa allergic angiitis and granulomatosis, polyangntis Vv egener's granulomatosis, lymphomatoid granulomatosis, and giant cell arteπtis Miscellaneous vascuhtides include mucocutaneous lymph node syndrome (MLΝS or Kawasaki's disease), isolated CΝS vascuhtis, Behet's disease, thromboangntis ob terans (Buerger's disease) and cutaneous necrotizmg venu tis The pathogenic mechanism of most of the types of vascuhtis listed is believed to be pnmanly due to the deposition of immunoglobulin complexes m the vessel wall and subsequent induction of an mflammatory response either via ADCC, complement activation, or both
Sarcoidosis is a condition of unknown etiology which is characteπzed by the presence of epithe oid granulomas in nearly any tissue in the body, involvement of the lung is most common The pathogenesis mvolves the persistence of activated macrophages and lymphoid cells at sites of the disease with subsequent chronic sequelae resultant from the release of locally and systemically active products released by these cell types
Autoimmune hemolytic anemia mcludmg autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal noctural hemoglobinuna is a result of production of antibodies that react with antigens expressed on the surface of red blood cells (and m some cases other blood cells including platelets as well) and is a reflection of the removal of those antibody coated cells via complement mediated lysis and/or ADCC/Fc-receptor- mediated mechanisms
In autoimmune thrombocytopenia including thrombocytopenic purpura, and immune-mediated thrombocytopenia in other clinical settings, platelet destruction/removal occurs as a result of either antibody or complement attaching to platelets and subsequent removal by complement lysis, ADCC or FC-receptor mediated mechanisms
Thyroiditis mcludmg Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis, are the result of an autoimmune response against thyroid antigens with production of antibodies that react with proteins present in and often specific for the thyroid gland Expenmental models exist mcludmg spontaneous models rats (BUF and BB rats) and chickens (obese chicken stram), inducible models immunization of animals with either thyroglobuhn, thyroid microsomal antigen (thyroid peroxidase) Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune destmction of pancreatic islet β cells, this destmction is mediated by auto-antibodies and auto-reactive T cells Antibodies to insulin or the insulin receptor can also produce the phenotype of lnsuhn-non-responsiveness
Immune mediated renal diseases, including glomeralonephπtis and tubulomterstitial nephritis, are the result of antibody or T lymphocv te mediated injury to renal tissue either directly as a result of the production of autoreactive antibodies or T cells against renal antigens or indirectly as a result of the deposition of antibodies and/or immune complexes in the kidney that are reactive against other, non-renal antigens Thus other immune- mediated diseases that result in the formation of immune-complexes can also induce immune mediated renal disease as an indirect sequelae Both direct and indirect immune mechanisms result in inflammatory response that produces/induces lesion development in renal tissues with resultant organ function impairment and m some cases progression to renal failure Both humoral and cellular immune mechanisms can be involved in the pathogenesis of lesions
Demyelmating diseases of the central and peripheral nervous systems, including Multiple Sclerosis, idiopathic demyelmating polv neuropathy or Guillam-Baπe syndrome, and Chronic Inflammatory Demyelmating Polyneuropathy are believed to have an autoimmune basis and result m nerve demyelmation as a result of damage caused to ohgodendrocytes or to myehn directly In MS there is evidence to suggest that disease induction and progression is dependent on T lymphocytes Multiple Sclerosis is a demyelmating disease that is T lymphocyte-dependent and has either a relapsing-remitting course or a chronic progressive course The etiology is unknown, however viral infections, genetic predisposition, environment, and autoimmumty all contnbute Lesions contain mfiltrates of predommantly T lymphocyte mediated, microglial cells and infiltrating macrophages, CD4+T lymphocytes are the predominant cell type at lesions The mechanism of ohgodendrocyte cell death and subsequent demyelmation is not known but is likely T lymphocyte driven
Inflammatory and Fibrotic Lung Disease, including Eosinophihc Pneumonias, Idiopathic Pulmonary Fibrosis and Hypersensitivity Pneumomtis may involve a deregulated immune- inflammatory response Inhibition of that response would be of therapeutic benefit
Autoimmune or Immune-mediated Skin Disease including Bullous Skin Diseases, Erythema Multiforme, and Contact Dermatitis are mediated by auto-antibodies, the genesis of which is T lymphocyte- dependent
Psonasis is a T lymphocyte-mediated mflammatory disease Lesions contain mfiltrates of T lymphocytes, macrophages and antigen processmg cells, and some neutrophils
Allergic diseases, mcludmg asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, and urticaπa are T lymphocyte dependent These diseases are predominantly mediated by T lymphocyte induced mflammation, IgE mediated-inflammation or a combination of both
Transplantation associated diseases, mcludmg Graft rejection and Graft- Versus-Host-Disease (GVHD) are T lymphocyte-dependent, inhibition of T lymphocyte function is ameliorative
Other diseases in which intervention of the immune and or inflammatory response have benefit are mfectious disease including but not limited to viral infection (including but not limited to AIDS, hepatitis A, B, C, D, E and herpes) bactenal mfection fungal mfections, and protozoal and parasitic infections (molecules (or denvatives/agonists) which stimulate the MLR can be utilized therapeutically to enhance the immune response to mfectious agents), diseases of immunodeficiency (molecules/denvatives/agomsts) which stimulate the MLR can be utilized therapeutically to enhance the immune response for conditions of inherited, acquired, infectious induced (as in HIV infection) or latrogenic (; e as from chemotherapy) immunodeficiency), and neoplasia
It has been demonstrated that some human cancer patients develop an antibody and/or T lymphocyte response to antigens on neoplastic cells It has also been shown in animal models of neoplasia that enhancement of the immune response can result in rejection or regression of that particular neoplasm Molecules that enhance the T lymphocyte response in the MLR have utility in vivo in enhancing the immune response against neoplasia Molecules which enhance the T lymphocyte prohferative response in the MLR (or small molecule agonists or antibodies that affected the same receptor in an agonistic fashion) can be used therapeutically to treat cancer Molecules that inhibit the lymphocyte response in the MLR also function in \ιvo during neoplasia to suppress the immune response to a neoplasm, such molecules can either be expressed by the neoplastic cells themselves or their expression can be induced by the neoplasm in other cells Antagonism of such inhibitory molecules (either with antibody, small molecule antagonists or other means) enhances immune-mediated tumor rejection
Additionally, inhibition of molecules with proinflammatory properties may have therapeutic benefit in reperfusion injury, stroke, myocardial infarction, atherosclerosis, acute lung injury, hemonhagic shock, burn, sepsis/septic shock, acute tubular necrosis, endometnosis, degenerative joint disease and pancreatis
The compounds of the present invention, e g , polypeptides or antibodies, are administered to a mammal, preferably a human m accord with known methods, such as intrav enous administration as a bolus or by contmuous infusion over a penod of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, lntra-articular intrasynovial, intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes Intravenous or inhaled administration of polypeptides and antibodies is prefened
In immunoadjuvant therapy, other therapeutic regimens, such administration of an anti-cancer agent, may be combined with the administration of the protems, antibodies or compounds of the instant invention For example, the patient to be treated with a the immunoadjuvant of the invention may also receive an anti-cancer agent (chemotherapeutic agent) or radiation therapy Preparation and dosing schedules for such chemotherapeutic agents may be used accordmg to manufacturers' instructions or as determmed empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed , M C Perry, Williams & Wilkins, Baltimore, MD (1992) The chemotherapeutic agent may precede, or follow administration of the immunoadjuvant or may be given simultaneously therewith Additionally, an anti-oestrogen compound such as tamoxifen or an anti-progesterone such as onapπstone (see, EP 616812) may be given m dosages known for such molecules
It may be desirable to also admmister antibodies agamst other immune disease associated or tumor associated antigens, such as antibodies which bmd to CD20, CDl la, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF) Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herem may be coadmmistered to the patient Sometimes, it may be beneficial to also admmister one or more cytokines to the patient In one embodiment, the PRO polypeptides are coadmmistered with a growth inhibitory agent For example, the growth inhibitory agent may be admmistered first, followed by a PRO polypeptide However, simultaneous administration or administration first is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the PRO polypeptide For the treatment or reduction in the seventy of immune related disease, the appropriate dosage of an a compound of the invention will depend on the type of disease to be treated as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician The compound is suitablv administered to the patient at one time or over a series of treatments For example, depending on the type and severity of the disease, about 1 μig/kg to 15 mg/kg (e g , 0 1 -20 mg/kg) of polypeptide or antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays O Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing mateπals (e g , comprising a PRO molecule) useful for the diagnosis or treatment of the disorders described above is provided The article of manufacture comprises a container and an instruction Suitable contamers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a variety of materials such as glass or plastic The contamer holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent in the composition is usually a polypeptide or an antibody of the invention An instruction or label on, or associated with, the container mdicates that the composition is used for diagnosmg or treating the condition of choice The article of manufacture may further compnse a second contamer compnsmg a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, mcludmg other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use
P Diagnosis and Prognosis of Immune Related Disease
Cell surface proteins, such as proteins which are overexpressed in certain immune related diseases, are excellent targets for dmg candidates or disease treatment The same proteins along with secreted proteins encoded by the genes amplified in immune related disease states find additional use m the diagnosis and prognosis of these diseases For example, antibodies directed against the protein products of genes amplified in multiple sclerosis, rheumatoid arthritis, or another immune related disease, can be used as diagnostics or prognostics
For example, antibodies, including antibody fragments, can be used to qualitatively or quantitatively detect the expression of protems encoded by amplified or overexpressed genes ("marker gene products") The antibody preferably is equipped with a detectable, e g , fluorescent label, and bindmg can be monitored by light microscopy, flow cytometry, fluonmetry, or other techniques known in the art These techniques are particularly suitable, if the overexpressed gene encodes a cell surface protein Such bmdmg assays are performed essentially as described above
In situ detection of antibody bmdmg to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy For this purpose, a histological specimen is removed from the patient, and a labeled antibodv is applied to it, preferably by overlaying the antibody on a biological sample This procedure also allows for determining the distribution of the marker gene product in the tissue examined It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection 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 refened to in the examples were used according to manufacturer's mstmctions 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 Unless otherwise noted the present invention uses standard procedures of recombinant DNA technology, such as those described heremabove and in the following textbooks Sambrook et al , Molecular Cloning A Laboratory Manual, Cold Sprmg Harbor Press N Y , 1989, Ausubel et al , Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N Y , 1989, Innis et al , PCR Protocols A Guide to Methods and Applications. Academic Press, mc , N Y , 1990, Harlow et al , Antibodies A Laboratory Manual, Cold Sprmg Harbor Press, Cold Sprmg Harbor, 1988, Gait, M J , Oligonucleotide Synthesis, IRL Press, Oxford, 1984, R I Freshney, Animal Cell Culture, 1987, Coligan et al , Current Protocols in Immunology, 1991
EXAMPLE 1 Isolation of cDNA clones Encodmg Human PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940, PRO6006 polypeptide
Various techniques were employed for isolating the cDNA clones descnbed below A general descnption of the methods employed follows immediately hereafter, whereas the details relating the specific sequences isolated is recited separately for each native sequence It is understood that the actual sequences of the PRO polypeptides are those which are contained within or encoded by the clone deposited with the ATCC - and that m the in event of any discrepancy between the sequence deposited and the sequence disclosed herem, the sequence of the deposit is the true sequence ECD Homology 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 mcluded public EST databases (e g , GenBank), a pnvate EST database (LIFESEQ* Incyte Pharmaceuticals, Palo Alto, CA), and propnetary ESTs from Genentech The search was performed usmg the computer program BLAST or BLAST2 [Altschul et al , Methods in Enzymology, 266 460-480 (1996)] as a companson of the ECD protem sequences to a 6 frame translation of the EST sequences Those compansons resulting in a BLAST score of 70 (or in some cases, 90) or greater that did not encode known protems were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)
Using various ESTs, drawing from both public and private databases, a consensus DNA sequence was assembled Oligonucleotides were then synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone encoding the particular native sequence PRO polypeptide identified herein
In order to screen several libraries for a source of a full-length, native sequence clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified below A positive library was then used to isolate clones encoding the particular native sequence PRO polypeptide using the probe oligonucleotide and one of the PCR primers
RNA for construction of the cDΝA libraries was isolated from various human tissue libraries, mcludmg, e , fetal lung, fetal liver, fetal brain, small intestine, smooth muscle cells, etc The cDΝA libraries used to isolated the cDΝA clones were constmcted by standard methods using commercially available reagents such as those from Invitrogen. San Diego, CA The cDΝA was primed with oligo dT containing a ΝotI site, linked with blunt to Sail hemikinased adaptors, cleaved with ΝotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)) in the unique Xhol and ΝotI sites The clones were sequenced using known and readily available methodology Amylase yeast screen
1 Preparation of oligo dT primed cDΝA library mRΝA was isolated from various tissues (e g , such as those indicated above under the ECD homology procedure) using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2) This RΝA was used to generate an oligo dT pπmed cDΝA library in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System) In this procedure, the double stranded cDΝA was sized to greater than 1000 bp and the Sall/Νotl nkered cDΝA was cloned into Xhol/Νotl cleaved vector pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restnction enzyme site preceding the Xhol/Νotl cDΝA cloning sites 2. Preparation of random primed cDΝA library A secondary cDΝA library was generated in order to preferentially represent the 5' ends of the primary cDΝA clones Sp6 RΝA was generated from the pnmary library (described above), and this RΝA was used to generate a random pπmed cDΝA library m the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Scπpt Plasmid System, referenced above) In this procedure the double stranded cDΝA was sized to 500-1000 bp, linkered with blunt to ΝotI adaptors, cleaved with Sfil, and cloned mto Sfil/Νotl cleaved vector pSST-AMY 0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDΝA 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, cDΝAs cloned mto this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropnately transfected yeast colonies 3 Transformation and Detection DNA from the hbrarv described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml) The bacteπa 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 ampicilhn and incubated foi 16 hours (37°C) Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet usmg standard protocols, e g , Cisco- gradient The purified DNA w as then earned 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. hιs3-l l, hιs3-15, MAL+, SUC~, GAL+ Preferably, yeast mutants can be employed that have deficient post-translational pathways Such mutants may have translocation deficient alleles in seel I, sec!2, sec62, with tmncated seclλ being most prefened Alternatively, antagonists (including antisense nucleotides and or ligands) which interfere with the normal operation of these genes, other protems implicated in this post translation pathway (e g , SEC61p, SEC72p, SEC62p, SEC63ρ, TDJlp or SSAlp-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 mto YEPD complex media broth (100 ml) and grown overnight at 30°C The YEPD broth was prepared as described m Kaiser et al , Methods in Yeast Genetics, Cold Sprmg Harbor Press, Cold Spnng Harbor, NY, p 207 (1994) The overnight culture was then diluted to about 2 x IO6 cells/ml (approx OD6oo = 0 1) into fresh YEPD broth (500 ml) and regrown to 1 x 107 cells/ml (approx OD600=0 4-0 5)
The cells were then harvested and prepared for transformation by transfer mto GS3 rotor bottles in a
Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended mto stenle water, and centrifuged again in 50 ml falcon tubes at 3,500 m m a Beckman GS-6KR centnfuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM Tns-HCI, 1 mM EDTA pH 7 5, 100 mM Lι20OCCH3), 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 bnefly by vortexing, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tns-HCI, 1 mM EDTA, 100 mM Lι2Ac, pH 7 5) was added This mixture was gently mixed and incubated at 30°C while agitatmg for 30 mmutes 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 Tns-HCI, 1 mM EDTA pH 7 5) followed by recentnfugation The cells were then diluted into TE (1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared m 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, NY, 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 ah, 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.
Isolation and sequencing by standard techniques identified a yeast EST fragment which served as the basis for additional database mining as described below. 4. Assembly
The yeast EST fragment identified above was used to search various expressed sequence tag (EST ) databases. The EST databases included public EST databases (e.g., GenBank, Merck Wash U) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul 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 sequence. Those comparisons resulting in 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, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using phrap. The consensus DNA sequence was extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above as well as EST sequences proprietary to Genentech.
Based on this consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone encoding the particular PRO polypeptide. 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.
RNA for constmction of the cDΝA libraries was isolated from various human tissues. The cDΝA libraries used to isolate the cDΝA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDΝA was primed with oligo dT containing a ΝotI site, linked with blunt to Sail hemikinased adaptors, cleaved with ΝotI, 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 Sfil site Holmes et al Science, 253 1278-1280 (1991)) in the unique Xhol and Notl sites Signal algoi ithm A proprietary signal sequence finding algorithm developed by Genentech, Ine was used upon
Expressed Sequence Tags (ESTs) and on clustered and assembled EST fragments from public (e g GenBank) and/or private (Lifeseq* , Incvte Pharmaceuticals, Ine , Palo Alto, C A) databases The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides sunoundmg 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 conespondmg amino acid sequences sunoundmg the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals
The above procedure resulted in the identification of EST sequences which were compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g GenBank) and a propnetary EST DNA database (LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA) The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al Methods in Enzymology 266 460-480 (1996)) Those compansons resulting m a BLAST score of 70 (or m some cases 90) or greater that did not encode known protems were clustered and assembled into a consensus DNA sequence with the program "phrap" (Phil Green, University of Washington, Seattle, Washington) This resulted in the identification of additional EST sequences which either conesponded to full-length clones, which were examined and sequenced or served as a template for the creation of cloning oligonucleotides which were then used to screen vanous tissue libraries resultmg m isolation of DNA encodmg a native sequence PRO polypeptide A Isolation of cDNA clones Encoding Human PRO 184 (UNO 158)
The cDNA DNA28500 (Figure 1, SEQ ID NO 1) which encodes the PR0184 protein of Figure 2 (SEQ ID NO 2) is publicly available as GenBank accession number Q92914 and is also descnbed in Smallwood, P M et al , Proc Natl Acad Sci US A 93 9850-9857 (1996) The sequence is alternatively known as FGF-11 or FHF-3
The entire nucleotide sequence of DNA28500 is shown in Figure 1 (SEQ ID NO 1) Clone DNA28500 (SEQ ID NO 1) contains a smgle open readmg frame with an apparent translation initiation site at nucleotide positions 731-733 and ending at the stop codon (TGA) at positions 1406-1408 (Fig 1, SEQ ID NO 1), as indicated in bolded underlme The predicted PRO 184 polypeptide precursor of Fig 2 (SEQ ID NO 2) is 225 ammo acids in length, has a calculated molecular weight of 25005 daltons and a pi of 10 14
Additional analysis of the PROl 84 polypeptide of Figure 2 (SEQ ID NO 2) reveals the presence of a a tyrosine kinase phosphorylation site at about amino acid residues 199-207, N-myπstylation sites at about residues 54-60, 89-95 and 131-137, HBGF/FGF family protein domains at about residues 80-96, 104-155, 171-198 and a fibroblast growth factor domain at about residues 71-200 B Isolation of cDNA clones Encoding Human PRQ212 (UNQ186) Use of the ECD homology procedure described above from a human fetal lung library resulted in the identification of the full-length DNA sequence for DNA30942-1 134 (Fig 3, SEQ ID NO 3) and the derived PR0212 protein sequence of Fig 4 (SEQ ID NO 4)
The PCR primers (forw ard and reverse) and probes used in the procedure were the following forward primer 5*-CACGCTGGTTTCTGCTTGGAG-3' (SEQ ID NO 5) reverse primer 5'-AGCTGGTGCACAGGGTGTCATG-3' (SEQ ID NO 6) hybridization probe (SEQ ID NO 7)
5'-CCCAGGCACCTTCTCAGCCAGCCAGCAGCTCCAGCTCAGAGCAGTGCCAGCCC-3'
The entire nucleotide sequence of DNA30942-1134 is shown m Figure 3 (SEQ ID NO 3) Clone DNA30942-1134 (SEQ ID NO 3) contains a single open reading frame with an apparent translation mitiation site at nucleotide positions 101-103 and ending at the stop codon (TGA) at positions 1001-1003 (Fig 3, SEQ ID NO 3), as indicated in bolded underline The predicted PR0212 polypeptide precursor of Fig 4 (SEQ ID NO 4) is 300 amino acids long, has a calculated molecular weight of 32680 daltons and a pi of 8 70 It is believed that the PR0212 sequence of Fig 4 (SEQ ID NO 4) lacks a transmembrane domam It is also believed that amino acids 1 to 215 of Fig 4 (SEQ ID NO 4) represents an ECD which includes four cysteine rich domains (CRDs) A cDNA clone containing DNA30942-1134 (SEQ ID NO 3) has been deposited with ATCC on September 16, 1997 and has been assigned ATCC deposit no 209254
Analysis of the PR0212 polypeptide of Figure 4 (SEQ ID NO 4) reveals the presence of a signal sequence at ammo acid residues 1 to about 23, an N-glycosylation site at about residues 173-177, cAMP-and cGMP-dependent protem kinase phosphorylation sites at about residues 63-67 and 259-263, tyrosine kmase phosphorylation site at about residues 28-37, N-myπstoylation sites at about residues 156-162, 178-184, 207- 213, 266-272 and 287-293, a TNFR NGFR family cysteine-πch region protein domam at about residues 48-60, 149-161 and 168-175 and a death domain protein profile domam at residues 141-157 C Isolation of cDNA Clones Encoding Human PRQ245 (UNQ219) Use of the ECD homology procedure descnbed above m a human fetal liver library resulted in the isolation of the full-length DNA sequence for DNA35658-1141 (Figure 5, SEQ ID NO 8) and the denved PR0245 native sequence protem of Figure 6 (SEQ ID NO 9)
The PCR primers (forward and reverse) and hybridization probes synthesized for use with the above- descnbed method were the following forward PCR nπmer 5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ ID NO 10) reverse PCR primer 5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID NO 11) hybridization probe (SEQ ID NO 12)
5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3'
The entire nucleotide sequence of DNA35638-1141 (SEQ ID NO 8) is shown in Figure 5 Clone DNA35638 contams a smgle open readmg frame with an apparent translation mitiation site at nucleotide positions 89-91 and endmg at the stop codon (TAG) at nucleotide positions 1025-1027 (Fig 5, SEQ ID NO 8) The predicted PR0245 polypeptide precursor of Figure 6 (SEQ ID NO 9) is 312 amino acids long, has a calculated molecular weight of 34,554 daltons and a pi of 9 39 A clone contammg DNA35638-1141 (SEQ ID NO 8) has been deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no 209265 Further analysis of the PR0245 polypeptide of Figure 6 (SEQ ID NO 9) reveals the presence of a signal peptide at amino acid residues 1 to about 20, a transmembrane domam at about amino acid residues 237- 258, an N-glycosylation site at about ammo acid residues 98-102, 187-191 , 236-240 and 277-281, N- myπstoylation sites at about ammo acids residues 82-188, 239-245, 255-261 , 257-263, 305-311, an amidation site at about amino acid residues 226-230 and an immunoglobulin domain at about ammo acid residues 148-216 D Isolation of cDNA clones Encoding Human PRQ266 (UNQ233)
Use of the ECD homology procedure descnbed above m a human fetal brain library resulted in the isolation of the full-length DNA sequence for DNA37150-1178 (Figure 7, SEQ ID NO 13) and the derived PR0266 native sequence protein of Figure 8 (SEQ ID NO 14) The PCR primer (forw ard and reverse) and hybridization probe synthesized were the following forward PCR primer 5'-GTTGGATCTGGGCAACAATAAC-3' (SEQ ID NO 15) reverse PCR primer 5'-ATTGTTGTGCAGGCTGAGTTTAAG-3' (SEQ ID NO 16) hybridization probe 5'-GGTGGCTATACATGGATAGCAATTACCTGGACACGCTGTCCCGGG-3' (SEQ ID NO 17) Clone DNA37150-1 178 (SEQ ID NO 13) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 167- 169 and ending at the stop codon (TAA) at nucleotide positions 2255-2257 (Figure 7) as indicated by bolded underline The predicted PR0266 precursor of Figure 8 (SEQ ID NO 14) is 696 amino acids long, has a calculated molecular weight of 77735 daltons and a pi of 6 36 A cDNA clone including DNA37150-1178 (SEQ ID NO 13) has been deposited with the ATCC on October 17, 1997 and has been assigned ATCC deposit no 209401
Analysis of the PR0266 polypeptide of Figure 8 (SEQ ID NO 14) reveals a signal peptide at about ammo acid residues 1 to about 15, a transmembrane at about residues 616-639, an N-glycosylation site at about residues 18-22, 253-257, 363-367, 416-420, 595-599, 655-659, a cAMP- and cGMP-dependent protein kinase phosphorylation site at about residues 122-126 and 646-650, N-myπstoylation sites at about residues 17-23, 67- 73, 100-106, 302-308, 328-334, 343-349, 354-360, 465-471, 493-499, 598-604, 603-609, a prokaryotic membrane lipoprotein lipid attachment site at about residues 337-348, an arthopod defensis protem domam at about residues 216-222, a leucine πch repeat at about residues 179-199 and a leucme rich repeat C-terminal domain at about residues 212-262 and 529-579 E Isolation of cDNA clones Encoding Human PRO306 (UNQ269) Use of the ECD homology procedure described above m a human fetal kidney library resulted in the isolation of the full-length DNA sequence for DNA39984-1221 (Fig 9, SEQ ID NO 18) and the derived PRO306 native sequence protein of Figure 10 (SEQ ID NO 19)
The PCR primers (forward and reverse) and hybndization probe synthesized were the following forward PCR pnmer ( fl ) 5'-CAGGTCGAACCCAGACCACGATGC-3' (SEQ ID NO 20) forward PCR pnmer ( f2) 5'-GCCACATGGCCCAGCTTG-3' (SEQ ID NO 21) forward PCR pnmer ( f3) 5'-GAGACGGAGGAAGCAGGC-3' (SEQ ID NO 22) forward PCR pnmer ( fla) 5'-GGCCACACTTACAGCTCTG-3' (SEQ ID NO 23) reverse PCR pnmer ( rl) 5'-AGCCGGCTTCTGAGGGCGTCTACC-3' (SEQ ID NO 24) hybndization probe 5'-TGGTGCTGCCGCTGCTGCTCCTGGCCGCGGCAGCCCTGGCCGAAG-3' (SEQ ID NO 25) Clone DNA39984-1221 (SEQ ID NO 18) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 199 201 and ending at the stop codon (TAG) at nucleotide positions 1471-1473 (Figure 9) as indicated by bolded underline The predicted PRO306 polypeptide precursor of Figure 10 (SEQ ID NO 19) is 424 am o acids long, has a calculated molecular weight of 46,832 daltons and a pi of 4 76 (Figure 10) A cDNA clone mcludmg DNA39984- 1221 (SEQ ID NO 18) has been deposited with the ATCC on November 7, 1997 and is assigned ATCC deposit number 209435
Analysis of the PRO306 polypeptide of Figure 10 (SEQ ID NO 19) reveals the presence of a signal sequence at residues 1 to about 22, an N-glycosylation site about residues 225-229, a glycosaminoglycan site at about residues 388-392, a tyrosine kinase phosphorylation site about residues 62-70, N-myπstoylation sites at about residues 28-34, 130-136, 201-207, 226-232, 237-243, 362-368, 372-378 and 387-393, a thyroglobu n type-1 repeat domain about residues 335-348, a kazal serine protease domain at about residues 140-162, an osteonectin domam protein signature at about residues 283 to 317 and a CTF-NF-I protein domain at residues 324-358 F Isolation of cDNA clones Encoding Human PRQ333 (UNQ294) Use of the ECD homology procedure in combmation with an in vivo cloning procedure resulted in the identification of the partial length sequence DNA41374-1312 (SEQ ID NO 26, Figure 11)
Clone DNA41374-1312 (SEQ ID NO 26) contains an incomplete open readmg frame with an apparent translation termination site (i e , stop codon, TGA) at nucleotide residues 1185-1187, as indicated m bolded underlme The predicted partial length PR0333 polypeptide of Figure 12 (SEQ ID NO 27) is 394 amino acids long, a calculate molecular weight of 43,725 daltons and a pi of 8 36
Analysis of the PR0333 (SEQ ID NO 27) polypeptide of Figure 12 reveals a signal sequence at about amino acid residues 1-14, a transmembrane domam at about residues 359-376, N-mynstoylation sites at about ammo acid residues 166-172, 206-212, 217-223, 246-252, 308-314, 312-318, 361-367 and an immunoglobulin and major histocompatibility complex protems signature at ammo acid residues 315-323. A cDNA clone contammg DNA41374-1312 has been deposited with the ATCC on and as assigned ATCC deposit number
G Isolation of cDNA clones Encoding Human PRQ526 (UNQ330)
Use of the ECD homology procedure descnbed above m a human fetal liver library resulted m the identification of the full-length DNA sequence DNA44184-1319 (Fig 13, SEQ ID NO 28) and the denved PR0526 native sequence protem of Figure 14 (SEQ ID NO 29)
The PCR primers (forward and reverse) and hybridization probes synthesized were the following forward PCR primer 5'-TGGCTGCCCTGCAGTACCTCTACC-3' (SEQ ID NO 30) reverse PCR pnmer 5'-CCCTGCAGGTCATTGGCAGCTAGG-3' (SEQ ID NO 31 ) hybndization probe (SEQ ID NO 32) 5'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCACAC-3'
Clone DNA44184-1319 (SEQ ID NO 28) contains a smgle open readmg frame with an apparent translation initiation site at nucleotide positions 514-516 and ending at the stop codon (TGA) at nucleotide positions 1933-1935 (Figure 13), as indicated by bolded underline The predicted PR0526 polypeptide precursor of Figure 14 (SEQ ID NO 29) is 473 ammo acids long The PR0526 (SEQ ID NO 29) protem shown in Figure 14 has an estimated molecular weight of about 50708 daltons and a pi of about 9 28 A cDNA clone containmg DNA44184-1319 has been deposited with the ATCC on 26 March 1998 and is assigned deposit number 209704
Analysis of the PR0526 polypeptide of Figure 14 (SEQ ID NO 29) reveals that the signal peptide sequence is at about ammo acids 1-26 A leucine zipper pattern is at about ammo acids 135-156 A glycosaminoglycan attachment is at about ammo acids 436-439 N-glycosylation sites are at about ammo acids 82-85, 179-182, 237-240 and 423-426 A von Willebrand factor (VWF) type C domam(s) is found at about amino acids 411-425 The skilled artisan can understand which nucleotides conespond to these amino acids based on the sequences provided herem H Isolation of cDNA clones Encoding Human PRQ381 (UNQ322) Use of the ECD homology procedure described above m a human fetal kidney library resulted in the identification of the full length DNA sequence DNA44194-1317 (Fig 15, SEQ ID NO 33) and the derived PR0381 native sequence protein of Figure 16 (SEQ ID NO 34)
The forward and reverse PCR primers and the hybridization probe used were the following Forward PCR primer (39651 fl) (SEQ ID NO 35) 5'-CTTTCCTTGCTTCAGCAACATGAGGC-3'
Reverse PCR primer (39651 rl ) (SEQ ID NO 36)
5'-GCCCAGAGCAGGAGGAATGATGAGC-3' hybndization probe (39651 pi) (SEQ ID NO 37)
5'-GTGGAACGCGGTCTTGACTCTGTTCGTCACTTCTTTGATTGGGGCTTTG-3' Clone DNA44194-1317 (SEQ ID NO 33) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 174-176 and ending at the stop codon (TAG) at nucleotide positions 807-809 (Fig 15), as indicated by bolded underline The predicted PR0381 polypeptide precursor of Figure 16 (SEQ ID NO 34) is 211 amino acids long, has a calculated molecular weight of 24,172 daltons and has a pi of 5 99 The PR0381 (SEQ ID NO 34) protem shown in Figure 16 has the following features a signal peptide from about ammo acid residues 1 to about 20, a potential N-glycosylation site at about ammo acid residue 156, potential casern kinase phosphorylation sites from about ammo acid residues 143 to about 146, about residues 156 to about 159, about residues 178 to about 181, about residues 200 to about 203, an endoplasmic reticulum targeting sequence from about amino acid residues 78 to about 114 and from about residues 118 to about 131, EF-hand calcium bmdmg domain from about ammo acid residues 140 to about 159, and an S-100/ICaBP type calcium bmdmg domam from about amino acid residues 183 to about 203 A cDNA clone containmg DNA44194-1317 (SEQ ID NO 33) has been deposited with the ATCC on Apnl 28, 1998 and is assigned deposit number 209808 I Isolation of cDNA clones Encoding Human PRQ364 (UNQ 19)
Use of the ECD homology procedure descnbed above m a human small mtestine library resulted m the identification of an expressed sequence tag (EST) (Incyte EST No 3003460) that encoded a polypeptide which showed homology to members of the tumor necrosis factor receptor (T FR) family of polypeptides
A consensus DNA sequence was then assembled relative to the Incyte 3003460 EST m a manner similar to that used in the ECD homology procedure which resulted in the isolation of the full-length DNA sequence DNA47365-1206 (Fig 17, SEQ ID NO 38) and the derived PR0364 native sequence protem of Figure 18 (SEQ ID NO 39) The PCR primers (forward and reverse) and hybridization probes synthesized for use in the above- described screening procedure were forward PCR primer (44825 f 1 ) 5'-CACAGCACGGGGCGATGGG-3' (SEQ ID NO 40) forward PCR primer (44825 f2) 5'-GCTCTGCGTTCTGCTCTG-3' (SEQ ID NO 4 ] ) forward PCR primer (44825 GITR f)
5'-GGCACAGCACGGGGCGATGGGCGCGTTT-3' (SEQ ID NO 42) reverse PCR primer (44825 rl ) 5'-CTGGTCACTGCCACCTTCCTGCAC-3' (SEQ ID NO 43) reverse PCR primer (44825 r2) 5'-CGCTGACCCAGGCTGAG-3' (SEQ ID NO 44) reverse PCR primer (44825 GITR r) 5'-GAAGGTCCCCGAGGCACAGTCGATACA-3' (SEQ ID NO 45) hybridization probe (44825 pi)
5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3' (SEQ ID NO 46) hybndization probe (44825 GITR p) 5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO 47) Clone DNA47365-1206 (SEQ ID NO 38) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 121-123 and ending at the stop codon (TGA) at nucleotide positions 844-846 (Figure 17). as indicated by bolded underline The predicted PR0364 polypeptide precursor of Figure 18 (SEQ ID NO 39) is 241 amino acids long The PR0364 (SEQ ID NO 39) protem shown m Figure 18 has an estimated molecular weight of about 26,000 daltons and a pi of about 6 34 A potential N- glycosylation sites exists between amino acids 146 and 149 of the amino acid sequence shown in Figure 18 A putative signal sequence is from ammo acids 1 to 25 and a potential transmembrane domain exists between amino acids 162 to 180 of the sequence shown in Figure 18 A cDNA clone containing DNA47365-1206 has been deposited with ATCC on November 7, 1997 and is assigned ATCC Deposit No ATCC 209436 J Isolation of cDNA clones Encoding Human PRQ356 (UNQ313)(NL4) An expressed sequence tag (EST) DNA database (LIFESEQ0*, Incyte Pharmaceuticals, Palo Alto, CA) was searched and an EST (#2939340) was identified which showed homology to human TIE-2 LI and TIE-2 L2
Based on the EST, a pair of PCR pnmers (forward and reverse), and a probe were synthesized NL4,5-1 5'-TTCAGCACCAAGGACAAGGACAATGACAACT-3' (SEQ ID NO 50) NL4,3-1 5'-TGTGCACACTTGTCCAAGCAGTTGTCATTGTC-3' (SEQ ID NO 51)
NL4,3-3 5'-GTAGTACACTCCATTGAGGTTGG-3' (SEQ ID NO 52)
Oligo dT pruned cDNA hbranes were prepared from uterus mRNA purchased from Clontech, Ine (Palo Alto, CA, USA, catalog # 6537-1) m the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System) pRK5D is a cloning vector that has an sp6 transcnption mitiation site followed by an Sfil restriction enzyme site precedmg the Xhol Notl cDNA cloning sites The cDNA was pnmed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized to greater than 1000 bp appropπately by gel electrophoresis, and cloned in a defined orientation mto XhoI/Notl-cleaved pRK5D In order to screen sev eral libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encodmg the PR0356 gene using the probe oligonucleotide and one of the PCR pnmers
DNA sequencing of the clones isolated as described above gave a full-length DNA sequence DNA47470-1130 (SEQ ID NO 48) and the derived PR0356 protem (SEQ ID NO 49) shown m Figure 20
The entire nucleotide sequence of DNA47470-1130 is shown m Figure 19 (SEQ ID NO 48) Clone DNA47470-1130 (SEQ ID NO 48) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 215-217, and a TAA stop codon at nucleotide positions 1038-1040, as indicated by bolded underline The predicted PR0356 polypeptide shown in Figure 20 is 346 ammo acids long (SEQ ID NO 49), has a calculated molecular weight of 40,018 daltons and a pi of 8 19 A cDNA clone containing DNA47470-1130 (SEQ ID NO 48) has been deposited with ATCC on October 28, 1997 and is assigned ATCC deposit no 209422
Further analysis of the PR0356 polypeptide of Figure 20 (SEQ ID NO 49) reveals a signal peptide at amino acid residues 1 to about 26, N-glycosylation sites at about residues 58-62, 253-257 and 267-271, glycosyaminoglycan attachment sites at residues 167-171, a cAMP- and cGMP-dependent protein kinase phosphorylation site at about residues 176-180, N-myπstoylation sites at about residues 168-174, 196-202, 241- 247, 252-258, 256-262, 327-333, a cell attachment sequence at about residues 199-202, and fibnnogen beta and gamma chains C-terminal domain proteins at about residues 160-198, 201-210, 219-256, 266-279, 283-313 K Isolation of cDNA clones Encoding Human PRQ719 (UNQ387) Use of the ECD homology procedure identified above in a human placenta tissue library resulted m the isolation of the full-length DNA sequence DNA49646-1327 (Fig 21, SEQ ID NO 53) and the deπved PR0719 native sequence protein of Figure 22 (SEQ ID NO 54)
The PCR primers (forward and reverse) and hybndization probe synthesized were forward PCR pnmer (44851 fl) 5'-GTGAGCATGAGCGAGCCGTCCAC-3' (SEQ ID NO 55) reverse PCR pnmer (44851 rl) 5'-GCTATTACAACGGTTCTTGCGGCAGC-3' (SEQ ID NO 56) hybndization probe (44851 pi) (SEQ ID NO 57)
5'-TTGACTCTCTGGTGAATCAGGACAAGCCGAGTTTTGCCTTCCAG-3'
Clone DNA49646-1327 (Fig 21, SEQ ID NO 53) contams a single open readmg frame with an apparent translation initiation site at nucleotide positions 223-225 and ending at the stop codon (TGA) at nucleotide positions 1285-1287 (Figure 21), as mdicated by bolded underline The predicted PR0719 polypeptide precursor of Figure 22 (SEQ ID NO 54) is 354 ammo acids long, has an estimated molecular weight of about 39,362 daltons and a pi of about 8 35 A clone containing DNA49646-1327 (SEQ ID NO 53) has been deposited with the ATCC on March 26, 1998 (under the designation DNA49646-1327) and has been assigned ATCC deposit number 209705 Analysis of the PR0719 protem sequence of Figure 22 (SEQ ID NO 54) reveals a signal peptide at ammo acid residues 1 to about 16, a hpase/senne active site at about residues 163-173, N-glycosylation sites at about residues 80-84 and 136-140, a cAMP- and cGMP-dependent protem kinase phosphorylation site at about residues 206-210 and 329-333 and N-myπstoylation sites at about residues 63-69, 96-102, 171-177, 191-197, 227-233, 251-157, 306-312 and 346-352 L Isolation of cDNA clones Encoding Human PRQ861 (UNQ423) The cDNA DNA50798 (Figure 23, SEQ ID NO 58) which encodes the PR0861 protein of Figure 24
(SEQ ID NO 59) is publicly available as GenBank accession number P22692 and is also described in Latour, D et al , Mol Endoci inol 4 1806-1814 (1990), Shimasaki, S et al Mol Endocronol 4 1451-1458 (1990),
Kiefer, M C et al J Biol Chem 266 9043-9049 (1991), Culouscou et al Cancer Res 51 2813-2819 (1991) The sequence is alternatively known as insulin-like growth factor binding protein 4 precursor
The entire nucleotide sequence of DNA50798 is shown in Figure 23 (SEQ ID NO 58) Clone
DNA50798 contains a single open reading frame with an apparent translation initiation site at nucleotide positions 265-267 and ending at the stop codon (TGA) at positions 1039-1041 (Fig 23, SEQ ID NO 58), as indicated in bolded underline The predicted PR0861 polypeptide precursor of Fig 24 (SEQ ID NO 59) is 258 ammo acids in length, has a calculated molecular weight of 27934 daltons and a pi of 7 23
Additional analysis of the PR0861 polypeptide of Figure 24 reveals the presence of a signal sequence at ammo acid residues 1 to about 21, an N-glycosylation site at about residues 125-129, a tyrosine kinase phosphorylation site at about residues 191-198, N-myπstoylation sites at about residues 52-58, 54-60, 64-70, 96-102 and 172-178, insulin-like growth factor binding protem domams at about residues 46-80, 201-229 and thyroglobuhn type-1 repeat domains at residues 52-100, 202-215 and 220-231 M Isolation of cDNA clones Encoding Mouse PRQ769 (UNQ407)
A public expressed sequence tag (EST) DNA databases (Merck/Washington University) was searched with the full-length murine m-FIZZl DNA (DNA 53517) cDNA and the EST W42069 was identified
The full-length clones coπesponding to the EST fragment W42069 was obtained from Incyte Pharmaceuticals (Palo Alto, California), and sequenced m the entirety, which ultimately resulted m the identification of the full length nucleotide sequence DNA54231-1366-1 (SEQ ID NO 60)
The nucleotide sequence conesponding to the full length, native sequence PR0769 clone is shown m Figure 25 This clone, designated DNA 54231-1366-1 (SEQ ID NO 60) contains a smgle open reading frame with an apparent translation mitiation site at nucleotide positions 75-77 and ending at the stop codon (TGA) at residues 417-419, as indicated by bolded underline (Fig 25) The predicted PR0769 polypeptide precursor (mcludmg a signal sequence of 10 ammo acιds)(SEQ ID NO 61) is 114 amino acids long, has a calculated molecular weight of 12,492 daltons and a pi of 8 19 Based on its homology to m-FIZZl (34%, using the ALIGN software) the protem was designated m-FIZZ3 A clone containing DNA54231-1366-1 (designated DNA54231-1366-1) has been deposited with ATCC on Apnl 23, 1998 and has been assigned ATCC deposit no 209804
Additional analysis of the PR0769 polypeptide of Figure 26 (SEQ ID NO 61) reveals the presence of a signal peptide at ammo acid residues 1 to about 20, a leucme zipper pattern at about residues 4-25, an N- glycosylation site at about residues 3-6 and a DNA polymerase family B protein domam at residues 39-48 Identification and clonmg of m-FIZZl (DNA53517) Mouse asthma model Female Balb/C mice, 6 to 8 weeks of age, were separated into two experimental groups controls and asthmatics The asthmatic group was immunized rntrapeπtoneally with 10 μg ovalbumin + 1 mg alum, while the control group was not Two weeks later, mice were exposed daily to an aerosol of 10 mg/ml ovalbumin in PBS aerosolized with a UltraNeb nebulizer (DeVilbiss) at the rate of 2 ml/mm for 30 mm each day, for 7 consecutive days One day after the last aerosol challenge, whole blood, serum and bronchoalveolar lav age (B AL) samples were collected and the lungs were harvested and preserved for histological examination lmmuno-histochemistry and in situ hybridization
Gel electrophoresis ofBAL sαwip/esExammation of the BAL samples by gel electrophoresis on a 16%
Tπcine gel shows that a low molecular weight protein is expressed in the BAL samples from asthmatic mice but not in the BAL samples from control mice This low molecular weight protem was termed m-FIZZl and was seen to co-migrate with a 8300 Dalton marker protein
Partial protein sequence The protein of interest was transfened upon a PVDF membrane and sequenced by Edman degradation This sequence served as a template for the preparation of various cloning o gos as described below Partial cDNA sequence We designed two degenerate oligonucleotide PCR primers conesponding to the putative DNA sequence for the first 7 and the last 7 ammo acids of the partial protem sequence
O go #1
5'-ACA AAC GCG TGA YGA RAC NAT HGA RAT-3' (SEQ ID NO 62)
Ohgo # 2 5'-TGG TGC ATG CGG RTA RTT NGC NGG RTT-3' (SEQ ID NO 63) cDNA prepared from the lungs of normal mice was used as a template for the PCR reaction which yielded an 88 bp product This 88 bp product contained 54 known base pairs, encoding the PCR primers, and 34 novel base pairs, and encoded another partial mFIZZ-1 sequence
Full length cDNA clone This second partial sequence was used to design primers which were ultimately successful in obtaining the full length FIZZ clone (DNA53517) by RT-PCR of mouse lung poly(A)+
RNA
Ohgo #3
5'-ACA AAC GCG TGC TGG AGA ATA AGG TCA AGG-3' (SEQ ID NO 64)
This ohgo was used as an RT-PCR primer in combination with 5' and 3' amp mers from Clontech Ohgo #4
5'-ACT AAC GCG TAG GCT AAG GAA CTT CTT GCC-3' (SEQ ID NO 65)
This ohgo was used as an RT-PCR pnmer in combmation with ohgo d(T)
N Isolation of cDNA clones Encoding Human PRQ788 (UNO430)
Use of the ECD homology procedure identified above resulted in the identification of the partial length EST sequence 2777282 Further analysis of the conesponding full-length sequence resulted m the identification of DNA56405-1357 (SEQ ID NO 66) and the derived native sequence PR0788 protein (SEQ ID NO 67) of
Figure 28
Clone DNA56405-1357 (SEQ ID NO 66) contains a single open reading frame with an apparent translation mitiation site at nucleotide positions 84-86 and endmg at the stop codon (TAG) at nucleotide positions 459-461 (Figure 27), as mdicated by bolded underline The predicted native sequence PR0788 polypeptide precursor (SEQ ID NO 67) is 125 amino acids long (Figure 28), has a calculated molecular weight of 13,115 daltons and a pi of 5 90 The PR0788 (SEQ ID NO 67) protem shown in Figure 28 has an estimated molecular weight of about 13115 and a pi of about 5 90 A clone containmg DNA56405-1357 (SEQ ID NO 66) has been deposited with the ATCC on May 6, 1998 and has been assigned deposit number 209849 In the event of a discrepancy m the nucleotide sequence of the deposit and the sequences disclosed herem, it is understood that the deposited clone contains the conect sequence It is further understood that the methodology of sequencing for the sequences provided herem are based on known sequencing techniques
Analysis of the PR0788 polypeptide (SEQ ID NO 67) shown in Figure 28 reveals a signal peptide at about amino acids 1-17 and an N-glycosylation site is at about amino acid 46 O Isolation of cDNA clones Encoding Human PRQ826 (UNQ467)
Use of the signal algorithm procedure described above resulted in the identification of an EST cluster sequence 47283 This sequence was then compared to a variety of various EST databases as described under the signal algorithm procedure above, and further resulted in the identification of Merck EST sequence W69233 Further exammation and sequencmg of the full-length clone conesponding to this EST sequence resulted in the isolation of the full-length DNA sequence DNA57694-1341 (Fig 29, SEQ ID NO 68) and the derived PR0826 native sequence protein of Figure 30 (SEQ ID NO 69)
Clone DNA57694-1341 (SEQ ID NO 68) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 13-15 and ending at the stop codon (TGA) at nucleotide positions 310-312 (Figure 29), as indicated by bolded underline The predicted PR0826 polypeptide precursor of Figure 30 (SEQ ID NO 69) is 99 amino acids long The PR0826 (SEQ ID NO 69) protein shown m Figure 30 has an estimated molecular weight of about 1 1050 daltons and a pi of about 747
Analysis of the full-length PR0826 sequence shown in Figure 30 (SEQ ID NO 69) evidences the presence of the following a signal peptide from about ammo acid 1 to about amino acid 22, potential N- mynstoylation sites from about ammo acid 22 to about ammo acid 27 and from about ammo acid 90 to about ammo acid 95 and an ammo acid sequence block having homology to peroxidase from about amino acid 16 to about ammo acid 48
A cDNA clone containing DNA57694-1341 has been deposited with ATCC on June 23, 1998 and is assigned ATCC deposit no 203017 P Isolation of cDNA clones Encoding Human PRQ982 (UNQ483) Use of the signal algorithm procedure descnbed above resulted in the identification of an EST cluster sequence no 43715 This sequence was then compared to a vanety of various EST databases as descnbed under the signal algonfhm procedure above, and further resulted in the identification of Merck EST No AA024389 Further exammation and sequencmg of the full-length clone conesponding to this EST resulted in the identification of the full-length sequence DNA57700-1408 (Fig 31, SEQ ID NO 70) and the derived PR0982 native sequence protem of Figure 32 (SEQ ID NO 71)
The DNA57700-1408 sequence of Figure 31 (SEQ ID NO 70) contams a smgle open reading frame with an apparent translation mitiation site at nucleotide positions 26-28 and ending at the stop codon (TAA) at nucleotide positions 401-403, as mdicated by bolded underline The prediced PR0982 polypeptide precursor of Figure 32 (SEQ ID NO 71) is 125 ammo acids in length, has a calculated molecular weight of approximately 14,198 daltons and an estimated pi of approximately 9 01 Further analysis of the PR0982 (SEQ ID NO 71) polypeptide of Figure 32 reveals a signal peptide from about amino acid residues 1 to about 21, N- mynstoylation sites at about residues 33-39 and 70-76 and a potential anaphylatoxin domam from about ammo acid residue 1 to about residue 59 A cDNA clone contammg DNA57700-1408 (SEQ ID NO 70) was deposited with the ATCC on January 12, 1999 under the designation DNA57700-1408 and is assigned ATCC deposit No 203583 Q Isolation of cDNA clones Encoding Human PRQ779 (UNQ455)(Apo-3)
Human fetal heart and human fetal lung lgtlO bacteπophage cDNA libraries (both purchased from Clontech) were screened by hybridization with synthetic oligonucleotide probes based on an EST (Genbank locus W71984), which showed some degree of homology to the intracellular domain (ICD) of human TNFR1 and CD95 W71984 is a 523 bp EST, which in its -1 reading frame has 27 identities to a 43 ammo acid long sequence in the ICD of human TNFR1 The oligonucleotide probes used in the screening were 27 and 25 bp long, respectively, with the following sequences 5'-GGCGCTCTGGTGGCCCTTGCAGAAGCC-3' (SEQ ID NO 74) 5'-TTCGGCCGAGAAGTTGAGAAATGTC-3' (SEQ ID NO 75)
Hybridization was done with a 1 1 mixture of the two probes overnight at room temperature in buffer containing 20% formamide, 5X SSC, 10% dextran sulfate, 0 1% NaPιP04, 0 05 M NaP04, 0 05 mg salmon sperm DNA, and 0 1% sodium dodecyl sulfate (SDS), followed consecutively by one wash at room temperature m 6X SSC, two washes at 37°C in IX SSC/0 1% SDS, two washes at 37°C m 0 5X SSC/0 1% SDS, and two washes at 37°C in 0 2X SSC/0 1% SDS One positive clone from each of the fetal heart (FH20A 57) and fetal lung (FL8A 53) libraries were confirmed to be specific by PCR using the respective above hybridization probes as pnmers Single phage plaques containing each of the positive clones were isolated by limiting dilution and the DNA was purified usmg a Wizard lambda prep DNA purification kit (Promega)
The cDNA inserts were excised from the lambda vector arms by digestion with EcoRI, gel-punfied, and subcloned mto pRK5 that was predigested with EcoRI The clones were then sequenced in entirety resultmg in the isolation and identification of the full-length clone DNA58801-1052 (Fig 33, SEQ ID NO 72), alternatively refened to as clone FH20 57, and the derived PRO770 native sequence protein of Figure 34 (SEQ ID NO 73)
Clone FH20A 57 (also refened to as Apo 3 clone FH20 57 deposited as ATCC 55820, as indicated below) contams a single open reading frame with an apparent translational initiation site at nucleotide positions 103-105 and ending at the stop codon (TGA) at nucleotide positions 1354-1356 (Figure 33), as mdicated by bolded underline The predicted PR0779 polypeptide precursor of Figure 34 (SEQ ID NO 73) is 417 amino acids long and has a calculated molecular weight of about 45385 daltons and a pi of about 6 4
A cDNA clone containing DNA58801-1052 (SEQ ID NO 72) has been deposited with the ATCC under the designation FH20 57 on Sept 5, 1996 and has been assigned ATCC deposit No 55820 In the event of a discrepancy between the sequence disclosed herem and the sequence of the deposit, it is understood that the deposited clone contams the conect sequence, and that the sequences provided herein are provided using known sequencmg techniques
Additional analysis of the PR0779 polypeptide of Figure 34 (SEQ ID NO 73) reveals a signal peptide at ammo acid residues 1 to about 24, a transmembrane domain at about residues 199-218, N-glycosylation site at about residues 67-71 and 106-110, cAMP- and cGMP-dependent protein kinase phosphorylation sites at about residues 157-161, a tyrosine kinase phosphorylation site at about residues 370-377, N-myπstoylation sites at about residues 44-50, 50-56, 66-72, 116-122, 217-223, 355-361, 391-397, 401-407, prokaryotic membrane lipoprotein lipid attachment site at about residues 177-188, a death domain at about residues 333-413 and a TNFR/NGFR family cysteine-nch region protem domain at residues 47-59 R Isolation of cDNA clones Encoding Human PRO 1068 (UNQ525)
Use of the signal algorithm procedure described above resulted in the identification of the Incyte cluster sequence no 141736 This sequence was then compared to a variety of various EST databases described under signal algorithm procedure above, and further resulted in the identification of Incyte EST clone no 004974 The full-length clone conesponding to this EST resulted in the identification of the full-length sequence DNA59214-1449 (Fig 35, SEQ ID NO 76) and the derived PRO 1068 native sequence protem of Figure 36 (SEQ ID NO 77)
The DNA59214-1449 sequence of Figure 35 (SEQ ID NO 76) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 42-44 and ending at the stop codon (TGA) at nucleotide positions 414-416, as indicated by bolded underline The predicted PRO1068 polypeptide precursor of Figure 36 (SEQ ID NO 77) is 124 amino acids long, has a calculated molecular weight of about 14,284 Daltons and an estimated pi of approximately 8 14
Further analysis of the PRO 1068 (SEQ ID NO 77) polypeptide of Figure 36 reveals a signal peptide from about ammo acid residues 1 to about 20, a urotension II signature sequence at about amino acids 118-124, a cell attachment sequence at about ammo acids 64-67, and a potential cAMP- and cGMP-dependent protein kinase phosphorylation site at about ammo acids 112-116 and N-myπstoylation sites at about residues 61-67 and 92-98 A cDNA clone containing DNA59214-1449 (SEQ ID NO 76) was deposited with the ATCC on July 1, 1998 under the designation DNA59214-1449 and is assigned ATCC deposit No 203046 S Isolation of cDNA clones Encodmg Human PRO 1031 (UNQ516) Use of the ECD homology procedure descnbed above resulted in the identification of the EST sequence Merck W74558 (clone 344649) The conesponding full-length clone was exammed and sequenced resultmg m the isolation of DNA sequencing gave the full-length DNA sequence DNA59294-1381 (Fig 37, SEQ ID NO 78) and the deπved PRO1031 native sequence protein of Figure 38 (SEQ ID NO 79)
Clone DNA59294-1381 (SEQ ID NO 78) contams a smgle open reading frame with an apparent translation mitiation site at nucleotide positions 42-44 and endmg at the stop codon (TGA) at nucleotide positions 582-584 (Figure 37), as mdicated by bolded underline The predicted PRO1031 polypeptide precursor of Figure 38 (SEQ ID NO 79) is 180 ammo acids long The PRO1031 protein (SEQ ID NO 79) shown m Figure 38 has an estimated molecular weight of about 20437 and a pi of about 9 58 A cDNA clone contammg DNA59294-1381 (SEQ ID NO 78) has been deposited with the ATCC on May 14, 1998 under the designation DNA59294-1381 and has been assigned deposit number 209866 In the event of a discrepancy between the sequence as disclosed herem and the sequence of the deposit, it is understood that the deposited clone contams the conect sequence, and the sequences provided herein were produced usmg known sequencmg techniques
Analysis of the ammo acid sequence of the PRO 1031 protem of Figure 38 (SEQ ID NO 79) reveals the putative signal peptide at about ammo acid residues 1-20, an N-glycosylation site is at about ammo acid residue 75-79 A region havmg sequence identity with IL-17 is at about ammo acid residues 96-180 T Isolation of cDNA clones Encodmg Human PROl 157 (U Q587)
Use of the signal algonthm procedure descnbed above resulted m the identification of an Incyte EST cluse sequence from the LIFESEQ® database, designated 65816 This sequence was then compared to a vanety of vanous EST databases descnbed under the signal algonthm procedure above, and further resulted m the identification of Merck EST No AA516481 Further examination and sequencmg of the full-length clone conesponding to this EST (No 955952) resulted in the identification of the full-length sequence DNA60292- 1506 (Fig 39, SEQ ID NO 80) and the deπved PROl 157 native sequence protein of Figure 40 (SEQ ID NO 81)
The cDNA DNA60292-1506 (SEQ ID NO 80) sequence shown m Figure 39 contams a smgle open reading frame with an apparent translation initiation site at nucleotide positions 56-58 and endmg at the stop codon (TGA) at nucleotide positions 332-334, as indicated by bolded underline The predicted PROl 157 polypeptide precursor shown in Figure 40 (SEQ ID NO 81) is 92 amino acids long, has a calculated molecular weight of approximately 9,360 Daltons and an estimated pi of approximately 9 17
Further analysis of the PROl 157 (SEQ ID NO 81) sequence reveals a signal peptide at amino acid residues 1 to about 18, a transmembrane domam at about residues 51-70, a glycosaminoglycan attachment site at about residues 40-44, N-myπstoylation sites at about residues 34-40, 37-43, 52-58 and a prokaryotic membrane lipoprotein lipid attachment site at about residues 29-40 A cDNA clone contammg DNA60292- 1506 (SEQ ID NO 80) was deposited with the ATCC on December 15, 1998 under the designation DNA60292- 1506 and has been assigned deposit number 203540 U Isolation of cDNA clones Encoding Human PROl 159 (UNQ589)
Use of the signal algonthm procedure described above resulted in the identification of EST cluster sequence 77245, which was then compared to a vanety of vanous EST databases as descnbed under the signal algonthm procedure above, and further resulted in the identification of Incyte EST no 376776 Analysis of the full-length clone conespondmg to this EST resulted in the identification of the full-length sequence DNA60627- 1508 (Fig 41, SEQ ID NO 82) and the denved PROl 159 native sequence protem of Figure 42 (SEQ ID NO 83)
Clone DNA60627-1508 (SEQ ID NO 82) contains a smgle open readmg frame with an apparent translation mitiation site at nucleotide positions 92-94 and endmg at the stop codon (TAG) at nucleotide positions 362-364 (Figure 41), as mdicated by bolded underline The predicted PROl 159 polypeptide precursor of Figure 42 (SEQ ID NO 83) is 90 ammo acids long The PROl 159 (SEQ ID NO 83) protem shown in Figure 42 has an estimated molecular weight of about 9,840 daltons and a pi of about 10 13
Analysis of the PROl 159 (SEQ ID NO 83) sequence shown in Figure 42 evidences the presence of the following a signal peptide from about ammo acid residue 1 to about residue 15 and a potential N-glycosylation site at about ammo acid residue 38 Clone DNA60627-1508 (SEQ ID NO 82) has been deposited with ATCC on August 4, 1998 and is assigned ATCC deposit no 203092
V Isolation of cDNA clones Encodmg Human PRO 1475 (UNQ746)
Use of the ECD homology procedure descnbed above m a human fetal bram tissue library resulted m the isolation of the full-length DNA sequence DNA61185-1646 (Fig 43, SEQ ID NO 84) and the denved PR01475 native sequence protem of Figure 44 (SEQ ID NO 85) The PCR pnmers (forward and reverse) and hybndization probe synthesized were forward PCR primer (45639 fl) 5'-GATGGCAAAACGTGTGTTTGACACG-3' (SEQ ID NO 86) forward PCR pnmer (45639 f2) 5'-CCTCAACCAGGCCACGGGCCAC-3' (SEQ ID NO 87) reverse PCR pnmer (45639 rl) 5'-CCCAGGCAGAGATGCAGTACAGGC-3' (SEQ ID NO 88) reverse PCR pnmer (45639 r2) 5'-CCTCCAGTAGGTGGATGGATTGGCTC-3' (SEQ ID NO 89) hybndization probe (45639 pi) (SEQ ID NO 90)
5'-CTCACCTCATGAGGATGAGGCCATGGTGCTATTCCTCAACATGGTAG-3' Clone DNA61185-1646 (Fig 43, SEQ ID NO 84) contams a single open reading frame with an apparent translation initiation site at nucleotide positions 130-132 and endmg at the stop codon (TGA) at nucleotide positions 21 10-2112 (Figure 43), as indicated by bolded underline The predicted PRO 1475 polypeptide precursor of Figure 44 (SEQ ID NO 85) is 660 amino acids long The PR01475 protein (SEQ ID NO 85) shown in Figure 44 has an estimated molecular weight of 75,220 Daltons and a pi of about 6 76 A clone containmg DNA61185-1646 (SEQ ID NO 84) has been deposited with the ATCC on November 17, 1998 (under the designation DNA61185-1646) and has been assigned ATCC deposit number 203464 Analysis of the PRO 1475 protein sequence of Figure 44 (SEQ ID NO 85) reveals a transmembrane domain at about ammo acid residues 38-55, N-myπstoylation sites at about residues 276-282, 309-315, 505-511, 606-612, amidation site at about residues 213-217 and a region homologous region to Mouse GNTl at about residues 229-660 W Isolation of cDNA clones Encoding Human PRO 1271 (UNQ641) Use of the signal algonthm procedure descnbed above resulted in the identification of an EST cluster sequence 312 This sequence was then compared to a vanety of various EST as described under the signal algonthm procedure above, and further resulted in the identification of the EST Merck AA625350 Further examination and sequence of the full-length clone conesponding to this EST sequence (clone 1047230) resulted m the isolation of the full-length DNA sequence DNA66309-1538-1 (Fig 45, SEQ ID NO 91) and the denved PR01271 native sequence protein of Figure 46 (SEQ ID NO 92)
The full-length clone shown in Figure 45 (DNA66309, SEQ ID NO 91) contams a smgle open readmg frame with an apparent translation initiation site at nucleotide positions 94-96 and endmg at the stop codon (TAA) at nucleotide positions 718-720, as indicated by bolded underline The predicted PRO 1271 polypeptide precursor of Figure 46 (SEQ ID NO 92) is 208 ammo acids long, has a calculated molecular weight of about 21,531 Daltons and an estimated pi of approximately 8 99 A cDNA clone contammg DNA66309-1538-1 (SEQ ID NO 91) was deposited with the ATCC on September 15, 1998 and is assigned ATCC deposit No 203235 Additional analysis of the PRO 1271 (SEQ ID NO 92) polypeptide reveals a signal peptide at ammo acid residues 1 to about 31, a transmembrane domain at about residues 166-187, N-glycosylation sites at about residues 46-50, 50-54, 64-68, 68-72, 83-87, 96-100, 106-110, 124-128, 138-142 and N-mynstoylation sites at about residues 4-10, 7-13, 42-48, 101-107, 167-173, 172-178
X Isolation of cDNA clones Encoding Human PRO 1343 (UNQ698)
Use of the amylase yeast screen procedure descnbed above on tissue isolated from human smooth muscle cell tissue resulted m an EST sequence which served as the template for the creation of the oligonucleotides below and screening as descnbed above m a human smooth muscle cell tissue library resulted m the isolation of the full length DNA sequence DNA66675-1587 (Fig 47, SEQ ID NO 93) and the denved PR01343 native sequence protem of Figure 48 (SEQ ID NO 94)
The oligonucleotide probes employed were as follows forward PCR pnmer (48921 fl) 5'-CAATATGCATCTTGCACGTCTGG-3' (SEQ ID NO 95) reverse PCR pnmer (48921 rl) 5'-AAGCTTCTCTGCTTCCTTTCCTGC-3' (SEQ ID NO 96) hybridization probe (48921 pi)
5'-TGACCCCATTGAGAAGGTCATTGAAGGGATCAACCGAGGGCTG-3' (SEQ ID NO 97)
The full length clone DNA66675-1587 (SEQ ID NO 93) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 71-73, and a stop signal (TAA) at nucleotide positions 812-814 (Figure 47), as indicated by bolded underline The predicted PRO 1343 polypeptide precursor of Figure
48 (SEQ ID NO 94) is 247 ammo acids long, has a calculated molecular weight of approximately 25,335 daltons and an estimated pi of approximately 7 0
Further analysis of the PROl 343 sequence shown in Figure 48 (SEQ ID NO 94) evidences the presence of the following a signal peptide from about amino acid 1 to about ammo acid 25, N-myπstolation sites at residues 17-23, 35-41, 39-45, 53-59, 57-63, 76-82, 89-95, 104-110, 118-124, 140-146, 152-158, 154-
160, 172-178, 190-196, 204-210, 215-221, 225-231 and a homologous region to circumsporozoite repeats from about amino acid 35 to about ammo acid 225 A cDNA clone containing DNA66675-1587 (SEQ ID NO 93), has been deposited with ATCC on September 22, 1998 and is assigned ATCC deposit no 203282
Alternatively, a comparison of the yeast EST sequence isolated from the amylase screen above was screened against vaπous EST databases, both public and pnvate (e g , see ECD homology procedure, above) resultmg m the identification of Incyte EST clone no 4701148 Further analysis and sequencing of the conespondmg full-length clone resulted in isolation of the DNA66675-1587 sequence (SEQ ID NO 93) shown in Figure 47
Y Isolation of cDNA clones Encodmg Human PRQ1375 (UNQ712) Use of the ECD homology procedure descnbe above in a human pancreas library resulted in the identification of the GenBank sequence AA143093 The conesponding full-length clone was examined and sequenced resultmg in the isolation of the full-length DNA sequence DNA67004-1614 (Fig 49, SEQ ID NO 98) and the deπved PR01375 native sequence protem of Figure 50 (SEQ ID NO 99)
Clone DNA67004-1614 (SEQ ID NO 98) contains a single open reading frame with an apparent translation mitiation site at nucleotide positions 104-106 and ending at the stop codon (TAA) at nucleotide positions 698-700 (Figure 49), as indicated by bolded underline The predicted PRO 1375 polypeptide precursor of Figur 49 (SEQ ID NO 99) is 198 amino acids long The PR01375 protein (SEQ ID NO 99) shown in Figure
50 has an estimated molecular weight of 22,531 and a pi of about 8 47 A cDNA clone containmg DNA67004-
1614 (SEQ ID NO 98) has been deposited with the ATCC on August 11, 1998 under the designation DNA67004-1614 and is assigned deposit number 203115 In the event of a discrepancy between the sequence as disclosed herem and the sequence of the deposit, it is understood that the deposited clone contains the conect sequence, and the sequences provided herem were produced using known sequencing techniques
Analysis of the ammo acid sequence of PRO 1375 polypeptide (SEQ ID NO 99) reveals transmembrane domams at about ammo acid residues 11-28 (type II) and at about residues 103-125, N-glycosylation site at about residues 60-64, a tyrosine kmase phosphorylation site at about residues 78-86 and an N-mynstoylation site at about residues 12-18
Z Isolation of cDNA clones Encoding Human PRQ1418 (UNQ732)
Use of the signal algonthm procedure described above resulted m the identification of an EST cluster sequence 10698 (Incyte cluster 121480) This sequence was then compared to a vanety of vanous EST databases (mcludmg those denved from a placenta tissue library) as descnbed under the signal algonthm procedure above, and further resulted in the identification of Incyte EST1306026 Further examination and sequencing of the full-length clone conesponding to this EST sequence resulted in the isolation of the full- length DNA sequence DNA68864-1629 (Fig 51, SEQ ID NO 100) and the deπved PR01418 native sequence protein of Figure 52 (SEQ ID NO 101) The full length clone shown in Figure 51 (DNA68864-1629, SEQ ID NO 100) contains a single open reading frame with an apparent translation initiation site at nucleotide positions 138-140 and ending at the stop codon (TAA) found at nucleotide positions 1 188-1190, as indicated by bolded underline The predicted PR01418 polypeptide precursor of Figure 52 (SEQ ID NO 101) is 350 ammo acids long with a signal peptide at about amino acids 1-19, a calculated molecular weight of approximately 39003 daltons, an estimated pi of approximately 5 59, N-glycosylation sites at about ammo acids 128-132, N-myπstoylation sites at about residues 189-195 and an amidation site at about residues 110-114 A cDNA clone containmg DNA68864-1629 (SEQ ID NO 184) was deposited with the ATCC on September 22, 1998 and is assigned ATCC deposit no 203276 AA Isolation of cDNA clones Encoding Human PRO 1474 (UNQ745) Use of the ECD homology procedure described above resulted in the identification of the Incyte EST sequence 1843692 Further analysis and sequencmg of the conesponding full-length clone resulted in the isolation of the full-length DNA sequence DNA73739-1645 (Fig 53, SEQ ID NO 102) and the denved PR01474 native sequence protein of Figure 54 (SEQ ID NO 103)
The full-length clone DNA73739-1645 (SEQ ID NO 102) shown in Figure 53 contains a smgle open readmg frame with an apparent translation mitiation site at nucleotide positions 45-47 and a stop codon (TAA) at nucleotide positions 300-302, as indicated by bolded underline The predicted PR01474 polypeptide precursor of Figure 54 (SEQ ID NO 103) is 85 amino acids long, has a calculated molecular weight of 9232 Daltons and has a pi of about 7 94 A cDNA contammg DNA73739-1645 (SEQ ID NO 102) has been deposited with the ATCC under the designation DNA73739-1645 on September 22, 1998 and is assigned ATCC deposit No 203270
Further analysis of the PR01474 polypeptide (SEQ ID NO 103) of Figure 53 reveals a signal peptide at about amino acids residues 1-19, a kazal serine protease inhibitor family signature at about residues 45-68, a tyrosine kmase phosphorylation site at about residues 28-36 and an integnn alpha cham protein domam at about residues 32-42 AB Isolation of cDNA clones Encoding Human PRQ1917 (UNO900)
Use of the signal algonthm procedure descnbed above resulted in the identification of an EST cluster sequence 85496 This sequence was then compared to a vanety of various EST databases as descnbed under the signal algonthm procedure above, and further resulted in the identification of Incyte EST3255033 This EST was denved from an ovanan tumor library Further examination and sequencmg of the full-length clone conesponding to this EST sequence resulted in the isolation of the full-length DNA sequence DNA76400-2528 (Fig 55, SEQ ID NO 104) and the derived PR01917 native sequence protem of Figure 56 (SEQ ID NO 105)
The full length clone DNA76400-2528 (SEQ ID NO 104) shown in Figure 55 contains a smgle open readmg frame with an apparent translation initiation site at nucleotide positions 6 to 9 and endmg at the stop codon (TGA) found at nucleotide positions 1467 to 1469 as indicated by bolded underline The predicted PR01917 polypeptide precursor of Figure 56 (SEQ ID NO 105) is 487 amino acids long, has a calculated molecular weight of approximately 55,051 daltons and an estimated pi of approximately 8 14 Additional features include a signal peptide at about ammo acid residues 1-30 potential N-glycosylation sites at about amino acid residues 242 and 481, protein kinase C phosphorylation sites at about amino acid residues 95-97, 182-184, and 427-429, N-mynstoylation sites at about amino acid residues 107-112, 1 13-118, 117-122, 1 18- 123, and 128-133, and an endoplasmic reticulum targeting sequence at about amino acid residues 484-487 A cDNA clone containing DNA76400-2528 (SEQ ID NO 104) has been deposited with the ATCC on January 12, 1999 and is assigned ATCC deposit No 203573 AC Isolation of cDNA clones Encoding Human PRQ5723 (1972)
The cDNA DNA82361 (Figure 57, SEQ ID NO 106) which encodes the PR05723 protein of Figure 58 (SEQ ID NO 107) is publicly available as GenBank accession number P78310 and is also descnbed m
Bergelson, J M et al . Science 275 1320-1323 (1997), Tomko, R P et al , Proc Natl Acad Sci USA 94 3352-
3356 (1997) and Bowles, K R. et al Hum Genet 105 354-359 (1999) The sequence is alternatively known as human coxsackievirus and adenovims receptor precursor
The entire nucleotide sequence of DNA82361 is shown in Figure 57 (SEQ ID NO 106) Clone DNA82361 contains a single open reading frame with an apparent translation mitiation site at nucleotide positions 71-73 and endmg at the stop codon (TAA) at positions 1133-1135 (Fig 57 SEQ ID NO 106), as mdicated by bolded underline The predicted PR05723 polypeptide precursor of Fig 58 (SEQ ID NO 107) is
352 ammo acids in length, has a calculated molecular weight 38938 and a pi of 7 86
Additional analysis of the PR05723 polypeptide of Figure 58 (SEQ ID NO 107) reveals the presence of a signal sequence at about amino acid residues 1-19, a transmembrane domain at about ammo acid residues 235-256, N-glycosylation sites at about amino acid residues 106-110, 201-205, 298-302, tyrosine kmase phosphorylation sites at about residues 31-39, 78-85, 263-270, N-myπstoylation sites at about residues 116-122, 208-214, 219-225, 237-243, 241-247, 245-251, 296-302, a myelm P0 protein at about residues 96-125, 229-283 and an immunoglobulin domam at about residues 34-122 and 155-214 AD Isolation of cDNA clones Encoding Human PRO4405 (UNO 1930)
Use of the ECD homology procedure descnbed above m a human fetal kidney library resulted m the isolation of the full-length DNA sequence DNA84920-2614 (Fig 59, SEQ ID NO 108) and the deπved PRO4405 native sequence protem of Figure 60, SEQ ID NO 109)
The PCR pnmers (forward and reverse) and hybndization probe synthesized were forward PCR pnmer 5'-CGGGACTTTCGCTACCTGTTGC-3' (SEQ ID NO 110) reverse PCR pnmer 5'-CATCATATTCCACAAAATGCTTTGGG-3' (SEQ ID NO 111) hybndization probe (SEQ ID NO 112)
5'-CCTTCGGGGATTCTTCCCGGCTCCCGTTCGTTCCTCTG-3' Clone DNA84920-2614 (Fig 59, SEQ ID NO 108) contains a smgle open readmg frame with an apparent translation mitiation site at nucleotide positions 79-81 and endmg at the stop codon (TAG) at nucleotide positions 1009-1011 (Figure 59), as mdicated by bolded underline The predicted PRO4405 polypeptide precursor of Figure 60 (SEQ ID NO 109) is 310 ammo acids long, has an estimated molecular weight of 33,875 Daltons and a pi of about 7 08 A clone contammg DNA84920-2614 (SEQ ID NO 108) has been deposited with the ATCC on Apnl 27, 1999 and has been assigned ATCC deposit number 203966 Further analysis of the PRO4405 polypeptide of Figure 60 (SEQ ID NO 109) reveals a signal peptide at amino acid residues 1 to about 34, a transmembrane domain at about residues 58-76, N-glycosylation sites at about residues 56-60 and 194-198, N-myπstoylation sites at about residues 6-12, 52-58, 100-106, 125-131, 233- 239, 270-276, 275-281, 278-284, an amidation site at residues 154-158 and a cell attachment sequence at residues 205-208
AE Isolation of cDNA clones Encoding Human PRO4302 (UNO 1866)
Use of the amylase screen procedure described above on tissue isolated from human tissue resulted in an EST sequence which was then compared against vaπous EST databases to create a consensus sequence by a methodology as descnbed above under the amylase yeast screen procedure and/or the ECD homology procedure Further analysis of this consensus sequence resulted in the identification of Incyte EST no 2408081H1 Analysis of the full-length clones conesponding to EST no 2408081H1 resulted in the isolation of the full length native sequence clones DNA92218-2554 (Fig 61, SEQ ID NO 113) and the derived PRO4302 full-length native sequence protem of Figure 62 (SEQ ID NO 114)
The full length clone DNA92218-2554 (SEQ ID NO 113) shown in Figure 61 has a single open readmg frame with an apparent translational initiation site at nucleotide positions 174-176 and a stop signal (TAG) at nucleotide positions 768-770, as indicated by bolded underline The predicted PRO4302 polypeptide precursor of Figure 62 (SEQ ID NO 114) is 198 amino acids long, has a calculated molecular weight of approximately 22,285 daltons and an estimated pi of approximately 9 35 Analysis of PRO4302 of Figure 62 (SEQ ID NO 114) reveals a signal peptide from about ammo acid residue 1 to about residue 23, a transmembrane domam from about amino acid residue 1 11 to about residue 130, a cAMP and cGMP-dependent protem kmase phosphorylation sites at residues 26-30, casern kmase II phosphorylation sites at residues 44-47 and 58-61, a tyrosine kinase phosphorylation site at residues 36-43 and N-mynstoylation sites at residues 124- 130, 144-150 and 189-195
A cDNA clone contammg DNA92218-2554 (SEQ ID NO 113) was deposited with the ATCC on March 9, 1999 and has been assigned deposit number 203834
AF Isolation of cDNA clones Encoding Human PRO9940 (UNQ889)
The cDNA DNA92282 (Figure 63, SEQ ID NO 115) which encodes the PRO9940 protem of Figure 63 (SEQ ID NO 115) is publicly available as GenBank accession number NM_013371 or AF192498 The sequence is alternatively known as human IL-19 The entire nucleotide sequence of DNA92282 (SEQ ID NO 115) is shown in Figure 63 (SEQ ID
NO 115) Clone DNA92282 contams a smgle open reading frame with an apparent translation mitiation site at nucleotide positions 33-35 and endmg at the stop codon (TGA) at positions 564-566 (Fig 63, SEQ ID NO 115), as mdicted by bolded underline The predicted PRO9940 polypeptide precursor of Fig 64 (SEQ ID NO 116) is 177 ammo acids m length, has a calculated molecular weight of 20,452 and a pi of 8 00 Additional analysis of the PRO9940 polypeptide of Figure 64 (SEQ ID NO 116) reveals the presence of a signal sequence at about ammo acid residues 1-18, N-glycosylation sites at about residues 56-60, 135-139, cAMP- and cGMP-dependent protein kmase phosphorylation site at about residues 102-106, N-mynstoylation site at about residues 24-30 and an actinin-type actin-bmding domam signature 1 at about residues 159-169 AG Isolation of cDNA clones Encoding Human PRO6006 (UNQ2516)
Use of the ECD homology procedure descnbed above resulted in the isolation of the full-length DNA sequence DNA105782-2693 (Fig 65, SEQ ID NO 117) and the derived PRO6006 native sequence protein of Figure 66 (SEQ ID NO 118) The PCR primer (forward and reverse) and hybridization probe synthesized were forward PCR pnmer (43028 fl) 5'-TGAGCAGGAGTCACAGCACGAAGAC-3' (SEQ ID NO 119) reverse PCR pnmer (43028 rl) 5'-TGAGTTGCATGCTTGAGGGCTGG-3' (SEQ ID NO 120) hybndization probe (43028 pi) (SEQ ID NO 121)
5'-CTCCATCCTGACTGCTCCTCCTAAGAGAGATGGCACCGGCCAGAGCAGGATT-3' Clone DNA105782-2693 (Fig 65, SEQ ID NO 117) contams a smgle open readmg frame with an apparent translation initiation site at nucleotide positions 100-102 and endmg at the stop codon (TAG) at nucleotide positions 568-570 (Figure 65), as indicated by bolded underline The predicted PRO6006 polypeptide precursor shown in Figure 66 (SEQ ID NO 118) is 156 ammo acids long, has a calculated molecular weight of 17,472 Daltons and a pi of 10 01 A clone containing DNA105782-2693 (SEQ ID NO 117) has been deposited with the ATCC on July 20, 1999 and has been assigned ATCC deposit number 387-PTA
Further analysis of the PRO6006 polypeptide of Figure 66 (SEQ ID NO 118) reveals a signal peptide at about amino acid residues 1 to 22, N-glycosylation sites at about residues 127-131, cAMP- and cGMP- dependent protein kmase phosphorylation site at about residues 139-143, N-mynstoylation sites at about residues 18-24, 32-38 and pancreatic nbonuclease family signature domams at about residues 65-72 and 49-93
EXAMPLE 2 Stimulatory Activity m Mixed Lymphocyte Reaction (MLR) Assay (no 24) This example shows that the polypeptides of the mvention are active as a stimulator of the proliferation of stimulated T-lymphocytes Compounds which stimulate proliferation of lymphocytes are useful therapeutically where enhancement of an immune response is beneficial A therapeutic agent may take the form of antagonists of the polypeptide of the invention, for example, murine-human chimenc, humanized or human antibodies agamst the polypeptide
The basic protocol for this assay is descnbed m Current Protocols in Immunology, unit 3 12, edited by J E Coligan, A M Kmisbeek, D H Marghes, E M Shevach, W Strober, National Institutes of Health, Published by John Wiley & Sons, Ine
More specifically, in one assay vanant, penpheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis (one donor will supply stimulator
PBMCs, the other donor will supply responder PBMCs) If desired, the cells are frozen in fetal bovme semm and DMSO after isolation Frozen cells may be thawed overnight m assay media (37°C, 5% CO2 )and then washed and resuspended to 3 x 10" cells/ml of assay media (RPMI, 10% fetal bovme semm, 1% penicillin/streptomycin, 1% glutamme, 1% HEPES, 1% non-essential ammo acids, l% pyruvate)
The stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads) The assay is prepared by platmg m tnp cate wells a mixture of lOOμl of test sample diluted to 1% or to 0 1%, 50 μl of nradiated stimulator cells and 50 μl of responder PBMC cells 100 microhters of cell culture media or 100 micro ter of CD4-IgG is used as the control The wells are then incubated at 37°C 5% CO-> for 4 days On day 5 and each well is pulsed with tπtiated thvmidine (1 0 mC/well, Amersham) After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated
In another variant of this assay, PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice The cells are teased from freshly harvested spleens in assay media (RPMI,10% fetal bovine semm, 1% penicillin/streptomycin 1% glutamme, 1% HEPES, 1% non-essential ammo acids 1% pyravate) and the PBMCs are isolated by overla ing these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to lx 10 ' cells/ml of assav media The assay is then conducted as described above The results of this assay for compounds of the inv ention are shown below Positive increases over control are considered positive with increases of greater than or equal to 180% being prefened However, any value greater than control indicates a stimulatory effect for the test protein
Table 7 PRO PRO Concentration Percent Increase Over Control
PR0861 95 nM 247 2
PR0861 9 5 nM 170 5
PR0788 29 nM 189 9
PR0788 2 9 nM 126
PROl 159 110 55 nM 186 6
PROl 159 11 06 nM 103 1
PR01475 0 07 nM 137 5
PR01475 0 7 nM 243
PR01917 2 15 nM 115 2
PR01917 21 46 nM 196 3
PRO9940 80 15 nM 183 3
PRO9940 8 02 nM 123
PRO9940 8 02 nM 170
PRO9940 80 15 nM 244 6
PRO9940 2 15 nM 115 2
PRO9940 21 46 nM 196 3
PR05723 66 nM 187 8
PR05723 6 6 nM 83 3
PRO6006 26 6 nM 199 9
PRO6006 2 66 nM 138 2
EXAMPLE 3 Inhibitory Activity m Mixed Lymphocyte Reaction (MLR) Assav (no 67) This example shows that one or more of the PRO polypeptides are active as inhibitors of the proliferation of stimulated T-lymphocytes Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an immune response is beneficial
The basic protocol for this assay is described in Current Pr otocols in Immunology, unit 3 12, edited by J E Coligan, A M Kmisbeek D H Marghes, E M Shevach, W Strober National Institutes of Health, Published by John Wiley & Sons Ine More specifically, in one assay variant peripheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis (one donor will supply stimulator PBMCs, the other donor will supply responder PBMCs) If desired, the cells are frozen in fetal bovine seram and DMSO after isolation Frozen cells may be thawed overnight m assay media (37°C, 5% C02) and then washed and resuspended to 3xl06 cells/ml of assay media (RPMI, 10% fetal bovine seram, 1% penicillin streptomycin, 1% glutamme, 1% HEPES, 1% non-essential ammo acids 1% pyravate) The stimulator PBMCs are prepared by inadiating the cells (about 3000 Rads) The assay is prepared by plating in triplicate wells a mixture of 100 1 of test sample diluted to 1% or to 0 1%, 50 1 of madiated stimulator cells, and 50 1 of responder PBMC cells
100 microhters of cell culture media or 100 microliter of CD4-IgG is used as the control The wells are then incubated at 37°C, 5% C02 for 4 days On day 5, each well is pulsed with tπtiated thymidine (1 0 mC/well, Amersham) After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated
In another variant of this assay, PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice The cells are teased from freshly harvested spleens in assay media (RPMI, 10% fetal bovme serum, 1% penicillin/streptomycin, 1% glutamme, 1% HEPES, 1% non-essential amino acids, 1% pyravate) and the PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to lxlO7 cells/ml of assav media The assay is then conducted as described above Any decreases below control is considered to be a positive result for an inhibitory compound, with decreases of less than or equal to 80% bemg prefened However, any value less than control indicates an inhibitory effect for the test protem
Table 8 PRO PRO Concentration Percent Decrease Below Control PROl 84 1 00 % 67 4
PR0184 0 10 % 123 2
PROl 84 0 65 nM 52 8
PROl 84 6 50 nM 51 9
PROl 84 5 34 nM 0 PROl 84 5 34 nM 37 3
PR0184 5 34 nM 50 7
PROl 84 5 34 nM 60 7
PR0184 5 34 nM 77 6
PR0184 5 34 nM 82 7 PRO 184 53 39 nM 0 PR0184 53 39 nM 5 8
PR0184 53 40 nM 5
PR0184 53 40 nM 7 2
PR0184 53 40 nM 10 9 PR0184 53 40 nM 14 1
PRO306 1 64 nM 77 1
PRO306 16 41 nM 63 5
PR0779 4 2 nM 50 4
PR0779 0 42 nM 56 4 PR01271 0 56 nM 27 3
PR01271 5 6 nM 60 3
PR01375 21 5 nM 74 2
PR01375 215 nM 59 9
PR01474 64 nM 22 8 PR01474 6 4 nM 21
EXAMPLE 4 Inhibition of co-stimulation of CD4+ enriched lymphocytes (ASY121) This assay shows that one or more of the PRO polypeptides are active as inhibitors of the stimulation of CD4+ emiched lymphocytes Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an mflammatory immune response is beneficial This assay is a variation of the MLR assay above wherem the PRO polypeptide is exammed for its inhibitory effect upon the co- stimulation of CD4+ enriched lymphocytes with both antι-CD3 and antι-CD28 The inhibition of the stimulatory effect of antι-CD3 and antι-CD28 on PBMCs is proposed to conelate with a general antiproliferative effect similar to the engagement of the TCR with a costimulatory signal
The basic protocol for the isolation of PBMCs used in this assay is descnbed in Current Protocols in Immunology, unit 3 12, edited by J E Coligan, A M Kmisbeek, D H Marghes, E M Shevach, W Strober, National Institutes of Health, Published by John Wiley & Sons, Inc.
More specifically, in one assay vanant, penpheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis Cells are isolated and emiched usmg negative selection If desired, the enπched cells are frozen m 90% fetal bovine serum and 10% DMSO Frozen cells may be thawed overnight m assay media (37°C, 5% C02) and then washed and resuspended to lxlO6 cells/ml of assay media (RPMI, 10%o fetal bovme seram, 1% penicillin/streptomycin, 1% glutamme, 1% HEPES, 1% non-essential ammo acids, 1% pyravate) The assay is prepared by plating m tπp cate wells a mixture of
100 ul of test sample diluted to mdicated concentration lOO ul of cells
50 ul of antι-CD3 (50 ng/ml, Amac 0178) and 50 ul antι-CD28 (100 ng/ml, Biodesign P42235M) is added to a 96 well plate for an overnight coat at 4'C pnor to the addition of cells and test sample 100 microhters of cell buffer control or 100 microhter of Hu-IgG is used as the control in place of the test sample
The wells are then incubated at 37°C, 5% C02 for about 3 days On day 4. each well is pulsed with tritiated thymidine (1 0 mC/ ell Amersham) After 6 hours, the plate is harvested and then the uptake of the label is evaluated
A result which shows an inhibitory effect (i e , 3[H] -thymidine incoφoration) less than 70%> of that observed in the control is considered to be a positive result
In another variant of this assay, CD4+ splenocytes are isolated from the spleens of Balb/c mice The cells are teased from freshlv harvested spleens in assay media (RPMI, 10% fetal bovine serum, 1% penicillin streptomycin, 1%> glutamme, 1% HEPES, 1% non-essential ammo acids 1%> pyravate) and the splenocytes are isolated by ov erlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 m for 20 mmutes, collecting and washing the mononuclear cell layer in assay media, negative selection and resuspendmg the cells to lxl 07 cells/ml of assay media The assay is then conducted as described above
Table 9
PRO concentration inhibition
PR0184 53 39 nM +
PR0212 4 64 nM +
PRO306 16 41 nM + PR0333 21 34 nM +
PR0364 17 nM +
PR0381 16 95 nM +
PR0982 8 44 nM +
PRO1068 144 nM + PROl 157 497 92 nM +
PRO 1343 45 29 nM +
PRO4302 135 57 nM +
PRO4405 0 5 nM +
EXAMPLE 5
Stimulation of penpheral blood mononuclear cells (PBMCs) or CD4+ cells with anti CD3 and PRO protem (ASY99) This assay shows that one or more of the PRO polypeptides are active as enhancers of the stimulation of PBMCs or CD4+ cells CD4+ cells are ennched by negative selection using MACs beads after LSM separation The ability of the PRO polypeptide to replace antι-CD28 is exammed to determine the stimulatory effect
Antι-CD3 and antι-CD28 are known to stimulate PBMCs The basic protocol for the isolation of PBMCs used in this assay is descnbed in Current Protocols in Immunology, unit 3 12, edited by J E Coligan, A M Kmisbeek, D H Marghes, E M Shevach, W Strober, National Institutes of Health, Published by John Wiley & Sons, Ine More specifically, in one assay vanant, penpheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis If desired, the cells are enriched for CD4+ cells, then frozen m 90% fetal bovine semm and 10% DMSO after isolation Frozen cells may be thawed overnight in assay media (37°C 5% C02) and then washed and resuspended to 0 5xl06 cells/ml of assay media (RPMI, 10% fetal bovine semm 1% penicillin/ streptomycin, 1% glutamme, 1% HEPES, 1% non-essential amino acids, 1% pyravate)
The assay is prepared by plating in triplicate wells a mixture of 200 ul of cells after the overnight coat of anti CD3 and SPDI protein
50 ul of antι-CD3 (50 ng/ml, Amac 0178) and 50 ul of 1% of SPDI protein are coated on a 96 well plate in PBS 4'C overnight 50 ul Hu-IgG is used as the control in place of the SPDI protein
The wells are then incubated at 37°C, 5% C02 for about 3 days On day 4, each well is pulsed with tπtiated thymidine (1 0 mC/well Amersham) After 6 hours the cells are harvested and then the uptake of the label is evaluated
A result which indicates a stimulatory effect (i e , 3[H] -thymidine incoφoration) greater than 200% of the control is considered to be positive stimulatory result.
In another variant of this assay, PBMCs or CD4 + splenocytes are isolated from the spleens of Balb/c mice The cells are teased from freshly harvested spleens in assay media (RPMI, 10% fetal bovine seram, 1% penicillin/streptomycin, 1% glutamme, 1% HEPES, 1% non-essential amino acids, 1% pyravate) and the PBMCs are isolated by overlaymg these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 φm for 20 mmutes, collecting and washing the mononuclear cell layer in assay media CD4+ cells are ennched by negative selection using beads, washed m media and resuspended the cells to lxlO7 cells/ml of assay media The assay is then conducted as descnbed above
Table 10
PRO concentration stimulation (+)/ιnhrbιtιon (-)
PR0245 3 5 nM +
PR0266 1 8 nM +
PRO306 5 46 nM +
PR0333 9 2 nM +
PR0356 1 I nM +
PR0364 27 23 nM +
PR0381 145 nM +
PR0526 15 6 nM +
PR0719 1 07 nM +
PR0719 1 07 nM +
PR0769 6 84 nM +
PR0826 9 03 nM +
PRO 1031 5 6 nM +
PRO1069 16 72 nM +
PR01343 176 nM + PRO 1343 176 nM +
PR01375 215 nM +
PR01418 63 98 nM +
EXAMPLE 6
In situ Hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences withm cell or tissue preparations It may be useful, for example, to identify sites of gene expression, analyze the tissue distnbution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping
In situ hybndization w as performed following an optimized version of the protocol by Lu and Gillett,
Cell Vision 1 169-176 (1994) using PCR-generated ^^P-labeled πboprobes Briefly, formalin-fixed, paraffin- embedded human tissues were sectioned, deparaffinized, deprotemated in proteinase K (20 g/ml) for 15 minutes at 37°C, and further processed for in situ hybridization as described by Lu and Gillett, supra A [33P] UTP- labeled antisense πboprobe was generated from a PCR product and hybridized at 55°C overnight The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks p-Rιboprobe synthesis
6 0 μl (125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried To each tube containing dπed33P-UTP, the following ingredients were added 2 0 μl 5x transcription buffer, 1 0 μl DTT (100 mM), 2 0 μl NTP mix (2 5 mM 10 μl, each of 10 mM GTP, CTP & ATP + 10 μl H20), 1 0 μl UTP
(50 μM), 1 0 μl Rnasin, 1 0 μl DNA template (lμg), 1 0 μl H20
The tubes were incubated at 37 °C for one hour 1 0 μL RQ1 DNase were added, followed by incubation at 37°C for 15 mmutes 90 μL TE (10 mM Tπs pH 7 6/lmM EDTA pH 8 0) were added, and the mixture was pipetted onto DE81 paper The remaining solution was loaded m a Mιcrocon-50 ultrafiltration unit, and spun using program 10 (6 mmutes) The filtration unit was inverted over a second tube and spun using program 2 (3 minutes) After the final recovery spin, 100 μL TE were added 1 μL of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II
The probe was ran on a TBE/urea gel 1-3 μL of the probe or 5 μL of RNA Mrk III were added to 3 μL of loading buffer After heating on a 95°C heat block for three minutes, the gel was immediately placed on ice The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 mmutes The gel was wrapped m saran wrap and exposed to XAR film with an intensifying screen m -70°C freezer one hour to overnight
33P-Hvbndιzatιon
Pretreatment of frozen sections The slides were removed from the freezer, placed on alummum trays and thawed at room temperature for 5 minutes The trays were placed m 55 °C incubator for five minutes to reduce condensation The slides were fixed for 10 mmutes in 4% paraformaldehyde on ice in the fume hood, and washed m 0 5 x SSC for 5 mmutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H2O) After deprote nation in 0 5 μg/ml proteinase K for 10 minutes at 37°C (12 5μL of 10 mg/ml stock m 250 ml prewarmed RNase-free RNAse buffer) the sections were washed in 0 5 x SSC for 10 minutes at room temperature The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each
Pr eti eatment of par affin embedded sections The slides were deparaffimzed, placed m SQ H2O, and rinsed twice in 2 x SSC at room temperature, for 5 mmutes each time The sections were deproteinated in 20 μg/ml proteinase K (500 μL of 10 mg/ml in 250 ml RNase-free RNase buffer, 37C, 15 minutes ) - human embryo, or 8 x proteinase K (100 μL in 250 ml Rnase buffer, 37°C, 30 minutes) - formalin tissues Subsequent rinsing in 0 5 x SSC and dehydration were performed as described above
Prehybndization The slides were laid out in plastic box lined with Box buffer (4 x SSC, 50%o formamide) - saturated filter paper The tissue was covered with 50 μL of hybridization buffer (3 75g Dextran Sulfate + 6 ml SQ H2O), vortexed and heated in the microwave for 2 mmutes with the cap loosened After cooling on ice, 18 75 ml formamide, 3 75 ml 20 x SSC and 9 ml SQ H2O were added, the tissue was vortexed well, and incubated at 42 °C for 1-4 hours
Hybridization 1 0 x 10^ cp probe and 1 0 μL RNA (50 mg/ml stock) per slide were heated at 95°C for 3 minutes The slides were cooled on ice, and 48 μL hybridization buffer were added per slide After vortexmg, 50 μL 33P mix were added to 50 μL prehybndization on slide The slides were incubated overnight at 55C
Washes Washing was done 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0 25M EDTA, Vf=4L) followed by RNaseA treatment at 37°C for 30 minutes (500 μL of 10 mg/ml in
250 ml Rnase buffer - 20 μg/ml), The slides were washed 2x10 minutes with 2 x SSC, EDTA at room temperature The stnngency wash conditions were as follows 2 hours at 55°C, 0 1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, V^L)
Alternatively, multi-tissue blots containing poly A RNA (2 μg per lane) from vaπous human tissues were purchased from Clontech (Palo Alto, CA) DNA probes were labeled with [α- ? P]dCTP by random priming DNA labelmg Beads (Pharmacia Biotech) Hybndization was performed with Expresshyb (Clontech) at 68°C for 1 hr The blots were then washed with 2X SSC/0 05% SDS solution at room temperature for 40 mm, followed by washes in 0 IX SSC/0 1%SDS solution at 55°C for 40 mm with one change of fresh solution
The blots were exposed in a phosphonmager
EXAMPLE 7 In situ Hybndization in Cells and Diseased Tissues
The in situ hybndization method of Example 6 is used to determine gene expression, analyze the tissue distnbution of transcnption, and follow changes m specific mRNA synthesis for the genes/DNAs and the protems of the invention in diseased tissues isolated from human individuals suffering from a specific disease These results show more specifically where in diseased tissues the genes of the mvention are expressed and are more predictive of the particular localization of the therapeutic effect of the inhibitory or stimulatory compounds of the mvention (and agonists or antagonists thereof) m a disease Hybridization is performed according to the method of Example 6 usmg one or more of the following tissue and cell samples (a) lymphocytes and antigen presenting cells (dendritic cells, Langherhans cells, macrophages and monocytes, NK cells),
(b) lymphoid tissues normal and reactive lymph node, thymus, Bronchial Associated Lymphoid Tissues, (BALT), Mucosal Associated Lymphoid Tissues (MALT), (c) human disease tissues
• Synovium and joint of patients with Arthritis and Degenerative Joint Disease,
• Colon from patients with Inflammatory Bowel Disease mcludmg Ulcerative Colitis and Crohns' disease,
• Skm lesions from Psoriasis and other forms of dermatitis, • Lung tissue including BALT and tissue lymph nodes from chronic and acute bronchitis, pneumonia, pneumomtis, pleuntis,
• Lung tissue including BALT and tissue lymph nodes from Asthma,
• nasal and sinus tissue from patients with rhinitis or sinusitis,
• Brain and Spinal cord from Multiple Sclerosis Alzheimer's Disease and Stroke, • Kidney from Nephπtis, Glomeralonephπtis and Systemic Lupus Erythematosis,
• Liver from Infectious and non-infectious Hepatitis and acetaminophen-induced liver cmhosis,
• Tissues from Neoplasms/Cancer
Expression is observed in one or more cell or tissue samples indicating localization of the therapeutic effect of the compounds of the mvention (and agonists or antagonists thereof) m the disease associated with the cell or tissue sample.
The sequences of the oligonucleotides used, where expression overlaps with the non-diseased tissue distnbution reported earlier is recited in Example 6
DNA67004- IS99-109.
DNA67004 (SEQ ID NO 98) has weak diffuse signal in a single section of tonsil There was weak some signal in the mucosal epithelium in colitis samples and as well as chronic asthma There was also focal expression in an area of hypeφlastic epidermis in a one section of psoπatic skin The probes used for the above procedures were the following
DNA67004.pl- (SEQ ID NO:122)
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GGG AGA GGG GAG GGA TGC-3' DNA67004.p2: (SEQ ID NO.123)
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GAC CGG AAA TGC TGA CAA ATG-3'
EXAMPLE 8 Use of the PRO polypeptides as a hybndization 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-occumng variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries
Hybridization and washing of filters contammg either library DNAs is perfomied under the following high stringency conditions Hybridization of radiolabeled PRO-denved probe to the filters is performed in a solution of 50%) formamide, 5x SSC, 0 1%> SDS, 0 1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6 8, 2x 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 lx SSC and 0 1%> SDS at 42°C
DNAs having a desired sequence identity with the DNA encodmg full-length native sequence PRO can then be identified using standard teclmiques known in the art
EXAMPLE 9 Expression of the PRO polvpeptide in E coli This example illustrates preparation of an unglycosylated form of PRO by recombmant expression in
The DNA sequence encoding PRO is initially amplified using selected PCR pnmers The primers should contain restriction enzyme sites which conespond to the restriction enzyme sites on the selected expression vector A vanety 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 ampicilhn and tetracychne 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 tφ promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcnptional terminator, and an argU gene The hgation mixture is then used to transform a selected E coli strain using the methods described m
Sambrook et αl , supiα 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 moculate a larger scale culture The cells are then grown to a desired optical density, dur g which the expression promoter is turned on
After cultuπng the cells for several more hours, the cells can be harvested by centrifugation The cell pellet obtamed by the centnfugation can be solubilized using vanous agents known in the art, and the solubilized PRO protem can then be punfied usmg a metal chelating column under conditions that allow tight bmdmg of the protem
PRO may be expressed E coli in a poly-His tagged form, using the following procedure The DNA encodmg PRO is initially amplified using selected PCR pnmers The pnmers will contain restnction enzyme sites which conespond to the restnction enzyme sites on the selected expression vector, and other useful sequences providmg for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase The PCR-amphfied, 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) Ion galE φoHts(htpRts) clpP(lacIq) Transformants are first grown m LB containmg 50 mg/ml carbenicillm 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 bv mixing 3 57 g (NH4)2S04, 0 71 g sodium cιtrate«2H20 1 07 g KC1, 5 36 g Difco yeast extract, 5 36 g Sheffield hvcase SF in 500 mL water, as well as 110 mM MPOS, pH 7 3, 0 55% (w/v) glucose and 7 mM MgS04) 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) m 7 M guanidine, 20 mM Tris, pH 8 buffer Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 IM and 0 02 M, respectively, and the solution is stined overnight at 4°C This step results in a denatured protem with all cysteine residues blocked by sulfitohzation The solution is centπfuged at 40,000 φm 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 clanfied extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer The column is w ashed with additional buffer containing 50 mM lmidazole (Calbiochem, Utrol grade), pH 7 4 The protein is eluted with buffer contammg 250 mM lmidazole Fractions containing the desired protein are pooled and stored at 4°C Protein concentration is estimated by its absorbance at 280 nm usmg the calculated extinction coefficient based on its ammo acid sequence The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consistmg of 20 mM Tris, pH 8 6, 0 3 M NaCl, 2 5 M urea, 5 mM cysteine, 20 mM glycme and 1 mM EDTA Refolding volumes are chosen so that the final protem concentration is between 50 to 100 micrograms/ml The refoldmg solution is stined 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 punfication 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 Rl/H reversed phase column usmg 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 containmg homogeneous refolded protein are pooled Generally, the properly refolded species of most protems are eluted at the lowest concentrations of acetonitrile smce those species are the most compact with their hydrophobic interiors shielded from mteraction with the reversed phase resin Aggregated species are usually eluted at higher acetonitrile concentrations In addition to resolving misfolded forms of protems from the desired form, the reversed phase step also removes endotoxin from the samples
Fractions contammg 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 chlonde and 4%> mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resms equilibrated in the formulation buffer and stenle filtered
Many of the PRO polypeptides disclosed herein were successfully expressed as descnbed above EXAMPLE 10 Expression of the PRO polypeptides 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 March 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 hgation methods such as described m Sambrook et al , supi a 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 m tissue culture plates in medium such as DMEM supplemented with fetal calf seram 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 Tns-HCI, 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 NaP04, and a precipitate is allowed to form for 10 mmutes 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 semm 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 containmg 200 μCi ml 5S-cysteme and 200 μCi/ml 35S-methιonιne 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 dned and exposed to film for a selected period of time to reveal the presence of PRO polypeptide The cultures contammg transfected cells may undergo further incubation (in seram free medium) and the medium is tested m selected bioassays
In an alternative technique, PRO may be mtroduced mto 293 cells transiently using the dextran sulfate method descnbed 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 transfernn After about four days, the conditioned media is centrifuged and filtered to remove cells and debns The sample contammg 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 m CHO cells The pRK5-PRO can be transfected mto CHO cells using known reagents such as CaP04 or DEAE-dextran As described above, the cell cultures can be mcubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S- methiomne After determining the presence of PRO polypeptide, the culture medium may be replaced with semm free medium Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium contammg the expressed PRO can then be concentrated and punfied by any selected method Epitope-tagged PRO mav also be expressed m host CHO cells The PRO may be subcloned out of the pRK5 vector The subclone insert can undergo PCR to fuse m frame with a selected epitope tag such as a polyhis tag into a Baculovims 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 abo e) 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 -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 constmct (immunoadhesin), in which the coding sequences for the soluble forms (e g extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hmge, CH2 and CH2 domams and/or is a poly-His tagged form
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector usmg standard techniques as descnbed in Ausubel et al Current Protocols of Molecular Biology, Unit 3 16, John Wiley and Sons (1997) CHO expression vectors are constmcted to have compatible restnction sites 5' and 3' of the DNA of mterest to allow the convenient shuttling of cDNA's The vector used expression in CHO cells is as descnbed m 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 mtroduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper0* or Fugene® (Boehπnger Mannheim) The cells are grown as descnbed in Lucas et al supra Approximately 3 x 10 7 cells are frozen in an ampule for further growth and production as descnbed below The ampules containmg the plasmid DNA are thawed by placement into water bath and mixed by vortexmg The contents are pipetted into a centrifuge tube containing 10 mLs of media and centnfuged at 1000 φm for 5 mmutes The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0 2 m filtered PS20 with 5% 0 2 μm diafiltered fetal bovme semm) The cells are then ahquoted into a 100 mL spinner contammg 90 mL of selective media After 1-2 days, the cells are transfened into a 250 mL spinner filled with 150 mL selective growth medium and mcubated at 37°C After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 x 105 cells/mL The cell media is exchanged with fresh media by centnfugation and resuspension m production medium Although any suitable CHO media may be employed, a production medium descnbed m U S Patent No 5,122,469, issued June 16, 1992 may actually be used A 3L production spinner is seeded at 1 2 x IO6 cells/mL On day 0, the cell number pH ιe determmed On day 1, the spinner is sampled and spargmg 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% polydunethylsiloxane emulsion, Dow Coming 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 centnfugation and filtering through a 0 22 m filter The filtrate was either stored at 4°C or immediately loaded onto columns for purification For the poly-His tagged constmcts, the proteins are purified usmg 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/mm at 4°C After loading, the column is washed with additional equilibration buffer and the protem eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein is subsequently desalted into a storage buffer containmg 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-contaming) 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 loadmg, 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 containmg 275 L of 1 M Tris buffer, pH 9 The highly purified protem is subsequently desalted into storage buffer as described above for the poly-His tagged protems The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal am o acid sequencing by Edman degradation Many of the PRO polypeptides disclosed herein were successfully expressed as described above
EXAMPLE 11 Expression of PRO in Yeast The following method descnbes recombinant expression of PRO in yeast First, yeast expression vectors are constmcted for intracellular production or secretion of PRO from the
ADH2/GAPDH promoter DNA encodmg PRO and the promoter is inserted into suitable restriction enzyme sites m the selected plasmid to direct intracellular expression of PRO For secretion, DNA encodmg PRO can be cloned into the selected plasmid, together with DNA encodmg 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 descnbed above and cultured in selected fermentation media The transformed yeast supernatants can be analyzed by precipitation with 10% tnchloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain Recombmant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centnfugation and then concentrating the medium using selected cartridge filters The concentrate containmg PRO may further be punfied using selected column chromatography resms
Many of the PRO polypeptides disclosed herein were successfully expressed as descnbed above
EXAMPLE 12 Expression of PRO in Baculoviras-Infected Insect Cells The following method descnbes recombmant expression of PRO in Baculovirus-infected insect cells The sequence codmg for PRO is fused upstream of an epitope tag contamed withm a baculovims expression vector Such epitope tags include poly-his tags and unmunoglobulm tags (like Fc regions of IgG) A variety of plasmids may be employed including plasmids derived from commercially av ailable 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 protem is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions The 5 pnmer may incoφorate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector
Recombinant baculovims is generated by co-transfecting the above plasmid and BaculoGold™ vims
DNA (Pharmingen) mto Spodopter a fr ugiper da ("Sf9") cells (ATCC CRL 171 1) using pofectin (commercially available from GIBCO-BRL) After 4 - 5 days of incubation at 28°C, the released vimses are harvested and used for further amplifications Viral infection and protem expression are performed as described by O'Reilley et al , Baculovirus expr ession vectors A Laboratory Manual, Oxford Oxford University Press (1994)
Expressed poly-his tagged PRO can then be purified, for example, by Nι2+-chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by
Rupert et al , Natwe, 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7 9, 12 5 mM MgCL, 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 centnfugation, and the supernatant is diluted 50-fold m loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7 8) and filtered through a 0 45 m filter A Nι2+-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 loadmg buffer The filtered cell extract is loaded onto the column at 0 5 mL per mmute 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 stammg or Western blot with Nι2+-NTA-conjugated to alkalme phosphatase (Qiagen) Fractions containmg the eluted Hιs,o-tagged PRO are pooled and dialyzed agamst loadmg buffer
Alternatively, punfication of the IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, mcludmg for instance, Protein A or protem G column chromatography
Many of the PRO polypeptides disclosed herein were successfully expressed as descnbed above
EXAMPLE 13 Preparation of Antibodies that Bind PRO This example illustrates preparation of monoclonal antibodies which can specifically bmd PRO Techniques for producmg the monoclonal antibodies are known in the art and are described, for mstance, in Godmg, supra Imrnunogens that may be employed mclude purified PRO, fusion protems contammg PRO, and cells expressing recombinant PRO on the cell surface Selection of the lmmunogen 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 mjected subcutaneously or rntrapentoneally in an amount from 1-100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hmd 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 Semm 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 lme such as P3X63AgU 1, available from ATCC, No CRL 1597 The fusions generate hybndoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopteπn, and thymidine) medium to inhibit proliferation of non- fused cells, myeloma hybrids, and spleen cell hybrids
The hybndoma cells will be screened in an ELISA for reactivity against PRO Determination of "positive" hybndoma cells secretmg the desired monoclonal antibodies against PRO is withm the skill in the art The positive hybndoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containmg the anti-PRO monoclonal antibodies Alternatively, the hybndoma cells can be grown m 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 14 Punfication of PRO Polypeptides Using Specific Antibodies
Native or recombmant PRO polypeptides may be purified by a variety of standard techniques m the art of protem purification For example, pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide is punfied by lmmunoaffinity chromatography usmg antibodies specific for the PRO polypeptide of interest In general, an lmmunoaffinity column is constmcted by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resm
Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by punfication on immobilized Protem 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 resm, the resm is blocked, and the denvative resm is washed according to the manufacturer's instructions
Such an lmmunoaffinity column is utilized in the purification of PRO polypeptide by prepanng a fraction from cells contammg PRO polypeptide m a soluble form This preparation is denved by solubihzation of the whole cell or of a subcellular fraction obtamed via differential centnfugation by the addition of detergent or by other methods well known m the art Alternatively, soluble PRO polypeptide contammg 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 lmmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e g , high ionic strength buffers m 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 thiocvanate ion) and PRO polypeptide is collected
EXAMPLE 15 Drag Screening
This invention is particularly useful for screening compounds by using PRO polypeptides or binding fragment thereof in any of a vanety of drag 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 dmg screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment Drags are screened against such transformed cells in competitive bindmg 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 drags or any other agents which can affect a PRO polypeptide-associated disease or disorder These methods compnse contactmg such an agent with an PRO polypeptide or fragment thereof and assaying (l) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (n) 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 bmding assays, the PRO polypeptide or fragment is typically labeled After suitable incubation, free PRO polypeptide or fragment is separated from that present m bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bmd to PRO polypeptide or to interfere with the PRO polypeptide/cell complex
Another technique for drag screenmg provides high throughput screening for compounds havmg suitable bindmg affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984 Bπefly 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 Punfied PRO polypeptide can also be coated directly onto plates for use in the aforementioned dmg screenmg 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 dmg screening assays in which neutralizing antibodies capable of bmdmg PRO polypeptide specifically compete with a test compound for bmdmg 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 antigemc determinants with PRO polypeptide
EXAMPLE 15 Rational Dmg Design The goal of rational drag design is to produce structural analogs of biologically active polypeptide of mterest (i e , a PRO polypeptide) or of small molecules with which they mteract, e g , agonists, antagonists, or inhibitors Any of these examples can be used to fashion drags 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 (cf, Hodgson, Bιo/Technologv, 9 19-21 ( 1991))
In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO polypeptide-mhibitor 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 sιte(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-hke molecules or to identify efficient inhibitors Useful examples of rational drag design may include molecules which have improved activity or stability as shown by Braxton and Wells,
Figure imgf000124_0001
3_1 :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, m principle, yields a pharmacore upon which subsequent drag 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 minor image of a minor 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 mvention, 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 ammo acid sequence provided herem will provide guidance to those employing computer modeling techniques m place of or in addition to x-ray crystallography
Deposit of Material
The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd , Manassas, VA 201 10-2209, USA (ATCC)
Material UNQ PRO ATCC # ATCC Deposit Date
DNA30942-1134 186 212 209254 September 16, 1997
DNA35638-1141 219 245 209265 September 16, 1997
DNA37150- 1178 233 266 209401 October 17, 1997
DNA39984-1221 269 306 209435 November 7, 1997
DNA41374-1312 294 333 DNA44184-1319 330 526 209704 March 26, 1998
DNA44194-1317 322 381 209808 April 28, 1998
DNA47365-1206 319 364 209436 November 7, 1997
DNA47470-1130 313 356 209422 November 28, 1997
DNA49646-1327 387 719 209705 March 26, 1998
DNA54231-1366 407 769 209804 April 23, 1998
DNA56405-1357 430 788 209849 May 6, 1998
DNA57694-1341 467 826 203017 June 23, 1998
DNA57700-1408 483 982 203583 January 12, 1999
DNA58801-1052 455 779 55820 July 5, 1996
DNA59214-1449 525 1068 203046 July 1, 1998
DNA59294-1381 516 1031 209866 May 14, 1998
DNA60292-1506 587 1157 203540 December 15, 1998
DNA60627-1508 589 1159 203092 August 4, 1998
DNA61185-1646 746 1475 203464 November 17, 1998
DNA66309-1538-1 641 1271 203235 September 15, 1998
DNA66675-1587 698 1343 203282 September 22, 1998
DNA67004-1614 712 1375 203115 August 11, 1998
DNA68864-1629 732 1418 203276 September 22, 1998
DNA73739-1645 745 1474 203270 September 22, 1998
DNA76400-2528 900 1917 203573 January 12, 1999
DNA84920-2614 1930 4405 203966 April 27, 1999
DNA92218-2554 1866 4302 203834 March 9, 1999
DNA105782-2693 2516 6006 PTA-387 July 20, 1999
These deposits was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Pmpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Ine and ATCC which 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 accordmg to 35 USC 122 and the Commissioner's rales 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 mateπals 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
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 descπption herem contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be constmed as limiting the scope of the claims to the specific illustrations that it represents Indeed, vanous modifications of the invention m addition to those shown and described herein will become apparent to those skilled in the art from the foregoing descnption and fall withm the scope of the appended claims

Claims

What is claimed
1 A composition useful for the treatment of immune related diseases comprising a PRO 184, PR0212, PR0245, PR0266, PRO306. PR0333, PR0526, PR0381 , PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031 , PROH57, PROH59, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, agonist or fragment thereof and a earner or excipient, having the properties of
(a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof,
(b) stimulating or enhancing an immune response in a mammal in need thereof,
(c) increasing the proliferation of T-lymphocytes in a mammal in need thereof m response to an antigen, or
(d) stimulating the activity of T-lymphocytes in a mammal in need thereof m response to an antigen
2 The composition of claim 1 comprising an effective amount of a PRO 184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788,
PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide , agonist, antagonist or fragment thereof
3 The composition of claim 2 further compnsmg a growth inhibitory agent, cytotoxic agent or chemotherapeutic agent
4 Use of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381,
PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723,
PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, agonist or a fragment thereof to prepare a composition havmg the properties of
(a) mcreasmg infiltration of mflammatory cells into a tissue of a mammal in need thereof,
(b) stimulatmg or enhancing an immune response in a mammal in need thereof, or (c) mcreasmg the proliferation of T-lymphocytes in a mammal in need thereof m response to an antigen, or (d) stimulatmg the activity of T-lymphocytes in a mammal m need thereof m response to an antigen
5 The use of claims 4 compnsmg an effective amount of a PR0184, PR0212, PR0245,
PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, agonist, antagonist or fragment thereof
6 The composition of claim 2 further comprising a growth inhibitory agent, cytotoxic agent or chemotherapeutic agent
7 A composition useful for the treatment of immune related diseases, comprising a PROl 84, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381 , PR0364, PR0356, PR0719, PR0861,
PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031 , PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375. PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, agonist or fragment thereof and a earner or excipient, having the properties of
(a) decreasing infiltration of mflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof,
(c) decreasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen, or
(d) decreasing the activity of T-lymphocytes in a mammal need thereof in response to an antigen
8 The composition of claim 2 compπsing an effective amount of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PROH59, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide , agonist, antagonist or fragment thereof
9 The composition of claim 8 further compnsmg a growth inhibitory agent, cytotoxic agent or chemotherapeutic agent
10 Use of a PR0184, PR0212. PR0245, PR0266, PRO306, PR0333, PR0526, PR0381,
PR0364, PR0356, PR0719, PR0861 , PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, agonist or a fragment thereof to prepare a composition havmg the properties of (a) decreasmg infiltration of mflammatory cells into a tissue of a mammal m need thereof,
(b) inhibiting or reducing an immune response m a mammal m need thereof, or
(c) decreasmg the proliferation of T-lymphocytes in a mammal in need thereof m response to an antigen, or
(d) decreasmg the activity of T-lymphocytes in a mammal in need thereof in response to an antigen
11 The use of claim 10 compnsmg an effective amount of a PR0184, PR0212, PR0245,
PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788,
PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, agonist antagonist or fragment thereof
12 The composition of claim 1 1 further comprising a growth inhibitory agent, cytotoxic agent or chemotherapeutic agent
13 A method of treating an immune related disorder in a mammal in need thereof, comprising administering to the mammal an effective amount of a PROl 84, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381 PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031 PROH57, PR01159, PR01475, PR01271 , PR01343, PR01375, PR01418, PR01474, PR01917, PR05723 PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, an agonist thereof or an antagonist thereto
14 The method of claim 13, wherein the disorder is selected from systemic lupus erythematosis, rheumatoid arthritis, osteoarthπtis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjogren's syndrome, systemic vascuhtis sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus immune-mediated renal disease, demyelmating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelmating polyneuropathy or Guillain-Bane syndrome, and chronic mflammatory demyelmating polyneuropathy, hepatobi ary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cinhosis, granulomatous hepatitis, and sclerosmg cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, lmmunologic diseases of the lung such as eosinophi c pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumomtis, transplantation associated diseases mcludmg graft rejection and graft -versus-host-disease
15 The composition or use of any of the preceding claims, wherein the agonist or antagonist is a monoclonal antibody
16 The composition or use of any of the precedmg claims, wherein the agonist or antagonist is an antibody fragment or a single-chain antibody
17 The composition or use of claims 15 or 16, wherein the antibody has nonhuman complementanty determining region (CDR) residues and human framework region (FR) residues
18 A method for determining the presence of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405 PRO4302, PRO9940 or PRO6006 polypeptide, compnsmg exposmg a cell suspected of contammg the polypeptide to an antι-PR0184, antι-PR0212, antι-PR0245, anti- PR0266, antι-PRO306, antι-PR0333, antι-PR0526. antι-PR0381 , antι-PR0364, antι-PR0356, antι-PR0719, antι-PR0861, antι-PR0769. antι-PR0788, antι-PR0826, antι-PR0982, antι-PR0779, antι-PRO1068, anti- PRO1031, antι-PROH57, antι-PROH59, antι-PR01475, antι-PR01271 , antι-PR01343, antι-PR01375, anti- PR01418, antι-PR01474, antι-PR01917, antι-PR05723, antι-PRO4405, antι-PRO4302. antι-PRO9940 or anti- PRO6006 antibody, respectively, and determining binding of the antibody to the cell
19 A method of diagnosing an immune related disease in a mammal, comprising detecting the level of expression of a gene encodmg a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) m a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level m the test sample as compared to the control indicates the presence of immune related disease in the mammal from which the test tissue cells were obtained
20 A method of diagnosing an immune related disease in a mammal, comprising (a) contacting an antι-PR0184. antι-PR0212, antι-PR0245, antι-PR0266, antι-PRO306, antι-PR0333, antι-PR0526, anti- PR0381, antι-PR0364, antι-PR0356, antι-PR0719, antι-PR0861, antι-PR0769, antι-PR0788, antι-PR0826, anti-PR0982, antι-PR0779, antι-PRO1068, antι-PRO1031, antι-PR01157, antι-PROH59, antι-PR01475, anti- PR01271, antι-PR01343, antι-PR01375, antι-PR01418, antι-PR01474, antι-PR01917, antι-PR05723, anti- PRO4405, antι-PRO4302, antι-PRO9940 or antι-PRO6006 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the polypeptide in the test sample; wherem the formation of a complex is indicative of the presence or absence of said disease
21 An immune related disease diagnostic kit, comprising an anti-PRO 184, antι-PR0212, anti-
PR0245, antι-PR0266, antι-PRO306, antι-PR0333, antι-PR0526, antι-PR0381, antι-PR0364, antι-PR0356, antι-PR0719, antι-PR0861, antι-PR0769, antι-PR0788, antι-PR0826, antι-PR0982, antι-PR0779. anti- PRO1068, antι-PRO1031, antι-PROH57, antι-PROH59, antι-PR01475, anti-PRO 1271, antι-PR01343, anti- PR01375, antι-PR01418, antι-PR01474, antι-PR01917, antι-PR05723, antι-PRO4405, antι-PRO4302, anti- PRO9940, antι-PRO6006 antibody or fragment thereof and a earner in suitable packaging
22 The kit of claim 21, further compnsmg instructions for using the antibody to detect a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide
23 An article of manufacture, compnsmg
(a) a composition of matter compnsmg a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031 , PROH 57, PROH59, PR01475, PR01271, PR01343, PROJ375, PR01418, PROH74, PR01917, PR05723, PRO4405. PRO4302, PRO9940 or PRO6006 polypeptide or agaonist or antagonist thereof,
(b) a container contammg said composition, and (c) an instruction affixed to said container, or a package insert included in said container refening to the use of said PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PRO86I, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROH59, PR01475, PR01271 , PR01343, PR01375, PROH18, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide or agaonist or antagonist thereof in the treatment of an lmune related disease
24 The article of manufacture of claim 23 wherein said active agent is an anti-PRO 184, anti- PR0212, antι-PR0245, antι-PR0266, antι-PRO306, antι-PR0333, antι-PR0526, antι-PR0381, antι-PR0364, antι-PR0356, antι-PR0719, antι-PR0861, antι-PR0769, antι-PR0788, antι-PR0826, antι-PR0982, anti- PR0779, antι-PRO1068, antι-PRO1031, antι-PROH57, antι-PROH59, antι-PR01475, antι-PR01271, anti- PR01343, antι-PR01375, antι-PR01418, antι-PR01474, antι-PR01917, antι-PR05723, antι-PRO4405, anti- PRO4302, antι-PRO9940 or antι-PRO6006 antibody
25 A method of diagnosing an immune-related disease in a mammal which comprises detecting the presence or absense of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381,
PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide in a test sample of tissue cells obtained from said mammal, wherein the presence or absence of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381 , PR0364. PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide in said test sample is indicative of the presence of an immun-related disease m said mammal
26 A method for identifying an agonist of a PR0184, PR0212, PR0245, PR0266, PRO306,
PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide compnsmg
(a) contactmg cells and a test compound to be screened under conditions suitable for the mduction of a cellular response normally mduced by a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526,
PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, and
(b) determining the mduction of said cellular response to determine if the test compound is an effective agaonist, herein the induction of said cellular response is indicative of said test compound bemg an effective agonist
27 A method for identifying an agonist of a PROl 84, PR0212, PR0245, PR0266, PRO306,
PR0333, PR0526, PR0381 , PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031 , PR01157, PR01159, PR01475, PR01271 , PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide comprising
(a) contacting cells and a test compound to be screened under conditions suitable for the stimulation of cell proliferation by a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861 , PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271 , PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, and
(b) measuring the proliferation of said cells to determine if the test compound is an effective agonist, wherein the stimulation of cell proliferation is indicative of said test compound bemg an effective agonist
28 A method for identifying a compound capable of inhibiting the expression and/or activity of a
PRO polypeptide by contacting a candidate compound with a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861 , PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PR01159, PR01475. PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide under conditions and for a time sufficient to allow these two components to mteract and determining whether the activity of the PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PR01157, PROH59, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide is inhibited
29 The method of claim 28 compnsmg the steps of
(a) contactmg cells and a test compound to be screened in the presence of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide under conditions suitable for the mduction of a cellular response normally induced by a PROl 84, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide; and
(b) determining the induction of said cellular response to determine if the test compound is an effective antagonist
30 The method of claim 28 compnsmg the steps of (a) contacting cells and a test compound to be screened in the presence of a PR0184, PR0212, PR0245, PR0266 PRO306 PR0333, PR0526 PR0381, PR0364 PR0356, PR0719, PRO86I , PR0769, PR0788, PR0826 PR0982 PR0779 PRO1068 PRO1031 , PROl 157 PROl 159, PR01475, PR01271, PR01343, PR01375, PROJ 418 PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide under conditions suitable for the stimulation of cell proliferation by a PRO 184, PR0212, PR0245, PR0266, PRO306, PR0333 PR0526, PR0381 , PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779 PRO1068, PRO1031, PR01157, PR01159, PR01475, PR01271 , PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide under conditions suitable for the stiulation of cell proliferation by a PRO 184 PR0212, PR0245, PR0266, PRO306, PR0333 PR0526, PR0381, PR0364, PR0356, PR0719, PROS61, PR0769, PR0788, PR0826, PR0982, PR0779 PRO1068, PRO1031, PROH57, PROH59, PROH75, PR01271, PR01343, PR01375, PR01418, PR01474 PR01917, PR05723, PRO4405, PRO4302 PRO9940 or PRO6006 polypeptide, and
(b) measuring the proliferation of the cells to determine if the test compound is an effective antagonist
31 A method for identifying a compound that inhibits the expression of a PROl 84, PR0212,
PR0245, PR0266, PRO306 PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide m cells that normally express the polypeptide, wherein the method compπses contacting the cells with a test compound and determining whether the expression of the PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PROH59, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide is inhibited
32 The method of claim 31 compπsing the steps of
(a) contacting cells and a test compound to be screened under conditions suitable for allowing expression of the PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, and
(b) determining the inhibition of expression of said polypeptide
33 A method for treating an immune-related disorder m a mammal that suffers therefrom compnsmg administenng to the mammal a nucleic acid molecule that codes for either (a) a PROl 84, PR0212,
PR0245, PR0266, PRO306 PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, (b) and agonist of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PR01157 PROl 159, PR01475 PR01271 , PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide or (c) an antagonist of a PR0184, PR0212, PR0245 PR0266, PRO306, PR0333, PR0526, PR0381 , PR0364 PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779 PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302 PRO9940 or PRO6006 polypeptide
34 The method of claim 33 wherein said agonist or antagonist is an antι-PR0184, antι-PR0212, antι-PR0245, antι-PR0266, antι-PRO306, antι-PR0333, antι-PR0526, antι-PR0381, antι-PR0364, anti- PR0356, antι-PR0719, antι-PR0861, antι-PR0769, antι-PR0788, antι-PR0826 antι-PR0982, antι-PR0779, antι-PRO1068, antι-PRO1031, antι-PR01157, antι-PROH59, antι-PR01475, antι-PR01271, antι-PR01343, antι-PR01375, antι-PR01418, antι-PR01474, antι-PR01917, antι-PR05723, antι-PRO4405, antι-PRO4302, antι-PRO9940 or antι-PRO6006 antibody
35 The method of claim 33 the nucleic acid is administered via ex vn o gene therapy
36 The method of claim 35 wherein the nucleic acid is comprised withm a vector
37 The method of claim 36 wherein the vector is selected from the group consisting of an adenoviral, a adeno-associated viral, a lentiviral and a retroviral vector
38 A retroviral vector consisting essentially of a promoter, nucleic acid encoding (a) a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364 PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROH57, PROH59, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006polypeptιde, (b) an agonist polypeptide of a PRO 184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, or (c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for cellular secretion of the polypeptide, wherem the retroviral vector is in association with retroviral stmctural proteins
39 An ex vivo producer cell compnsmg a nucleic acid construct that expresses retroviral structural protems and also compnses a retroviral vector consisting essentially of a promoter, nucleic acid encodmg (a) a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide, (b) an agonist polypeptide or a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418 PR01474, PR01917, PR05723, PRO4405, PRO4302 PRO9940 or PRO6006 polypeptide or (c) an antagonist polypeptide of a PRO polypeptide and a signal sequence for cellular secretion of the polypeptide, wherein said producer cell packages the retroviral vector in association with the structural protems to produce recombinant retroviral particles
40 A method of stimulating the proliferation of T-lymphocytes in a mammal compnsmg administering a therapeutically effective amount of a PR0861 , PR0788, PROl 159, PR01646, PR01475, PR01917, PRO9940, PR05723 or PRO6006 polypeptide, wherein the proliferation of T-lymphocytes in the mammal is stimulated
41 A method of decreasing the proliferation of T-lymphocytes in a mammal compπsing administering a therapeutically effective amount of a PR0184, PRO306, PR0779, PR01271, PR01375 or PRO 1474 polypeptide, wherein the proliferation of T-lymphocytes in the mammal is decreased
42 A method of stimulating the activity of T-lymphocytes compnsmg administering a therapeutically effective amount of a PR0245, PR0266, PRO306, PR0333, PR0356, PR0364, PR0381, PR0526, PR0719, PR0769, PR0826, PRO1031, PRO1069, PR01343, PR01375 or PR01418 polypeptide, wherein the activity of T-lymphocytes is mcreased
43 A method of descreasrng the activity of T-lymphocytes compnsmg admmistenng a therapeutically effective amount of a PR0184, PR0212, PRO306, PR0333, PR0364, PR0381, PR0982, PRO1068, PROl 157, wherem the activity of T-lymphocytes is decreased
44 A method of affecting the proliferation of T-cells comprising contacting PBMC cells with an effective amount of a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364,
PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157,
PR01159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405,
PRO4302, PRO9940, PRO6006 polypeptide and measuπng the change in proliferation from control levels
45 A method of stimulatmg the activity of T-cells compnsmg contactmg CD4+ cells or PBMC cells with an effective amount of a PROl 84, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940, PRO6006 polypeptide in combination with an effective amount of antι-CD3 antibody and measuring the change m activity from control levels
46 A method of inhibiting the activity of T-cells comprising contacting CD4+ cells which have been previously stimulated by treatment with antι-CD3 and antι-CD28 antibodies, with an effective amount of
PR0184, PR0212, PR0245, PR0266, PRO306, PR0333, PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475 PR01271, PR01343 PR01375, PR01418, PR01474, PR01917 PR05723, PRO4405, PRO4302, PRO9940, PRO6006 polypeptide and measuring the change in activity from control levels
47 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an ammo acid sequence selected from the group consisting of the ammo acid sequence shown in Figure 1 (SEQ ID NO 1), Figure 3 (SEQ ID NO 3), Figure 5 (SEQ ID NO 8), Figure 7 (SEQ ID NO 13), Figure 9 (SEQ ID NO 18), Figure 1 1 (SEQ ID NO 26), Figure 13 (SEQ ID NO 28), Figure 15 (SEQ ID NO 33), Figure 17 (SEQ ID NO 38), Figure 19 (SEQ ID NO 48), Figure 21 (SEQ ID NO 53), Figure 23 (SEQ ID NO 58), Figure 25 (SEQ ID NO 60), Figure 27 (SEQ ID NO 66), Figure 29 (SEQ ID NO 68), Figure 31 (SEQ ID NO 70), Figure 33 (SEQ ID NO 72), Figure 35 (SEQ ID NO 76), Figure 37 (SEQ ID NO 78), Figure 39 (SEQ ID NO 80) Figure 41 (SEQ ID NO 82), Figure 43 (SEQ ID NO 84), Figure 45 (SEQ ID NO 91), Figure 47 (SEQ ID NO 93), Figure 49 (SEQ ID NO 98), Figure 51 (SEQ ID NO 100), Figure 53 (SEQ ID NO 102), Figure 55 (SEQ ID NO 104), Figure 57 (SEQ ID NO 106), Figure 59 (SEQ ID NO 108), Figure 61 (SEQ ID NO 113), Figure 63 (SEQ ID NO 115) or Figure 65 (SEQ ID NO 1 17)
48 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown m Figure 1 (SEQ ID NO 1) Figure 3 (SEQ ID NO 3), Figure 5 (SEQ ID NO 8), Figure 7 (SEQ ID NO 13), Figure 9 (SEQ ID NO 18), Figure 11 (SEQ ID NO 26), Figure 13 (SEQ ID NO 28), Figure 15 (SEQ ID NO 33), Figure 17 (SEQ ID NO 38), Figure 19 (SEQ ID NO 48), Figure 21 (SEQ ID NO 53), Figure 23 (SEQ ID NO 58), Figure 25 (SEQ ID NO 60), Figure 27 (SEQ ID NO 66), Figure 29 (SEQ ID NO 68), Figure 31 (SEQ ID NO 70), Figure 33 (SEQ ID NO 72), Figure 35 (SEQ ID NO 76), Figure 37 (SEQ ID NO 78), Figure 39 (SEQ ID NO 80), Figure 41 (SEQ ID NO 82), Figure 43 (SEQ ID NO 84), Figure 45 (SEQ ID NO 91), Figure 47 (SEQ ID NO 93), Figure 49 (SEQ ID NO 98), Figure 51 (SEQ ID NO 100), Figure 53 (SEQ ID NO 102), Figure 55 (SEQ ID NO 104), Figure 57 (SEQ ID NO 106), Figure 59 (SEQ ID NO 108), Figure 61 (SEQ ID NO 113), Figure 63 (SEQ ID NO 115) or Figure 65 (SEQ ID NO 117)
49 Isolated nucleic acid havmg at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown m Figure 1 (SEQ ID NO 1), Figure 3 (SEQ ID NO 3), Figure 5 (SEQ ID NO 8), Figure 7 (SEQ ID NO 13), Figure 9 (SEQ ID NO 18), Figure 11 (SEQ ID NO 26), Figure 13 (SEQ ID NO 28), Figure 15 (SEQ ID NO 33), Figure 17 (SEQ ID NO 38), Figure 19 (SEQ ID NO 48), Figure 21 (SEQ ID NO 53), Figure 23 (SEQ ID NO 58), Figure 25 (SEQ ID NO 60), Figure 27 (SEQ ID NO 66), Figure 29 (SEQ ID NO 68), Figure 31 (SEQ ID NO 70), Figure 33 (SEQ ID NO 72), Figure 35 (SEQ ID NO 76), Figure 37 (SEQ ID NO 78), Figure 39 (SEQ ID NO 80), Figure 41 (SEQ ID NO 82), Figure 43 (SEQ ID NO 84), Figure 45 (SEQ ID NO 91), Figure 47 (SEQ ID NO 93), Figure 49 (SEQ ID NO 98), Figure 51 (SEQ ID NO 100), Figure 53 (SEQ ID NO 102), Figure 55 (SEQ ID NO 104), Figure 57 (SEQ ID NO 106), Figure 59 (SEQ ID NO 108), Figure 61 (SEQ ID NO 113), Figure 63 (SEQ ID NO 115) or Figure 65 (SEQ ID NO 117)
50 Isolated nucleic acid havmg at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession number 209254, 209265, 209401, 209435,
-, 209704, 209808. 209436. 209422, 209705. . 209849, 203017. 203583, 55820, 203046. 209866,
203540, 203092, 203464. 203235, 203282, 203115, 203276, 203270, 203573, 203966, 203834 or 387-PTA
51 A vector compnsmg the nucleic acid of any one of Claims 47 to 50
52 The vector of Claim 51 operably linked to control sequences recognized by a host cell transformed with the vector
53 A host cell comprising the vector of Claim 51
54. The host cell of Claim 53 wherem said cell is a CHO cell
55 The host cell of Claim 53, wherem said cell is an E coli
56 The host cell of Claim 53, wherem said cell is a yeast cell
57 A process for producing a PR0184, PR0212, PR0245, PR0266, PRO306, PR0333,
PR0526, PR0381, PR0364, PR0356, PR0719, PR0861, PR0769, PR0788, PR0826, PR0982, PR0779, PRO1068, PRO1031, PROl 157, PROl 159, PR01475, PR01271, PR01343, PR01375, PR01418, PR01474, PR01917, PR05723, PRO4405, PRO4302, PRO9940 or PRO6006 polypeptide compnsmg cultunng the host cell of Claim 53 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
58 An isolated polypeptide havmg at least 80% ammo acid sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO-2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO- 14), Figure 10 (SEQ ID NO- 19), Figure 12 (SEQ ID N0.27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID N0.34), Figure 18 (SEQ ID N0.39), Figure 20 (SEQ ID NO-49), Figure 22 (SEQ ID NO:54), Figure 24 (SEQ ID N0.59), Figure 26 (SEQ ID NO:61), Figure 28 (SEQ ID NO:67), Figure 30 (SEQ ID NO:69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO:73), Figure 36 (SEQ ID N0.77), Figure 38 (SEQ ID NO:79), Figure 40 (SEQ ID NO-81), Figure 42 (SEQ ID N0.83), Figure 44 (SEQ ID N0.85), Figure 46 (SEQ ID NO:92), Figure 48 (SEQ ID NO-94), Figure 50 (SEQ ID NO:99), Figure 52 (SEQ ID NO: 101), Figure 54 (SEQ ID NO: 103), Figure 56 (SEQ ID NO:105), Figure 58 (SEQ ID NO:107), Figure 60 (SEQ ID NO.109), Figure 62 (SEQ ID NO 114), Figure 64 (SEQ ID NO:l 16) or Figure 66 (SEQ ID NO: 118)
59 An isolated polypeptide scoring at least 80%> positives when compared to an amino acid sequence selected from the group consisting of the ammo acid sequence shown in Figure 2 (SEQ ID NO:2),
Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO: 19), Figure 12 (SEQ ID N0.27), Figure 14 (SEQ ID NO:29), Figure 16 (SEQ ID NO:34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49), Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67), Figure 30 (SEQ ID NO 69) Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99) Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 114), Figure 64 (SEQ ID NO 116) or Figure 66 (SEQ ID NO 118)
60 An isolated polypeptide having at least 80% ammo acid sequence identity to an am o acid sequence encoded by the full-length codmg sequence of the DNA deposited under ATCC accession number
209254, 209265, 209401 , 209435, , 209704, 209808, 209436, 209422, 209705, , 209849,
203017, 203583, 55820, 203046, 209866, 203540, 203092, 203464, 203235, 203282, 203115, 203276, 203270, 203573, 203966, 203834 or 387-PTA
61 A chimeπc molecule compnsmg a polypeptide according to any one of Claims 58 to 60 fused to a heterologous ammo acid sequence
62 The chimeric molecule of Claim 61 , wherein said heterologous amino acid sequence is an epitope tag sequence
63 The chimenc molecule of Claim 61, wherein said heterologous amino acid sequence is a Fc region of an unmunoglobulm
64 An antibody which specifically bmds to a polypeptide according to any one of Claims 58 to 60
65 The antibody of Claim 64, wherein said antibody is a monoclonal antibody a humanized antibody or a single-chain antibody
66 Isolated nucleic acid havmg at least 80% nucleic acid sequence identity to
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4
(SEQ ID NO 4), Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 19), Figure 12 (SEQ ID NO 27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID NO 34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49), Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67), Figure 30 (SEQ ID NO 69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99), Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 114), Figure 64 (SEQ ID NO 116) or Figure 66 (SEQ ID NO 118) lacking its associated signal peptide, (b) a nucleotide sequence encoding an extracellular domain of the pohpeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4) Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 19), Figure 12 (SEQ ID NO 27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID NO 34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49) Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67), Figure 30 (SEQ ID NO 69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99), Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 114), Figure 64 (SEQ ID NO 116) or Figure 66 (SEQ ID NO 118) with its associated signal peptide, or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 19), Figure 12 (SEQ ID NO 27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID NO 34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49), Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67), Figure 30 (SEQ ID NO 69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99), Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 114), Figure 64 (SEQ ID NO 116) or Figure 66 (SEQ ID NO 118) lacking its associated signal peptide
67 An isolated polypeptide having at least 80% ammo acid sequence identity to
(a) the polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 19), Figure 12 (SEQ ID NO 27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID NO 34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49), Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67), Figure 30 (SEQ ID NO 69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99), Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 114), Figure 64 (SEQ ID NO 116) or Figure 66 (SEQ ID NO 118) lacking its associated signal peptide,
(b) an extracellular domam of the polypeptide shown m Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 19), Figure 12 (SEQ ID NO 27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID NO 34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49), Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67), Figure 30 (SEQ ID NO 69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99), Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 1 14), Figure 64 (SEQ ID NO 1 16) or Figure 66 (SEQ ID NO 1 18). with its associated signal peptide, or
(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2) Figure 4 (SEQ
ID NO 4), Figure 6 (SEQ ID NO 9), Figure 8 (SEQ ID NO 14), Figure 10 (SEQ ID NO 19), Figure 12 (SEQ ID NO 27), Figure 14 (SEQ ID NO 29), Figure 16 (SEQ ID NO 34), Figure 18 (SEQ ID NO 39), Figure 20 (SEQ ID NO 49), Figure 22 (SEQ ID NO 54), Figure 24 (SEQ ID NO 59), Figure 26 (SEQ ID NO 61), Figure 28 (SEQ ID NO 67). Figure 30 (SEQ ID NO 69), Figure 32 (SEQ ID NO 71), Figure 34 (SEQ ID NO 73), Figure 36 (SEQ ID NO 77), Figure 38 (SEQ ID NO 79), Figure 40 (SEQ ID NO 81), Figure 42 (SEQ ID NO 83), Figure 44 (SEQ ID NO 85), Figure 46 (SEQ ID NO 92), Figure 48 (SEQ ID NO 94), Figure 50 (SEQ ID NO 99), Figure 52 (SEQ ID NO 101), Figure 54 (SEQ ID NO 103), Figure 56 (SEQ ID NO 105), Figure 58 (SEQ ID NO 107), Figure 60 (SEQ ID NO 109), Figure 62 (SEQ ID NO 1 14), Figure 64 (SEQ ID NO 116) or Figure 66 (SEQ ID NO 118), lacking its associated signal peptide
PCT/US2000/015264 1994-09-08 2000-06-02 Compositions and methods for the treatment of immune related diseases WO2000073452A2 (en)

Priority Applications (1058)

Application Number Priority Date Filing Date Title
AU54601/00A AU5460100A (en) 1999-06-02 2000-06-02 Compositions and methods for the treatment of immune related diseases
AU6802801A AU6802801A (en) 2000-03-01 2000-11-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP00983846A EP1250426A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding same
EP06000586A EP1688497A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000589A EP1661997A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA2709291A CA2709291A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000581A EP1666494A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002490853A CA2490853A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001542531A JP2004522404A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
EP06000588A EP1690873A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP10005292A EP2228446A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptieds and nucleic acids encoding the same
PCT/US2000/032678 WO2001040466A2 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000587A EP1690872A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
EP06000583A EP1686134A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000584A EP1669371A3 (en) 1999-12-01 2000-12-01 Composition and methods for the diagnosis of tumours
CA002496312A CA2496312A1 (en) 1999-12-01 2000-12-01 Colon tumour marker pro4799 polypeptides and nucleic acids encoding the same
CA002492070A CA2492070A1 (en) 1999-12-01 2000-12-01 Lung tumor marker pro4329 polypeptides and nucleic acids encoding the same
AU20554/01A AU2055401A (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000585A EP1661996A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491610A CA2491610A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491433A CA2491433A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002490909A CA2490909A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP06000582A EP1666495A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002491258A CA2491258A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002391455A CA2391455A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002492049A CA2492049A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05025102A EP1672070A3 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002494705A CA2494705A1 (en) 1999-12-01 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
ES00989401T ES2333772T3 (en) 1999-12-23 2000-12-20 HOMOLOGICAL POLIPEPTIDES OF IL-17 AND IL-17R AND THEIR THERAPEUTIC USES
DK07016900.8T DK1897944T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and their therapeutic use
ES07016902T ES2388748T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
EP10009491.1A EP2258848B1 (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptide and therapeutic uses thereof
PCT/US2000/034956 WO2001046420A2 (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
PT07016901T PT1897945E (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
EP07016900A EP1897944B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
EP07016899A EP1897943B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
PT07016900T PT1897944E (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
EP09009972.2A EP2163625B1 (en) 1999-12-23 2000-12-20 IL-17 and IL-17R homologous polypeptides and therapeutic uses thereof
DK00989401.5T DK1240325T3 (en) 1999-12-23 2000-12-20 IL-17 and IL-17R homologous polypeptides and therapeutic use thereof
EP07016903A EP1897947B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
CA002391374A CA2391374A1 (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
PT07016903T PT1897947E (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
EP07016902A EP1897946B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
ES09009972.2T ES2506665T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 and IL-17R polypeptides and therapeutic uses thereof
ES07016899T ES2379101T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 polypeptides and therapeutic uses thereof
PT00989401T PT1240325E (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
EP10009490A EP2290081A3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptide and therapeutic uses thereof
AU25909/01A AU2590901A (en) 1999-12-23 2000-12-20 Il-17 homologous polypeptides and therapeutic uses thereof
DK07016903.2T DK1897947T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and their therapeutic uses
ES07016901T ES2380958T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 polypeptides and therapeutic uses thereof
AT07016899T ATE537258T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
AT07016903T ATE541931T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
DK07016901.6T DK1897945T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof.
AT07016901T ATE541930T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
EP00989401A EP1240325B1 (en) 1999-12-23 2000-12-20 Il-17 and il-17r homologous polypeptides and therapeutic uses thereof
JP2001546918A JP2003527104A (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and their therapeutic uses
ES10009491.1T ES2458349T3 (en) 1999-12-23 2000-12-20 Homologous IL-17 polypeptides and therapeutic uses thereof
DE60043069T DE60043069D1 (en) 1999-12-23 2000-12-20 IL-17 AND IL-17R HOMOLOGOUS POLYPEPTIDES AND THEIR THERAPEUTIC USES
AT00989401T ATE444361T1 (en) 1999-12-23 2000-12-20 IL-17 AND IL-17R HOMOLOGUE POLYPEPTIDES AND THERAPEUTIC USES
AT07016900T ATE519847T1 (en) 1999-12-23 2000-12-20 IL-17 HOMOLOGUE POLYPEPTIDES AND THEIR THERAPEUTIC USE
EP07016901A EP1897945B1 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US09/747,259 US6569645B2 (en) 1999-05-14 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
ES07016903T ES2380812T3 (en) 1999-12-23 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
CA002533831A CA2533831A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002401448A CA2401448A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2001/006520 WO2001068848A2 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002534030A CA2534030A1 (en) 2000-03-01 2001-02-28 Screted and transmembrane polypeptides and nucleic acids encoding the same
AU2001268028A AU2001268028A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002539214A CA2539214A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002534186A CA2534186A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2001567332A JP2004508805A (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding them
CA002534391A CA2534391A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002534018A CA2534018A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002533903A CA2533903A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002533991A CA2533991A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002531917A CA2531917A1 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP01945919A EP1259614A2 (en) 2000-03-01 2001-02-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/816,744 US6579520B2 (en) 1998-05-15 2001-03-22 IL-17 related mammalian cytokine polypeptides (IL-17E)
AU2001265019A AU2001265019A1 (en) 2000-05-30 2001-05-25 Compositions and methods for the treatment of immune related diseases
US09/866,034 US20030170864A1 (en) 2000-05-30 2001-05-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2001/017092 WO2001092331A2 (en) 2000-05-30 2001-05-25 Compositions and methods for the treatment of immune related diseases
AU6531101A AU6531101A (en) 2000-06-02 2001-05-31 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591841A CA2591841A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591930A CA2591930A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024028A EP1686174A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024033A EP1666493A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids ancoding the same
PCT/US2001/017800 WO2001093983A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024031A EP1666492A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002585822A CA2585822A1 (en) 2000-06-02 2001-06-01 Breast, rectal, colon and lung tumour marker pro19628 polypeptide and encoding nucleic acid
EP05024035A EP1666594A3 (en) 2000-06-02 2001-06-01 Polypeptide, nucleic acid encoding it, and their use for the diagnosis of cancer
CA002591929A CA2591929A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591814A CA2591814A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024037A EP1700867A3 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024038A EP1702928A3 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024030A EP1666491A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP04005726A EP1489095A1 (en) 2000-06-02 2001-06-01 Polypeptide, nucleic acid encoding it, and their use for the diagnosis of cancer
CA002594659A CA2594659A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024023A EP1683864A3 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024034A EP1666497A3 (en) 2000-06-02 2001-06-01 Polypeptide, nucleic acid encoding it, and their use for the diagnosis of cancer
CA002410162A CA2410162A1 (en) 2000-06-02 2001-06-01 Breast, rectal, colon and lung tumor marker pro 4332 polypeptide and encoding nucleic acid
EP05024025A EP1666597A3 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2002501551A JP2004510409A (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding them
EP01939834A EP1286749A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591656A CA2591656A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591590A CA2591590A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024027A EP1666596A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591583A CA2591583A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
CA002591630A CA2591630A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP05024032A EP1659177A3 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids ancoding the same
AU2001265311A AU2001265311A1 (en) 2000-06-02 2001-06-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/874,503 US20020177188A1 (en) 1998-05-15 2001-06-05 IL-17 homologous polypeptides and therapeutic uses thereof
US09/902,572 US20030108983A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,903 US20030044839A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,979 US20030113718A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,736 US20030049676A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,853 US20020192659A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,634 US20030082540A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,759 US20030077654A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,692 US20030054400A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,615 US20030092002A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/902,713 US20030082541A1 (en) 1997-09-17 2001-07-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,925 US20030096233A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,119 US20030049621A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids enconding the same
US09/903,806 US20030130489A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,749 US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US09/904,011 US20030003530A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,520 US20030054401A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,786 US20030044793A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,640 US7208308B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,603 US20030148419A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,562 US6965015B2 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,943 US20030054349A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/903,823 US20030104381A1 (en) 1997-09-17 2001-07-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,088 US20030073077A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,766 US20030152999A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,125 US6664376B2 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,056 US20030054441A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,992 US20030135025A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,786 US7557192B2 (en) 1997-09-17 2001-07-12 Anti-PRO335 antibodies
US09/905,291 US20020160374A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,859 US20030036060A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,938 US20030211569A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,877 US20030186358A1 (en) 1997-09-17 2001-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,820 US20030036094A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,920 US6806352B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,449 US6965011B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,348 US20030064923A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,553 US20030059828A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,075 US7169906B2 (en) 1997-09-17 2001-07-13 PRO211 polypeptides
US09/904,532 US7151160B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/905,381 US6818746B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,462 US6878807B2 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,485 US20030064367A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,838 US20030148370A1 (en) 1997-09-17 2001-07-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/904,956 US20030049622A1 (en) 1995-12-01 2001-07-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,700 US6723535B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,777 US20030148371A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,815 US7094567B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,722 US6946262B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,742 US20030023054A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,838 US7070979B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,618 US6828146B2 (en) 1997-09-17 2001-07-16 Nucleic acid encoding PRO229 polypeptides
US09/906,760 US20030096340A1 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/906,646 US6852848B2 (en) 1997-09-17 2001-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,728 US20030190611A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,794 US6635468B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,652 US20030104469A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,942 US7087738B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,979 US20030082542A1 (en) 1994-09-08 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,841 US7033825B2 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,613 US20030027145A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,925 US20030054352A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,824 US20020197671A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,093 US20030017498A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/907,575 US20030073079A1 (en) 1997-09-17 2001-07-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,064 US6818449B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,088 US20020146709A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/908,827 US20030054442A1 (en) 1998-05-15 2001-07-18 IL-17 homologous polypeptides and therapeutic uses thereof
US09/908,576 US20040005553A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,204 US20030036061A1 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/909,320 US7074592B2 (en) 1997-09-17 2001-07-18 Secreted and transmembrane polypeptides nucleic acid encoding
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/931,836 US7435793B2 (en) 1998-05-15 2001-08-16 Peptides that induce chondrocyte redifferentiation
US09/941,992 US20030082546A1 (en) 1996-11-06 2001-08-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/946,374 US20030073129A1 (en) 1998-09-01 2001-09-04 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,295 US20020156006A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,189 US6972325B2 (en) 1997-10-17 2001-10-15 PRO273 polypeptides
US09/978,194 US20030195333A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,191 US20030050239A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,298 US20030134785A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,192 US20020177553A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,299 US20030199435A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,193 US20030073624A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,188 US20030139328A1 (en) 1997-10-17 2001-10-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
US09/978,802 US20030199674A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20050124789A9 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,403 US20030050240A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,643 US20030104998A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,608 US20030045462A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,544 US20030199436A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,665 US7294700B2 (en) 1997-10-17 2001-10-16 Anti-PRO846 antibodies
US09/978,681 US20030195148A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US20030054986A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/981,915 US7285623B2 (en) 1997-10-17 2001-10-16 PRO337 polypeptides
US09/978,423 US20030069178A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,585 US20030049633A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,757 US20030083248A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,388 US20040223964A1 (en) 1998-03-17 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/143,029 US7105640B2 (en) 1997-10-17 2001-10-19 Anti-pro792 antibodies
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/166,709 US20030104536A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,929 US20030194781A1 (en) 1998-03-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,089 US7208575B2 (en) 1998-10-07 2001-10-19 PRO531 polypeptides
US10/160,502 US7220835B2 (en) 1998-07-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,829 US20030194780A1 (en) 1998-04-29 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,728 US20030186368A1 (en) 1998-05-13 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/145,124 US20030190701A1 (en) 1998-04-30 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/165,036 US20050227342A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,083 US20030148376A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,086 US7122375B2 (en) 1997-10-17 2001-10-24 PRO274 nucleic acids
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US09/999,834 US20030064407A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,084 US20030203402A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US10/002,967 US20030148373A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,830 US20030077700A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,081 US20030049684A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,833 US6916648B2 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US20020192706A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,829 US20030195344A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,918 US20030211091A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,920 US20040006219A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,925 US7037710B2 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/013,917 US7029874B2 (en) 1998-03-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,928 US20030215905A1 (en) 1998-10-07 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,926 US7074593B2 (en) 1998-04-01 2001-10-25 PRO 703 nucleic acids
US10/000,157 US20020182673A1 (en) 1998-05-15 2001-10-30 IL-17 homologous polypedies and therapeutic uses thereof
US09/990,562 US20030027985A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,442 US20020132252A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,854 US20030059780A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,436 US20020198148A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,687 US20020198149A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,583 US7074897B2 (en) 1997-06-16 2001-11-14 Pro943 polypeptides
US09/990,443 US20030054987A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,521 US20030083461A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,073 US20020127576A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/992,598 US6956108B2 (en) 1997-06-16 2001-11-14 PRO1184 antibodies
US09/990,711 US20030032023A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,438 US20030027754A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,163 US20020132253A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,748 US20030069403A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,469 US20030068623A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,440 US20030060407A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,444 US6930170B2 (en) 1997-06-16 2001-11-14 PRO1184 polypeptides
US09/990,456 US20020137890A1 (en) 1997-03-31 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,726 US20030054359A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/993,667 US20030022187A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,427 US20030073809A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US20030027163A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,333 US6953836B2 (en) 1997-06-16 2001-11-15 PRO844 polypeptides
US09/997,542 US20030068647A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/998,156 US20030044806A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US20030119001A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/002,796 US20030032057A1 (en) 1997-08-26 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,514 US7019116B2 (en) 1997-06-16 2001-11-15 PRO 1387 polypeptides
US09/997,601 US20030054404A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,614 US20030124531A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,653 US7034122B2 (en) 1997-06-16 2001-11-15 Antibodies to PRO1159 polypeptides
US09/997,529 US20030134284A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US20030224358A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US20030087305A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,384 US7119177B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US7189814B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,559 US20030054403A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,857 US20030064375A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,628 US20030059782A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,641 US7112656B2 (en) 1997-06-16 2001-11-15 PRO1312 polypeptides
US09/997,428 US20030027162A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/998,041 US7309775B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,683 US20030059783A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US7166282B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,440 US20030059833A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US7309761B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,573 US20030049682A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,666 US7244816B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,585 US20030119055A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/991,150 US20030194760A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,172 US20030050457A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,157 US7101687B2 (en) 1997-06-16 2001-11-16 Nucleic acids encoding PRO943
US09/990,437 US20030045463A1 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,862 US20030130182A1 (en) 1997-11-05 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,721 US20020142961A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,328 US7056736B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,734 US7491529B2 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,279 US7083978B2 (en) 1997-06-16 2001-11-19 Nucleic acid encoding PRO1111 polypeptides
US09/989,723 US20020072092A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,727 US20020072497A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,722 US20020072067A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,732 US7037679B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO1184 polypeptides
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,731 US20020103125A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,728 US7029873B2 (en) 1997-06-16 2001-11-20 Nucleic acids to PRO1387 polypeptides
US09/989,725 US20030139329A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,730 US7157247B2 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/001,054 US20020192209A1 (en) 1997-09-17 2001-11-30 Methods and compositions for inhibiting neoplastic cell growth
US10/006,172 US7081514B2 (en) 1998-09-01 2001-12-06 PRO1347 polypeptides
US10/006,856 US7538086B2 (en) 1998-09-01 2001-12-06 PRO1303 polypeptides
US10/006,867 US7160985B2 (en) 1997-10-29 2001-12-06 Pro180 polypeptide
US10/006,485 US7026448B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,116 US20030082626A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,818 US20030054406A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,117 US7071304B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,041 US6951921B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/007,194 US7041805B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,063 US20030114652A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/007,236 US7034123B2 (en) 1998-09-01 2001-12-06 Anti-PRO1347 antibodies
US10/006,130 US7098312B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,768 US6936697B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,746 US7026449B2 (en) 1999-01-05 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,149 US7038019B2 (en) 1998-09-01 2001-12-07 Antibodies to PRO1410
US10/012,754 US7375184B2 (en) 1998-09-01 2001-12-07 PRO1382 polypeptides
US10/012,101 US20030187239A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,231 US6924355B2 (en) 1998-09-01 2001-12-07 PRO1343 polypeptides
US10/011,833 US6951920B2 (en) 1998-09-01 2001-12-07 PRO1340 polypeptides
US10/012,753 US7488796B2 (en) 1998-09-01 2001-12-07 PRO1269 polypeptides
US10/011,671 US20030096954A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,121 US7022817B2 (en) 1998-09-01 2001-12-07 PRO1325 polypeptides
US10/012,755 US20030096955A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,064 US6953841B2 (en) 1999-01-05 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,795 US7012131B2 (en) 1998-09-01 2001-12-07 Pro1410 polypeptides
US10/012,137 US20030187189A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,752 US7026455B2 (en) 1998-09-01 2001-12-07 Anti-pro 1343 antibodies
US10/012,754 US20030187191A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,692 US20030109672A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,237 US20030191281A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,906 US20030191282A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,907 US20030064925A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,913 US20030083462A1 (en) 1999-01-05 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,822 US20030130491A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,915 US20030204053A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,430 US20030092883A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,910 US7057018B2 (en) 1999-01-05 2001-12-10 Pro 1474 polypeptides
US10/013,909 US20030186318A1 (en) 1999-01-05 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,911 US20030187193A1 (en) 1998-09-01 2001-12-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,389 US6936436B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,519 US7033785B2 (en) 1998-09-01 2001-12-11 PRO1347 nucleic acids
US10/015,393 US6951737B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,499 US20030065142A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,869 US7189530B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,653 US20030187195A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,869 US20030073130A1 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,671 US6946263B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,480 US7074912B2 (en) 1998-09-01 2001-12-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,390 US20030216562A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,388 US20030191299A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,385 US20030195347A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,391 US20030120053A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,715 US7033786B2 (en) 1998-09-01 2001-12-12 Pro1340 nucleic acids
US10/015,386 US7022498B2 (en) 1998-09-01 2001-12-12 Pro 1325 nucleic acids
US10/015,387 US20030135034A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,867 US20030180792A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,306 US20030170718A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/020,063 US20030119097A1 (en) 1999-01-05 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,527 US20030082628A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,610 US20030113795A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/017,407 US20030125535A1 (en) 1998-09-01 2001-12-13 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/035,977 US20030134327A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/036,160 US7125959B2 (en) 1998-05-15 2001-12-26 PRO4405 polypeptides
US10/036,150 US7256039B2 (en) 1998-05-15 2001-12-26 PRO4405 nucleic acids
US10/035,855 US7105639B2 (en) 1998-05-15 2001-12-26 Anti-PRO 4405 antibodies
US10/036,041 US20020192751A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/035,958 US7241862B2 (en) 1998-05-15 2001-12-26 Polypeptides that induce cell proliferation or induce fetal hemoglobin
US10/035,719 US20030036114A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/036,063 US20030092063A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/036,342 US7193045B2 (en) 1998-05-15 2001-12-26 Polypeptides that induce cell proliferation
US10/036,214 US20030032061A1 (en) 1998-05-15 2001-12-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,244 US20020192668A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,301 US20020098506A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/032,996 US20030054447A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,435 US20030027256A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,326 US20020098507A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,223 US20020164646A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,396 US20030077657A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,167 US20020182618A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,245 US20020160392A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/032,990 US20030032060A1 (en) 1998-08-04 2001-12-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/033,246 US20020098505A1 (en) 1998-08-04 2001-12-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/052,586 US20020127584A1 (en) 1997-09-18 2002-01-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/053,107 US20020192752A1 (en) 1998-09-09 2002-01-17 Compositions and methods for the treatment of immune related diseases
US09/978,187 US20030096744A1 (en) 1997-10-17 2002-01-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,273 US7317092B2 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,494 US20030032063A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,211 US20030044844A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,269 US20030040014A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,500 US20020177165A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding
US10/066,203 US20030180796A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,198 US20030170721A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/066,193 US20030044902A1 (en) 1997-08-26 2002-02-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,045 US20030073210A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,042 US20030096386A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,047 US20030077778A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,041 US20030077776A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,046 US20030194791A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,051 US20030092147A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,044 US20030190717A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,059 US20030190721A1 (en) 1997-03-31 2002-04-11 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,052 US20030199052A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,054 US20030199054A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,055 US20030190718A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,062 US20030077779A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,049 US20030022239A1 (en) 1997-06-18 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,050 US20030054516A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,063 US20030199055A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,048 US20030199051A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,053 US20030199053A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,061 US20030082761A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,060 US20030190722A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,057 US20030190719A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/121,058 US20030190720A1 (en) 1997-03-31 2002-04-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,235 US20030082762A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US20030199057A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,292 US20030073211A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,322 US20030199059A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,214 US7343721B2 (en) 1997-03-31 2002-04-15 PRO4406 polypeptide
US10/123,109 US20030190723A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,771 US20030199060A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,155 US20030068794A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,156 US20030194792A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,154 US20030190724A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,108 US7635478B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,291 US20030199058A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,236 US20030068795A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,157 US20030190725A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/123,912 US20030100087A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,906 US20030190726A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,903 US20030073212A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,902 US20030077781A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,910 US7329404B2 (en) 1997-03-31 2002-04-16 Antibodies against PRO1310
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,913 US20030203462A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,824 US20030077659A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,817 US20030077786A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,821 US20030199023A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,822 US7109305B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,704 US7357926B2 (en) 1997-03-31 2002-04-17 Antibodies against PRO1879 and the use thereof
US10/124,820 US20030190729A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,816 US20030190728A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,813 US7312307B2 (en) 1997-03-31 2002-04-17 PRO1056 polypeptides
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/124,823 US20030199062A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,814 US7105335B2 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,795 US7304131B2 (en) 1997-03-31 2002-04-17 PRO1483 polypeptides
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,924 US7342097B2 (en) 1997-03-31 2002-04-19 PRO1309 polypeptides
US10/125,927 US20030190731A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/127,838 US20030082691A1 (en) 1998-11-17 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/127,836 US7432345B2 (en) 1998-11-17 2002-04-22 PRO1475 polypeptide
US10/128,693 US7355006B2 (en) 1998-08-31 2002-04-23 Antibodies against the PRO1271 polypeptides
US10/128,686 US7345146B2 (en) 1998-08-31 2002-04-23 PRO1271 Polypeptides
US10/128,689 US20030087365A1 (en) 1997-03-31 2002-04-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/131,817 US7291701B2 (en) 1997-03-31 2002-04-24 PRO1777 polypeptides
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/063,510 US7109292B2 (en) 1999-03-08 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,520 US20030187196A1 (en) 1998-12-30 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,521 US20030190669A1 (en) 1998-12-30 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,517 US7232889B2 (en) 1999-03-08 2002-05-01 PRO300 antibodies
US10/063,518 US7465785B2 (en) 1999-03-08 2002-05-01 Polypeptide encoded by a nucleic acid over-expressed in melanoma
US10/063,519 US20030009013A1 (en) 1998-12-30 2002-05-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,540 US20030181667A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,540 US7193061B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in esophageal tumor and melanoma
US10/063,544 US20030027212A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,553 US20030045684A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,548 US20030187228A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,549 US7253256B2 (en) 1999-03-08 2002-05-02 Polypeptides encoded by a nucleic acid are expressed in esophageal and kidney tumor
US10/063,532 US7202336B2 (en) 1999-03-08 2002-05-02 Polypeptide encoded by a nucleic acid overexpressed in kidney tumor and underexpressed in lung tumor
US10/063,554 US7223841B2 (en) 1999-03-08 2002-05-02 PR0994 antibodies
US10/063,567 US20030069394A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,537 US7276586B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid under-expressed in stomach and lung tumor
US10/063,546 US7435798B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid overexpressed in normal stomach, normal skin and kidney tumor
US10/063,536 US20030181696A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,524 US7205391B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide suppressed in stomach tumors
US10/063,560 US7232882B2 (en) 1999-03-08 2002-05-02 Polypeptide uder-expressed in melanoma
US10/063,523 US20030181636A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,541 US20030060601A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,547 US20020182638A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,569 US7378491B2 (en) 1999-03-08 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,570 US7507404B2 (en) 1999-03-08 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,565 US20030180904A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,527 US20030181637A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,568 US20030181668A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,538 US7253255B2 (en) 1999-03-08 2002-05-02 Polypeptide encoded by a nucleic acid underexpressed in esophageal tumor and melanoma
US10/063,566 US20030073821A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,564 US20030180794A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,563 US20030060602A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,569 US20030018168A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,536 US7259238B2 (en) 1999-03-08 2002-05-02 Polypeptide encoded by a nucleic acid under-expressed in stomach and lung tumor
US10/063,523 US7220830B2 (en) 1999-03-08 2002-05-02 Polypeptide underexpressed in stomach tumors
US10/063,534 US7193060B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptides encoded by a nucleic acid overexpressed in kidney tumor and underexpressed in lung tumor
US10/063,547 US7294690B2 (en) 1999-03-08 2002-05-02 Polypeptides encoded by a nucleic acid differentially expressed in lung tumor
US10/063,561 US7271247B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid differentially expressed in melanoma
US10/063,528 US20030181666A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,525 US20030036634A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,555 US20030065143A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,548 US7232892B2 (en) 1999-03-08 2002-05-02 Antibodies to a polypeptide encoded by a nucleic acid differentailly expressed in stomach, lung and melanoma tumor
US10/063,553 US7235630B2 (en) 1999-03-08 2002-05-02 PRO994 polypeptides
US10/063,526 US20030171550A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,551 US20020183494A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,545 US7256261B2 (en) 1999-03-08 2002-05-02 Polypeptides encoded by a nucleic acid over expressed in normal stomach normal skin and kidney tumor
US10/063,524 US20030027992A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,551 US7214777B2 (en) 1999-03-08 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,545 US20020183505A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,530 US7193059B2 (en) 1999-03-08 2002-05-02 Antibodies that recognize a polypeptide overexpressed in rectal tumors
US10/063,562 US20030181697A1 (en) 1998-12-30 2002-05-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,577 US7423130B2 (en) 1999-03-08 2002-05-03 PRO1158 polypeptides
US10/063,588 US20030130483A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,611 US20030181677A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,867 US20030207349A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,587 US7244428B2 (en) 1998-09-10 2002-05-03 PRO1357 antibodies
US10/063,606 US20030181675A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,865 US20030032155A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,607 US7345145B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,581 US7189803B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,615 US7405269B2 (en) 1998-10-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,610 US7371814B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,578 US7417125B2 (en) 1999-03-08 2002-05-03 Antibodies that bind a PRO1158 polypeptide
US10/063,604 US7390876B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,597 US7227000B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,617 US7423119B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,591 US20030180906A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,592 US20030181672A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,602 US7230082B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,583 US7189804B2 (en) 1999-03-08 2002-05-03 PRO1335 polypeptides
US10/063,593 US7189805B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,612 US7399828B2 (en) 1998-09-24 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,579 US20030181638A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,610 US20030180907A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,609 US7402661B2 (en) 1998-10-06 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,592 US7220841B2 (en) 1998-10-27 2002-05-03 Antibody to PRO1557 polypeptide encoded by a nucleic acid highly expressed in esophageal and kidney tumors
US10/063,618 US7468424B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,600 US7230076B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,616 US7358339B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,582 US7205389B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,611 US7378501B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,596 US7285624B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,591 US7196174B2 (en) 1998-10-27 2002-05-03 PRO1557 polypeptide encoded by a nucleic acid highly expressed in esophageal and kidney tumors
US10/063,598 US7223838B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,614 US7393931B2 (en) 1999-03-08 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,589 US20030181641A1 (en) 1998-12-30 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,023 US20030207416A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,963 US7288625B2 (en) 1997-03-31 2002-05-06 PRO4395 polypeptides
US10/140,024 US20040058424A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,020 US20030207415A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,470 US20030022331A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,474 US20030032156A1 (en) 1997-03-31 2002-05-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,649 US20030181652A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,650 US7217787B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in rectal tumor
US10/063,664 US7256262B2 (en) 1999-03-08 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,676 US20030180843A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,661 US7193047B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in esophageal tumor
US10/063,677 US20030187242A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,673 US20030180908A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
US10/063,646 US20030181681A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,928 US20030068798A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,647 US7193046B2 (en) 1998-10-20 2002-05-07 Polypeptide encoded by a nucleic acid overexpressed in esophageal and lung tumor, and underexpressesd in kidney tumor and melanoma
US10/063,657 US7193063B2 (en) 1999-03-08 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US10/063,669 US20030180838A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,670 US20030180839A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,638 US7101970B2 (en) 1999-03-08 2002-05-07 PRO1570 polypeptides
US10/140,865 US20030207420A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,654 US7202337B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US10/063,644 US7196167B2 (en) 1998-10-20 2002-05-07 Polypeptide underexpressed in lung tumor
US10/063,671 US20030180840A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,642 US20030181650A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,644 US20030181651A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,864 US20030207419A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,665 US7427664B2 (en) 1999-03-08 2002-05-07 Antibodies to polypeptides that stimulate TNF-α release
US10/063,643 US20030181680A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,805 US20030207417A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,808 US7425621B2 (en) 1997-03-31 2002-05-07 Antibodies against the PRO4401 polypeptide
US10/063,666 US7411037B2 (en) 1999-03-08 2002-05-07 Polypeptides encoded by a nucleic acid underexpressed in melanoma
US10/063,668 US20030191290A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,647 US20030187197A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,653 US7238787B2 (en) 1999-03-08 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in melanoma
US10/063,651 US7193057B2 (en) 1997-10-29 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in rectal tumor
US10/063,646 US7189821B2 (en) 1998-10-20 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in lung tumor
US10/063,674 US20030180841A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,860 US7307151B2 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,639 US7081520B2 (en) 1998-10-08 2002-05-07 Anti-pro 1570 antibodies
US10/063,659 US7186801B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in stomach tumor and lung tumor
US10/063,652 US7230077B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in melanoma
US10/063,675 US20030180842A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,662 US20030180795A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,660 US7189822B2 (en) 1999-03-08 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid underexpressed in stomach and lung tumor
US10/063,640 US7354997B2 (en) 1999-03-08 2002-05-07 Polypeptide encoded by a nucleic acid underexpressed in stomach tumor
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,672 US20030181700A1 (en) 1998-12-30 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,648 US7193062B2 (en) 1998-10-20 2002-05-07 Antibodies to a polypeptide encoded by a nucleic acid over expressed in esoprageal and lung tumor, and under expressed in kidney tumor and melanoma
US10/063,714 US20030180913A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,705 US7220850B2 (en) 1999-03-08 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,693 US20030180847A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,735 US20030138882A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,713 US20030180855A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,722 US20030180918A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,721 US20030181702A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,694 US20030180848A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,710 US20030180910A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,703 US20030207357A1 (en) 1999-03-10 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,730 US20030180858A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,726 US20030180919A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,709 US7189564B2 (en) 1999-03-08 2002-05-08 PRO1335 nucleic acids
US10/063,699 US20030180850A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,711 US20030180911A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,716 US20030180915A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,717 US20030180916A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,756 US7488586B2 (en) 1997-03-31 2002-05-08 PRO4409 polypeptides
US10/063,684 US20030186407A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,692 US20030180846A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/063,732 US20030180922A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,728 US20030180920A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,723 US20030181703A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,698 US20030180849A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,718 US20030190698A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,689 US20030180845A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,731 US20030180921A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,724 US20030180856A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,707 US20030180853A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,685 US20030180909A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,702 US7193074B2 (en) 1999-03-08 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,744 US20030180863A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,720 US20030180917A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,715 US20030180914A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/063,686 US20030180844A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,703 US7189563B2 (en) 1999-03-08 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,701 US20030207421A1 (en) 1997-03-31 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,734 US20030180859A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,712 US20030180912A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,736 US20030180860A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,682 US20030181701A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,688 US20030186408A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,727 US20030180857A1 (en) 1998-12-30 2002-05-08 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,743 US20030180862A1 (en) 1998-12-30 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,745 US20040058411A1 (en) 1998-12-30 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 polypeptides
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,742 US7189532B2 (en) 1999-03-08 2002-05-09 Nucleic acid underexpressed in stomach tumor and lung tumor
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/063,741 US7118887B2 (en) 1999-03-08 2002-05-09 Nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 polypeptides
US10/142,423 US20030049817A1 (en) 1997-03-31 2002-05-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,431 US7285629B2 (en) 1997-03-31 2002-05-10 Pro5005 polypeptides
US10/142,419 US7153941B2 (en) 1997-03-31 2002-05-10 Antibodies that bind PRO4994 polypeptides
US10/146,792 US20030207428A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/146,730 US20030207427A1 (en) 1997-03-31 2002-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,528 US20030219885A1 (en) 1997-03-31 2002-05-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,519 US20030077791A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,536 US20040077064A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,490 US20030166089A1 (en) 1998-11-17 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,492 US20030082765A1 (en) 1997-03-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/147,482 US20030157627A1 (en) 1998-08-31 2002-05-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/152,395 US7189534B2 (en) 1997-03-31 2002-05-21 PRO4320 polynucleotide
US10/153,934 US20030129695A1 (en) 1997-03-31 2002-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/156,843 US20030207805A1 (en) 1997-06-18 2002-05-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,782 US20030077792A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/157,786 US20030208055A1 (en) 1997-03-31 2002-05-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,782 US20030082766A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/158,791 US20030207429A1 (en) 1997-03-31 2002-05-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,691 US20030166106A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,694 US20030166107A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,689 US20030166104A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,707 US20030166110A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,705 US20030032103A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,690 US20030166105A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,703 US20030170794A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,695 US20030032101A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,697 US20030032102A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,693 US20030073169A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,692 US20030166188A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,698 US20030166108A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,706 US20030022293A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,696 US20030082767A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,702 US20030170793A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,701 US20030104538A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,699 US20030166109A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,704 US20030170795A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/173,700 US20030027262A1 (en) 1997-09-18 2002-06-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,588 US20030027266A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,590 US20030008352A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,572 US20030027263A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,583 US7211645B2 (en) 1997-09-18 2002-06-18 PRO268 polypeptides
US10/174,578 US20030073170A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,579 US20030027264A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,582 US20030027265A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,587 US20030166113A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,585 US20030032105A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,586 US20030032106A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,591 US20030166115A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,576 US7125962B2 (en) 1997-09-18 2002-06-18 Anti-Pro268 antibodies
US10/174,589 US20030166114A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,581 US7153939B2 (en) 1997-09-18 2002-06-18 PRO354 antibodies
US10/174,569 US20030166111A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,570 US20030211572A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/174,574 US20030170796A1 (en) 1997-09-18 2002-06-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,743 US20030027269A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,745 US20030166120A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,744 US20030166119A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,738 US20030022294A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,741 US20030073171A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,746 US20030027270A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,750 US20030073172A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,735 US20030082715A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,742 US20030166118A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,753 US20030077732A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,749 US20050196832A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,747 US20030032107A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,752 US20030022295A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,754 US20030166123A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,739 US20030027267A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,751 US20030166122A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,748 US20030166121A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/175,736 US20030166117A1 (en) 1997-09-18 2002-06-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,913 US20030022298A1 (en) 1997-09-15 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,917 US20030044918A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,759 US20030166128A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,484 US20030059876A9 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,754 US7709602B2 (en) 1997-09-18 2002-06-20 PRO1078 polypeptides
US10/176,914 US20030017543A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,749 US20030017542A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,747 US20030027273A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,989 US20030170803A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,482 US20030022296A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,918 US7495083B2 (en) 1997-09-18 2002-06-20 PRO940 antibodies
US10/176,491 US20030087373A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,753 US20030044917A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,483 US20030017541A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,746 US20030068680A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,988 US20030170802A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,485 US20030032109A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,981 US20030170800A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,911 US20030032113A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,920 US20030166129A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,490 US20030170798A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,479 US20030040054A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,993 US20030027280A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,757 US7317082B2 (en) 1997-09-18 2002-06-20 PRO1018 polypeptides
US10/176,493 US20030032111A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,921 US20030027276A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,487 US20030032110A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,919 US20030032114A1 (en) 1997-09-18 2002-06-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,978 US20030032116A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,924 US20030166131A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,992 US20030027279A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,752 US20030170799A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,489 US20030166125A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,750 US20030027274A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,983 US20030170801A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,756 US20030032112A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,751 US20030036117A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,492 US20030027272A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,925 US20030032115A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,979 US20030087374A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,481 US20030032108A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,922 US20030166130A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,985 US20030027277A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,480 US20030166124A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,991 US20030027324A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,915 US20030017544A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,923 US20030068681A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,755 US20030166127A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,987 US20030027278A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,982 US20030044919A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,760 US7339033B2 (en) 1998-06-26 2002-06-21 Pro1481
US10/176,758 US20030008353A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,748 US20030040055A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,986 US20030073173A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,486 US7354999B2 (en) 1997-09-18 2002-06-21 PRO1481 polypeptides
US10/176,916 US20030040056A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/176,488 US20030027271A1 (en) 1997-09-18 2002-06-21 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,513 US20030044921A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,510 US20030032117A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,512 US20030166134A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,516 US20030040058A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,526 US20030100061A1 (en) 1998-06-26 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,509 US20030207392A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,517 US20030170805A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,515 US20030166135A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,511 US20030104539A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,507 US20030040057A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,519 US7339024B2 (en) 1997-09-18 2002-06-24 PRO1772 polypeptides
US10/179,508 US20030166133A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,518 US20030104540A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,525 US20030040060A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,521 US20030170806A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,523 US20030215909A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,514 US20030044922A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,522 US20030044923A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,506 US20030044920A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/179,520 US20030096353A1 (en) 1997-09-18 2002-06-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,546 US20030032120A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,551 US20030036123A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,544 US20030032119A1 (en) 1998-06-26 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,554 US20050202526A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,543 US20030032118A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,545 US20030040062A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,556 US7355000B2 (en) 1997-09-18 2002-06-25 PRO1380 polypeptides
US10/180,553 US7365156B2 (en) 1997-09-18 2002-06-25 PRO1316 polypeptides
US10/180,547 US20030032121A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,550 US20030064440A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,557 US20030022301A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,541 US20030036120A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,552 US7348415B2 (en) 1997-09-18 2002-06-25 PRO1316 antibodies
US10/180,540 US20030040061A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,549 US20030032122A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,548 US7696319B2 (en) 1997-09-18 2002-06-25 PRO1772 antibodies
US10/180,542 US20030036121A1 (en) 1998-06-26 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,560 US20030044925A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,555 US20030032123A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,559 US20030032124A1 (en) 1997-09-18 2002-06-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,008 US20030040064A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,002 US20030054454A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,998 US7087421B2 (en) 1997-09-18 2002-06-26 Pro1278 polypeptides
US10/183,011 US20030068682A1 (en) 1998-06-26 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,005 US7317093B2 (en) 1997-09-18 2002-06-26 PRO1339 antibodies
US10/183,013 US7309769B2 (en) 1997-09-18 2002-06-26 PRO1487 polypeptides
US10/183,019 US7425605B2 (en) 1997-09-18 2002-06-26 PRO1486 polypeptides
US10/181,000 US7319137B2 (en) 1997-09-18 2002-06-26 PRO1339 polypeptides
US10/183,001 US7084255B2 (en) 1997-09-18 2002-06-26 PRO1278 polypeptides
US10/183,009 US7339034B2 (en) 1997-09-18 2002-06-26 PRO1305 antibodies
US10/183,006 US7297776B2 (en) 1997-09-18 2002-06-26 PRO1374 antibodies
US10/183,010 US20030032126A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,017 US20030040065A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,012 US7718770B2 (en) 1997-09-18 2002-06-26 PRO1305-polypeptides
US10/183,003 US20030082716A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,015 US20030044926A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/180,999 US7297767B2 (en) 1997-09-18 2002-06-26 PRO1374 polypeptides
US10/183,016 US20030082717A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,014 US20030064441A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/183,018 US20030104541A1 (en) 1997-09-18 2002-06-26 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,614 US20030032128A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,651 US7291704B2 (en) 1997-09-18 2002-06-27 PRO1758 polypeptides
US10/184,630 US7304143B2 (en) 1997-09-18 2002-06-27 PRO1571 antibodies
US10/184,654 US7378486B2 (en) 1997-09-18 2002-06-27 PRO1482 antibodies
US10/184,627 US7282569B2 (en) 1997-09-18 2002-06-27 PRO1508 antibodies
US10/184,631 US20030036134A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,652 US20030032134A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,641 US20030073174A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,633 US20030068683A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,618 US7393917B2 (en) 1997-09-18 2002-06-27 PRO1482 polypeptides
US10/184,615 US20030044927A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,628 US7309770B2 (en) 1997-09-18 2002-06-27 PRO1757 polypeptides
US10/184,630 US20030036133A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,642 US7332573B2 (en) 1997-09-18 2002-06-27 PRO1571 polypeptides
US10/184,613 US20030119105A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,627 US20030040070A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,619 US20030049738A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,612 US20030036127A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,640 US7271250B2 (en) 1998-06-26 2002-06-27 PRO1757 antibodies
US10/184,638 US20030054456A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,616 US20030036128A1 (en) 1997-09-18 2002-06-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,644 US20030044930A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,655 US20030040073A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,632 US20030036135A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,634 US20030068684A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,620 US20030044928A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,645 US7291718B2 (en) 1998-06-26 2002-06-28 PRO1758 antibodies
US10/184,657 US20030104543A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,643 US20030044929A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,658 US20030027281A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,624 US20030104542A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,626 US20030040069A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,623 US20030032129A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,647 US20030032133A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,650 US20030036138A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,636 US20030036136A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,617 US20030036129A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,621 US20030054455A1 (en) 1998-06-26 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,646 US20030032132A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,656 US20030044931A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,637 US20030032131A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,629 US20030036132A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,635 US20030032130A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,625 US20030040068A1 (en) 1997-09-18 2002-06-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/184,622 US20030036130A1 (en) 1997-09-18 2002-06-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,739 US7291706B2 (en) 1998-06-26 2002-07-01 PRO4352 polypeptides
US10/187,598 US20030036142A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,747 US7291707B2 (en) 1997-09-18 2002-07-01 PRO1337 polypeptides
US10/187,601 US7291705B2 (en) 1997-09-18 2002-07-01 PRO19645 polypeptides
US10/187,886 US7291708B2 (en) 1997-09-18 2002-07-01 PRO1785 polypeptides
US10/187,884 US20030036155A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,887 US7285645B2 (en) 1997-09-18 2002-07-01 PRO4356 antibodies
US10/187,594 US7294335B2 (en) 1998-06-26 2002-07-01 PRO19645 antibodies
US10/187,588 US7351795B2 (en) 1998-06-26 2002-07-01 PRO19563 polypeptides
US10/187,597 US20030036141A1 (en) 1997-09-18 2002-07-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,885 US20030032138A1 (en) 1998-06-24 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,600 US20030036143A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,596 US20030032136A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,781 US20030036160A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,603 US20030036146A1 (en) 1998-06-26 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,751 US20030036151A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,767 US7312310B2 (en) 1997-09-18 2002-07-02 PRO6015 polypeptides
US10/187,746 US20030036149A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,747 US20030036150A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,766 US7351804B2 (en) 1998-06-26 2002-07-02 Antibodies against PRO4421
US10/187,754 US20030036153A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,769 US20030036157A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,602 US20030036145A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,753 US20030036152A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,780 US7268217B2 (en) 1998-06-26 2002-07-02 PRO4421 polypeptides
US10/188,775 US20030040075A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,774 US20030040074A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,745 US7250490B2 (en) 1997-09-18 2002-07-02 PRO1480 polypeptides
US10/187,741 US20030036147A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/188,770 US7358340B2 (en) 1997-09-18 2002-07-02 PRO19563 antibodies
US10/188,773 US20030036159A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/187,757 US7276578B2 (en) 1997-09-18 2002-07-02 PRO4334 polypeptides
US10/187,743 US20030036148A1 (en) 1997-09-18 2002-07-02 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/192,010 US20030044932A1 (en) 1997-09-18 2002-07-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,461 US20030054459A1 (en) 1998-06-26 2002-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,365 US7381791B2 (en) 1998-06-26 2002-07-12 PRO9739 polypeptides
US10/194,462 US7388073B2 (en) 1998-06-26 2002-07-12 PRO9835 polypeptides
US10/194,361 US20030036161A1 (en) 1998-06-26 2002-07-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/194,423 US7339025B2 (en) 1998-06-26 2002-07-12 PRO6246 polypeptides
US10/195,902 US20030038826A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,894 US20030043176A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,889 US7534856B2 (en) 1998-06-26 2002-07-15 PRO19624 antibodies
US10/195,892 US7385033B2 (en) 1998-06-26 2002-07-15 PRO12970 polypeptides
US10/195,901 US20030036165A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,893 US20030206188A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,897 US20030036164A1 (en) 1997-09-18 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,888 US20060073545A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/195,883 US20060073544A1 (en) 1998-06-26 2002-07-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,760 US7408034B2 (en) 1998-06-26 2002-07-16 PRO20025 polypeptides
US10/196,743 US20030038827A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,745 US7423120B2 (en) 1997-09-18 2002-07-16 PRO19814 polypeptides
US10/196,756 US7304145B2 (en) 1998-06-26 2002-07-16 PRO19646 antibodies
US10/196,762 US20030040078A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/196,759 US20030071835A1 (en) 1998-06-26 2002-07-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/197,942 US20030175882A1 (en) 1998-09-10 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,462 US20030054468A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,464 US20030032140A1 (en) 1997-09-18 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/199,316 US20030068726A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/198,768 US20030049756A1 (en) 1998-06-26 2002-07-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/205,904 US20030073813A1 (en) 1998-06-26 2002-07-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/213,199 US7381809B2 (en) 1998-09-09 2002-08-05 Compositions and methods for the treatment of immune related diseases
US10/226,739 US7390879B2 (en) 1999-06-15 2002-08-23 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/232,225 US20030096969A1 (en) 2000-06-02 2002-08-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/237,471 US20030119113A1 (en) 1999-07-20 2002-09-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/238,283 US20030073190A1 (en) 1998-07-01 2002-09-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/238,325 US20030104560A1 (en) 1998-07-01 2002-09-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/245,055 US20030073192A1 (en) 1999-07-20 2002-09-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/245,875 US20030119135A1 (en) 1999-07-20 2002-09-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/272,051 US20030108544A1 (en) 1999-09-01 2002-10-16 Compositions and methods for the diagnosis and treatment of tumor
US10/439,249 US20030228664A1 (en) 1998-12-16 2003-05-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/972,317 US7208321B2 (en) 1998-06-02 2004-10-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/025,607 US20050181478A1 (en) 1998-09-01 2004-12-28 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/100,159 US7425613B2 (en) 1997-11-05 2005-04-05 PRO1375 polypeptides
JP2005143079A JP2005323597A (en) 1999-12-23 2005-05-16 Il-17 homologous polypeptide and therapeutic use thereof
JP2005171206A JP4239021B2 (en) 1999-12-23 2005-06-10 IL-17 homologous polypeptide and therapeutic uses thereof
JP2005171514A JP2006006327A (en) 1999-12-23 2005-06-10 Il-17-homologous polypeptide and remedial use thereof
JP2005171112A JP2005348735A (en) 1999-12-23 2005-06-10 Il-17 homologous polypeptide and its therapeutic use
JP2005171331A JP4452659B2 (en) 1999-12-23 2005-06-10 IL-17 homologous polypeptide and therapeutic uses thereof
JP2005171424A JP2006006326A (en) 1999-12-23 2005-06-10 Il-17-homologous polypeptide and remedial use thereof
US11/189,442 US20060246465A1 (en) 1998-06-04 2005-07-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005264294A JP2006081548A (en) 2000-03-01 2005-08-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005264293A JP2006068016A (en) 1999-12-01 2005-08-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/240,891 US20060246540A1 (en) 1997-08-26 2005-09-29 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/283,473 US20090142800A1 (en) 1998-08-04 2005-11-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US11/311,555 US7473763B2 (en) 1999-12-23 2005-12-20 Receptor for IL-17 homologous polypeptides and uses thereof
US11/311,561 US20060088917A1 (en) 1999-12-23 2005-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US11/323,117 US20070092941A1 (en) 1998-09-16 2005-12-29 PRO1298 polypeptides
JP2006000562A JP4688682B2 (en) 1999-12-23 2006-01-05 IL-17 homologous polypeptide and therapeutic uses thereof
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/518,609 US20070077623A1 (en) 1997-09-17 2006-09-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2006266069A JP2007049999A (en) 2000-06-02 2006-09-28 Secreted and transmembrane polypeptide and nucleic acid encoding the same
US11/529,324 US8273703B2 (en) 1998-05-15 2006-09-29 IL-17 receptor-like polypeptides and therapeutic uses thereof
US11/538,754 US20070098634A1 (en) 1999-09-01 2006-10-04 Compositions and methods for the diagnosis and treatment of tumor
US11/786,466 US20080182275A1 (en) 1998-12-22 2007-04-10 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007114868A JP2007291109A (en) 1999-12-23 2007-04-24 Il-17 homologous polypeptide and therapeutic use thereof
US11/796,725 US20090197301A1 (en) 1998-09-01 2007-04-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2007326613A JP2008161190A (en) 1999-12-01 2007-12-18 Secreted and transmembrane polypeptide and nucleic acid encoding the same
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JP2008153789A JP2008301822A (en) 2000-03-01 2008-06-12 Secreted and transmembrane polypeptide and nucleic acid encoding the same
HK08110078.6A HK1114638A1 (en) 1999-12-23 2008-09-10 Il-17 homologous polypeptides and therapeutic uses thereof
HK08110071.3A HK1114634A1 (en) 1999-12-23 2008-09-10 Il-17 homologous polypeptides and therapeutic uses thereof
HK08110077.7A HK1114637A1 (en) 1999-12-23 2008-09-10 Il-17 homologous polypeptides and therapeutic uses thereof
HK08110072.2A HK1114635A1 (en) 1999-12-23 2008-09-10 Il-17 homologous polypeptides and therapeutic uses thereof
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JP2009171169A JP5258691B2 (en) 1999-12-23 2009-07-22 IL-17 homologous polypeptide and therapeutic uses thereof
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HK11104834.9A HK1150856A1 (en) 1999-12-23 2011-05-17 Il-17 homologous polypeptide and therapeutic uses thereof il-17
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JP2014138440A JP2014239685A (en) 1999-12-23 2014-07-04 Il-17 homologous polypeptides and therapeutic uses thereof
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Applications Claiming Priority (62)

Application Number Priority Date Filing Date Title
USPCT/US99/12252 1999-06-02
PCT/US1999/012252 WO1999063088A2 (en) 1998-06-02 1999-06-02 Membrane-bound proteins and nucleic acids encoding the same
US14473299P 1999-07-20 1999-07-20
US14475899P 1999-07-20 1999-07-20
US60/144,758 1999-07-20
US60/144,732 1999-07-20
US14622299P 1999-07-28 1999-07-28
US60/146,222 1999-07-28
USPCT/US99/20111 1999-09-01
PCT/US1999/020111 WO2000012708A2 (en) 1998-09-01 1999-09-01 Further pro polypeptides and sequences thereof
PCT/US1999/021547 WO2000015797A2 (en) 1998-09-17 1999-09-15 Compositions and methods for the treatment of immune related diseases
USPCT/US99/21547 1999-09-15
USPCT/US99/21090 1999-09-15
PCT/US1999/021090 WO2000015796A2 (en) 1998-09-16 1999-09-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US16250699P 1999-10-29 1999-10-29
US60/162,506 1999-10-29
USPCT/US99/28313 1999-11-30
PCT/US1999/028313 WO2000032221A2 (en) 1998-12-01 1999-11-30 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US1999/028634 WO2000036102A2 (en) 1998-12-16 1999-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US99/28634 1999-12-01
PCT/US1999/028551 WO2000053750A1 (en) 1999-03-08 1999-12-02 Compositions and methods for the treatment of tumors
USPCT/US99/28551 1999-12-02
PCT/US1999/028565 WO2000037638A2 (en) 1998-12-22 1999-12-02 Methods and compositions for inhibiting neoplastic cell growth
USPCT/US99/28565 1999-12-02
US17026299P 1999-12-09 1999-12-09
US60/170,262 1999-12-09
PCT/US1999/030911 WO2000075316A1 (en) 1999-06-02 1999-12-20 Methods and compositions for inhibiting neoplastic cell growth
USPCT/US99/30911 1999-12-20
USPCT/US00/00219 2000-01-05
PCT/US2000/000219 WO2000053753A2 (en) 1999-03-08 2000-01-05 Promotion or inhibition of angiogenesis and cardiovascularization
USPCT/US00/00376 2000-01-06
PCT/US2000/000376 WO2000053755A2 (en) 1999-03-08 2000-01-06 Compositions and methods for the treatment of tumor
PCT/US2000/003565 WO2001053486A1 (en) 1999-03-08 2000-02-11 Compositions and methods for the treatment of tumor
USPCT/US00/03565 2000-02-11
PCT/US2000/004342 WO2000078961A1 (en) 1999-06-23 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/04341 2000-02-18
USPCT/US00/04342 2000-02-18
PCT/US2000/004341 WO2000053756A2 (en) 1999-03-08 2000-02-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/04414 2000-02-22
PCT/US2000/004414 WO2001004311A1 (en) 1999-07-07 2000-02-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/04914 2000-02-24
PCT/US2000/004914 WO2000075327A1 (en) 1999-06-02 2000-02-24 Methods and compositions for inhibiting neoplastic cell growth
USPCT/US00/05004 2000-02-24
PCT/US2000/005004 WO2000053757A2 (en) 1999-03-08 2000-02-24 Promotion or inhibition of angiogenesis and cardiovascularization
PCT/US2000/005601 WO2000056889A2 (en) 1999-03-23 2000-03-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/05601 2000-03-01
PCT/US2000/005841 WO2000053758A2 (en) 1999-03-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
USPCT/US00/05841 2000-03-02
US18720200P 2000-03-03 2000-03-03
US60/187,202 2000-03-03
USPCT/US00/06884 2000-03-15
PCT/US2000/006884 WO2001005972A1 (en) 1999-07-20 2000-03-15 Compositions and methods for the treatment of immune related diseases
PCT/US2000/007377 WO2001019991A1 (en) 1999-09-15 2000-03-20 Compositions and methods for the treatment of immune related diseases
USPCT/US00/07377 2000-03-20
USPCT/US00/07532 2000-03-21
PCT/US2000/007532 WO2000070050A1 (en) 1999-05-14 2000-03-21 Compositions and methods for the treatment of immune related diseases
PCT/US2000/008439 WO2000073454A1 (en) 1999-06-02 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/08439 2000-03-30
USPCT/US00/13705 2000-05-17
PCT/US2000/013705 WO2000073445A2 (en) 1999-06-02 2000-05-17 Interleukin-1-receptor associated kinase-3 (irak3)
PCT/US2000/014042 WO2000077037A2 (en) 1999-06-15 2000-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
USPCT/US00/14042 2000-05-22

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PCT/US2000/005841 Continuation-In-Part WO2000053758A2 (en) 1994-09-08 2000-03-02 Compositions and methods for the treatment of immune related diseases
PCT/US2000/007532 Continuation-In-Part WO2000070050A1 (en) 1997-03-31 2000-03-21 Compositions and methods for the treatment of immune related diseases
PCT/US2000/008439 Continuation-In-Part WO2000073454A1 (en) 1994-09-08 2000-03-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/014042 Continuation-In-Part WO2000077037A2 (en) 1994-09-08 2000-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/014042 Continuation WO2000077037A2 (en) 1994-09-08 2000-05-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/014941 Continuation-In-Part WO2000073348A2 (en) 1996-11-06 2000-05-30 Methods and compositions for inhibiting neoplastic cell growth

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US31183299A Continuation-In-Part 1997-09-15 1999-05-14
PCT/US2000/020710 Continuation-In-Part WO2001009327A2 (en) 1994-09-08 2000-07-28 Method of preventing the injury or death of retinal cells and treating ocular diseases
PCT/US2000/022031 Continuation-In-Part WO2001040464A1 (en) 1996-11-06 2000-08-11 Interleukin-1-receptor associated kinase-3 (irak3) and its use in promotion or inhibition of angiogenesis and cardiovascularization
US64484800A Continuation-In-Part 1997-09-15 2000-08-22
US64484800A Continuation 1997-09-15 2000-08-22
PCT/US2000/023522 Continuation-In-Part WO2001016319A2 (en) 1996-11-06 2000-08-23 Compositions and methods for the treatment of immune related diseases
PCT/US2000/023328 Continuation-In-Part WO2001016318A2 (en) 1994-09-08 2000-08-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
PCT/US2000/032678 Continuation-In-Part WO2001040466A2 (en) 1996-11-06 2000-12-01 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/747,259 Continuation-In-Part US6569645B2 (en) 1997-03-31 2000-12-20 IL-17 homologous polypeptides and therapeutic uses thereof
US09/866,034 Continuation US20030170864A1 (en) 1996-11-06 2001-05-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US87203501A Continuation-In-Part 1996-11-06 2001-06-01
US09/903,749 Continuation US7147853B2 (en) 1997-09-17 2001-07-11 Anti-pro211 polypeptide antibodies
US10/028,072 Continuation US20030004311A1 (en) 1997-03-31 2001-12-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/081,056 Continuation US20040043927A1 (en) 1997-09-19 2002-02-20 Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis
US10/119,480 Continuation US20040087769A1 (en) 1998-09-10 2002-04-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/197,942 Continuation US20030175882A1 (en) 1998-03-27 2002-07-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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US8338132B2 (en) 2000-03-21 2012-12-25 Genentech, Inc. Nucleic acids encoding receptor for IL-17 homologous polypeptides and therapeutic uses thereof
US7256264B2 (en) 2000-08-24 2007-08-14 Genentech, Inc. Receptor for IL-17 homologous polypeptides and uses thereof
US7544482B2 (en) 2000-08-24 2009-06-09 Genentech, Inc. Nucleic acids encoding receptor for IL-17 homologous polypeptides and uses thereof
US7456255B2 (en) 2000-10-06 2008-11-25 Yale University Nogo receptor homologs that decrease inhibition of axonal elongation
US7173118B2 (en) 2000-10-06 2007-02-06 Biogen Idec Ma Inc. Nogo receptor homologs
US8455217B2 (en) 2000-10-24 2013-06-04 Genentech, Inc. Nucleic acids encoding IL-17 homologous receptor-like polypeptides and therapeutic uses thereof
US7034132B2 (en) 2001-06-04 2006-04-25 Anderson David W Therapeutic polypeptides, nucleic acids encoding same, and methods of use
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US8030456B2 (en) 2002-08-10 2011-10-04 Yale University Nogo receptor antagonists
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US7507808B2 (en) 2002-12-12 2009-03-24 Isis Pharmaceuticals, Inc. Modulation of endothelial lipase expression
WO2005101013A3 (en) * 2004-04-15 2006-03-02 Reglia Ab Materials and methods for screening modulators of neural regneration
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