WO1999035170A2 - Compositions and methods for the treatment of tumor - Google Patents

Compositions and methods for the treatment of tumor Download PDF

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Publication number
WO1999035170A2
WO1999035170A2 PCT/US1999/000106 US9900106W WO9935170A2 WO 1999035170 A2 WO1999035170 A2 WO 1999035170A2 US 9900106 W US9900106 W US 9900106W WO 9935170 A2 WO9935170 A2 WO 9935170A2
Authority
WO
WIPO (PCT)
Prior art keywords
ant
pro
antibody
pro509
pro201
Prior art date
Application number
PCT/US1999/000106
Other languages
French (fr)
Other versions
WO1999035170A9 (en
WO1999035170A8 (en
Inventor
David Botstein
Audrey Goddard
Austin L. Gurney
Kenneth J. Hillan
David A. Lawrence
Margaret Ann Roy
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
Application filed by Genentech, Inc. filed Critical Genentech, Inc.
Priority to AU22122/99A priority Critical patent/AU2212299A/en
Publication of WO1999035170A2 publication Critical patent/WO1999035170A2/en
Publication of WO1999035170A8 publication Critical patent/WO1999035170A8/en
Publication of WO1999035170A9 publication Critical patent/WO1999035170A9/en
Priority to US09/918,585 priority patent/US20030060406A1/en
Priority to US09/941,992 priority patent/US20030082546A1/en
Priority to US09/946,374 priority patent/US20030073129A1/en
Priority to US09/978,188 priority patent/US20030139328A1/en
Priority to US09/978,192 priority patent/US20020177553A1/en
Priority to US09/978,189 priority patent/US6972325B2/en
Priority to US09/978,298 priority patent/US20030134785A1/en
Priority to US09/978,295 priority patent/US20020156006A1/en
Priority to US09/978,194 priority patent/US20030195333A1/en
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Priority to US09/978,191 priority patent/US20030050239A1/en
Priority to US09/978,299 priority patent/US20030199435A1/en
Priority to US09/981,915 priority patent/US20030054986A1/en
Priority to US09/978,423 priority patent/US20030069178A1/en
Priority to US09/978,757 priority patent/US20030083248A1/en
Priority to US09/978,824 priority patent/US20050124789A9/en
Priority to US09/978,643 priority patent/US20030104998A1/en
Priority to US09/978,681 priority patent/US20030195148A1/en
Priority to US09/978,802 priority patent/US20030199674A1/en
Priority to US09/978,608 priority patent/US20030045462A1/en
Priority to US09/978,375 priority patent/US7196165B2/en
Priority to US09/978,564 priority patent/US7195760B2/en
Priority to US09/978,544 priority patent/US20030199436A1/en
Priority to US09/978,665 priority patent/US7294700B2/en
Priority to US09/978,585 priority patent/US20030049633A1/en
Priority to US09/978,403 priority patent/US20030050240A1/en
Priority to US09/978,697 priority patent/US20020169284A1/en
Priority to US09/981,915 priority patent/US7285623B2/en
Priority to US09/978,824 priority patent/US20030055216A1/en
Priority to US10/152,388 priority patent/US20040223964A1/en
Priority to US10/210,028 priority patent/US20030203446A1/en
Priority to US10/165,036 priority patent/US20050227342A1/en
Priority to US10/164,929 priority patent/US20030194781A1/en
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Priority to US10/145,089 priority patent/US7208575B2/en
Priority to US10/162,521 priority patent/US7067628B2/en
Priority to US10/145,017 priority patent/US20030186365A1/en
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Priority to US09/978,187 priority patent/US20030096744A1/en
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Priority to US10/123,215 priority patent/US7291329B2/en
Priority to US10/123,108 priority patent/US7635478B2/en
Priority to US10/123,157 priority patent/US20030190725A1/en
Priority to US10/123,213 priority patent/US7193048B2/en
Priority to US10/123,212 priority patent/US7276577B2/en
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Priority to US10/123,907 priority patent/US7084258B2/en
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Priority to US10/160,498 priority patent/US20030073216A1/en
Priority to US10/176,913 priority patent/US20030022298A1/en
Priority to US10/213,199 priority patent/US7381809B2/en
Priority to US11/025,607 priority patent/US20050181478A1/en
Priority to US11/100,159 priority patent/US7425613B2/en
Priority to US11/341,175 priority patent/US7468427B2/en
Priority to US11/796,725 priority patent/US20090197301A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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

Definitions

  • cancers Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al , CA Cancel J Clin 43, 7 [ 1993])
  • Cancer is characterized by the increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis)
  • a cell proliferates under conditions in which normal cells would not grow Cancer manifests itself in a wide vdnety of forms, characterized by different degrees of invasiveness and aggressiveness Alteration of gene expression is intimately related to the uncontrolled cell growth and de- differentiationwhich are a common feature of all cancers
  • the genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which v ⁇ uld normally function to prevent malignant cell growth, and/or overexpression of certair dominant genes, such as oncogenes, that act to promote malignant growth
  • tumor suppression genes which v ⁇ uld normally function to prevent malignant
  • a well known mechanism of gene (e g oncogene) overexpression in cancer cells is gene amplification This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome ( Alitalo et al , Adv Cancer Res 47, 235-281 [1986]) It is believed that the overexpression of the gene parallels gene amplification, I e is proportionate to the number of copies made
  • Proto-oncogenesthat encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer
  • the human ErbB2 gene erbB2. also known as her2, or c-erbB-2
  • her2, or c-erbB-2 which encodes a 185-kd transmembrane glycoprote receptor (pl 85 HER2 , HER2) related to the epidermal growth factor receptor (EGFR)
  • EGFR epidermal growth factor receptor
  • rhuMAb HER2 or Herceptin ® has been clinically active in patients with ErbB2-overexpress ⁇ ng metastatic breast cancers that had received extensive prior anticancer therapy (Baselga et al , -/ Clin Oncol 14 737-744 [1996]) Summary of the Invention
  • the present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans
  • the present invention is based on the identification of genes that are amplified in the genome of tumor cells Such gene amplification is expected to be associated with the overexpression of the gene product and conc ⁇ bute to tumo ⁇ genesis Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevent ⁇ on)of certain cancers, and may act of predictors of the prognosis of tumor treatment
  • the present invention concerns an isolated antibody which binds a polypeptide which is designated PRO201 (UNQ175), PR0292 (UNQ255), PR0327 (UNQ288). PR01265 (UNQ636), PR0344 (UNQ303), PR0343 (UNQ302), PR0347 (UNQ306), PR0357 (UNQ314).
  • the antibody induces death of a cell overexpressing a PRO20I , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357.
  • the antibody is a monoclonal antibody, which preferably has nonhuman compiementa ⁇ tydeterm ⁇ n ⁇ ngreg ⁇ on (CDR) residues and human framework region (FR) residues
  • CDR nonhuman compiementa ⁇ tydeterm ⁇ n ⁇ ngreg ⁇ on
  • FR human framework region
  • the antibody may be labeled and may be immobilized on a solid support
  • the antibody is an antibody fragment, a single-chain antibody, or an anti-idiotypic antibody
  • the invention concerns a composition
  • a composition comprising an antibody which binds a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509 , PR0853 or PR0882 polypeptide m admixture with a pharmaceutically acceptable carrier
  • the composition comprises a therapeutically effective amount of the antibody
  • the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent
  • the composition is sterile
  • the invention concerns nucleic acid encoding an ant ⁇ -PRO201 , anti- PR0292. ant ⁇ -PR0327.ant ⁇ -PR01265,ant ⁇ -PR0344.ant ⁇ -PR0343,ant ⁇ -PR0347.ant ⁇ -PR0357,ant ⁇ -PR0715, ant ⁇ -PRO1017, anti-PROl 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody, and vectors and recombinant host cells comprising such nucleic acid
  • the invention concerns a method for producing an ant ⁇ -PRO201 , anti- PR0292. ant ⁇ -PR0327,ant ⁇ -PR01265,ant ⁇ -PR0344.ant ⁇ -PR0343,ant ⁇ -PR0347.ant ⁇ -PR0357 ant ⁇ -PR0715, ant ⁇ -PRO1017,ant ⁇ -PROl 1 12,ant ⁇ -PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody by culturing a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the antibody from the cell culture
  • the invention further concerns antagonists and agonists of a PRO201 , PR0292, PR0327.
  • the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides
  • the nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions
  • Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions
  • sequences can be used as part of ⁇ bozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes
  • the invention concerns a method for determimngthe presence of a PRO201,
  • the present invention concerns a method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypept ⁇ de(a) in 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 expression ievel in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained
  • the present invention concerns a method of diagnosing tumor in a mammal, comprising (a) contacting an ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, ant ⁇ -PR01265, ant ⁇ -PR0344, anti- PR0343. ant ⁇ -PR0347, ant ⁇ -PR0357. ant ⁇ -PR0715 ant ⁇ -PRO1017, anti-PROl 1 12, ant ⁇ -PRO509, anti- PR0853 or ant ⁇ -PR0882 antibody with a test sample of tissue cells obtained from the mammal , and (b) detecting the formation of a complex between the ant ⁇ -PRO201 , ant ⁇ -PR0292.
  • 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 sampie indicates the presence of tumor in the mammal from which the test tissue cells were obtained
  • the antibody preferably carries a detectable label Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluo ⁇ metry, or other techniques known in the art
  • test sample is usually obtained from an individual suspected to have neoplastic cell growth or proliferation (e g cancerous cells)
  • the present invention concerns a cancer diagnostic kit, comprising an anti-
  • PRO201 ant ⁇ -PRO292,ant ⁇ -PRO327.ant ⁇ -PRO1265,ant ⁇ -PRO344,ant ⁇ -PRO343,ant ⁇ -PRO347,ant ⁇ -PRO357, ant ⁇ -PR0715, ant ⁇ -PRO1017, anti-PROl 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody and a carrier (e g a buffer) in suitable packaging
  • the kit preferably contains instructions for using the antibody to detect the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide
  • the invention concerns a method for inhibitingthe growth of tumor cells comprising exposing a cell which overexpressesa PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017.
  • PROl 1 12, PRO509, PR0853 or PR0882 polypeptideto an effective amount of an agent inhibiting the expression and/or activity of the PR 201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347.
  • the agent preferably is an ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327. anti-PROl 265, anti- PR0344, ant ⁇ -PR0343, ant ⁇ -PR0347, ant ⁇ -PR0357, ant ⁇ -PR0715, ant ⁇ -PRO1017, anti-PROl 1 12, anti- PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody, a small organic and inorganic molecule, peptide, phosphopeptide, antisense or ⁇ bozyme molecule, or a triple helix molecule
  • the agent e g ant ⁇ -PRO201,ant ⁇ -PRO292,ant ⁇ -PRO327,ant ⁇ -PRO1265,ant ⁇ -PRO344.ant ⁇ -PRO343,ant ⁇ -PRO347,ant ⁇ - PRO357,ant ⁇ -PRO715,ant ⁇
  • the invention concerns an article of manufacture, comprising a container, a label on the container, and a composition comprising an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327, PR01265.
  • PR0882 polypeptide, and the active agent in the composition is an agent inhibiting the expression and/or activity of the PRO201 PR0292, PR0327, PR01265 PR0344, PR0343, PR0347 PR0357.
  • PR0715 is an agent inhibiting the expression and/or activity of the PRO201 PR0292, PR0327, PR01265 PR0344, PR0343, PR0347 PR0357.
  • the active agent is an ant ⁇ -PRO201. ant ⁇ -PR0292, ant ⁇ -PR0327. anti-PRO 1265,ant ⁇ -PR0344, ant ⁇ -PR0343. ant ⁇ -PR0347 anti-
  • PRO201 PRO0292, PR0327.
  • PRO 1265 PR0344, PR0343, PR0347, PR0357, PR0715.
  • PRO509, PR0853 or PR0882 polypeptide comprising contacting a candidate compound with a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12,
  • PRO509, PR0853 or PR0882 polypeptide under conditions and for a time sufficient to allow these two components to interact
  • the candidate compound or the PRO201 , PR0292,
  • PRO509 PR0853 or PR0882 polypeptide is immobilized on a solid support Brief Description of the Figures
  • Figure 1 is the nucleotide sequence of DNA30676 encoding a PRO201 (UNQ 175)
  • Figure 2 is the ammo acid sequence of the native human PRO201 (LTNQ175) polypeptide
  • Figure 3 is the nucleotide sequence of DNA35617 encoding a PR0292
  • Figure 4 is the amino acid sequence of the native human PR0292 (UNQ266) polypeptide
  • Figure 5 is the nucleotide sequence of DNA381 13 encoding a PR0327
  • Figure 6 is the ammo acid sequence of the native human PR0327 (UNQ288) polypeptide
  • Figure 7 is the nucleotide sequence of DNA60764 encoding a PRO 1265
  • Figure 8 is the ammo acid sequence of the native human PRO 1265 (UNQ636) polypeptide
  • Figure 9 is the nucleotide sequence of DNA40592 encoding a PR0344
  • Figure 10 is the ammo acid sequence of the native human PR0344 (UNQ303) polypeptide
  • Figure 1 1 is the nucleotide sequence of DN A43318 encoding a PR0343
  • Figure 12 is the ammo acid sequence of the native human PR0343 (UNQ302) polypeptide
  • Figure 13 is the nucleotide sequence of DNA44176 encoding a PR0347
  • Figure 14 is the ammo acid sequence of the native human PR0347 (UNQ306) polypeptide
  • Figure 15 is the nucleotide sequence of DNA44804 encoding a PR0357
  • Figure 16 is the ammo acid sequence of the native human PR0357 (UNQ314) polypeptide
  • Figure 17 is the nucleotide sequence of DNA52722 encoding a PR0715
  • Figure 18 is the amino acid sequence of the native human PR0715 (UNQ383) polypeptide
  • Figure 19 is the nucleotide sequence of DN A561 12 encoding a PRO 1017
  • Figure 20 is the amino acid sequence of the native human PRO 1017 (UNQ500) polypeptide
  • Figure 21 is the nucleotide sequence of DNA57702 encoding a PROl 1 12
  • Figure 22 is the ammo acid sequence of the native human PROl 1 12 (UNQ555)
  • Figure 23 is the nucleotide sequence of DNA50148 encoding a PRO509
  • Figure 24 is the amino acid sequence of the native human PRO509 polypeptide
  • Figure 25 is the nucleotide sequence of DNA48227 encoding a PRO1350
  • Figure 26 is the ammo acid sequence of the native human PRO 1350 polypeptide (UNQ419)
  • Figure 27 is the nucleotide sequence of DNA58125 encoding a PR0882
  • Figure 28 is the amino acid sequence of the native human PR0882 polypeptide (UNQ448)
  • Figure 29 is the nucleotide sequence of DNA28710 used in the cloning of DNA 30676 encoding PRO201
  • Figure 30 is the nucleotide sequence encoding EST 2452972
  • Figure 31 is the nucleotide sequence encoding EST2099855
  • Figure 32 is a map of chromosome 19 showing the mapping regions of DNA30676, DNA381 13 and DNA60764
  • Figure 33 is a map of chromosome 1 1 show ing the mapping region of DNA354617
  • Figure 34 is a map of chromosome 16 showing the mapping region of DNA58125 and DNA43318
  • Figure 35 is a map of chromosome 7 showing the mapping region of DNA561 12
  • Figure 36A is map of chromosome ' 7 showing the mapping region of DNA52722
  • Figure 36B is a map of chromosome 17 showing the mapping region of DNA48227
  • Figures 37-39 are in situ hybridizations as described in the example section
  • Figure 40 is a map of chromosome 16 showing the mapping region of DNA44804
  • gene amplification and “gene duplication ' are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line
  • the duplicated region (a stretch of amplified DNA) is often referred to as "amplicon " Usually. the amount of the messenger RNA (mRNA) produced, I e the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed "Tumor as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized bv unregulated cell growth
  • examples of cancer include but are not limited to, carcinoma, lymphoma blastoma.sarcoma, and leukemia More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastromtestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e g radiation and/or chemotherapy
  • the "pathology" of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc
  • “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, horses, cats, cows, etc Preferably, the mammal is human
  • Carriers as used herein include pharmaceuticallyacceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being e) ; ⁇ sed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin, or ⁇ mmunoglobul ⁇ ns,hydroph ⁇ l ⁇ c polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagme, arginine or lysine, monosaccha ⁇ des,d ⁇ saccha ⁇ des,and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counte ⁇ on
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells
  • the term is intended to include radioactive isotopes (e g I 13 ', I 125 , Y w and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer Examples of chemotherapeuticagents include ad ⁇ amycin doxorubicin.epirubicin, 5-fluorourac ⁇ l, cytosine arabinoside ("Ara-C”) cyclophosphamide.
  • thiotepa busulfan.
  • cytoxin cytoxin, taxoids, e g paclitaxel (Taxol Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate cisplatin.melphalan, vinblastine.bleomycin.etoposide. ifosfamide.mitomycinC, itoxantrone, vincristine. vinorelbine, carboplatm, teniposide, daunomycin, carminomycin, ammopte ⁇ n.
  • growth inhibitory 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 m vitro or in vivo
  • growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase) such as agents that induce Gl arrest and M-phase arrest
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorub ⁇ c ⁇ n,ep ⁇ rub ⁇ c ⁇ n,daunorub ⁇ c ⁇ n,etopos ⁇ de and bleomycin
  • doxorub ⁇ c ⁇ n,ep ⁇ rub ⁇ c ⁇ n,daunorub ⁇ c ⁇ n,etopos ⁇ de and bleomycin Those agents that arrest G 1 also spill over into S-phase arrest, for example, DNA alkylating
  • Doxorubicin is an athracycline antibiotic
  • the full chemical name of doxorubicin is (8S-c ⁇ s)- 10- [(3-am ⁇ no-2,3,6-t ⁇ deoxy-o.-L-lyxo-hexapyranosyI)oxy]-7,8,9, 10-tetrahydro-6,8.1 l -t ⁇ hydroxy-8- (hydroxyacety 1)- 1 -methoxy-5, 12-naphthacened ⁇ one
  • cytokme is . generic term for proteins released by one cell population which act on another cell as intercellular mediators
  • lymphokines include lymphokines. monokines, and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxm, prorelaxin, glycoprote hormones such as follicle stimulating hormone (FSH), thyroid st ⁇ mulat ⁇ nghormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor- ⁇ and - ⁇ , mullerian-inhibiting substance, mouse gonadotropm-associatedpeptide, inhibin, activin.
  • growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
  • parathyroidhormone parathyroidhormone
  • vascular endothelial growth factor ⁇ nteg ⁇ n,thrombopo ⁇ et ⁇ n (TPO)
  • nerve growth factors such as NGF- ⁇ , platelet-growth factor, transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ , insulin-like growth factor-I and -II, erythropoietin (EPO), osteomductive factors, interferons such as mterferon - ⁇ , - ⁇ , and - ⁇ , colonv 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- l ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9.
  • TGFs transforming growth factors
  • EPO erythropoie
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines
  • prodrug ' refers to a precursor or derivative form of a pharmaceutically active substance that is iess cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form See, e g Wilman, "Prodrugs in Cancer Chemotherapy ⁇ Biochemical Society Transactions.
  • the prodrugs of this invention include, but are not limited to, phosphate-contaimngprodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glysocyiated prodrugs, ⁇ -lactam- containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-contaming prodrugs, 5-fluorocytos ⁇ ne and other 5-fluorou ⁇ d ⁇ ne prodrugs which can be converted into the more active cytotoxic free drug
  • cytotoxic drugs that can be de ⁇ vatized into a prodrugs form for use in this invention include, but are not limited to, those chemotherapeutic agents described above
  • PRO201 polypeptide encompass both native sequence and active variants thereof Th PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic means The term specifically encompasses naturally-occurringtruncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms
  • PRO509, PR0853 or PR0882 polypeptide Fragments of the respective native polypeptides herein include, but are not limited, to polypeptide variants from which the native N-terminal signal sequence has been fully or partially deleted or replaced by another sequence, and extracellular domains of the respective native sequences, regardless whether such truncated (secreted) forms occur in nature Fragments are preferably sufficient in length for the production of an antibody specifically binding the corresponding native "PRO" polypeptide
  • the terms “UNQ 175", “UNQ255”, “UNQ288”, “UNQ636”, “UNQ303”, “UNQ302”, “UNQ306”, “UNQ314", “UNQ383”, “UNQ500”, “UNQ555”, “LTNQ329”, “UNQ419” and “LTNQ448” are used to refer to the native sequence polypeptide corresponding to the PRO designated sequences above appearing as a full-length native presequence or a mature form of a PRO201 polypeptide shown in Figure 2 (SEQ ID NO 2), a PR0292 polypeptide shown in Figure 4 (SEQ ID NO 4), a PR0327 polypeptide shown in Figure 6 (SEQ ID NO 6), a PRO 1265 polypeptide shown in Figure 8 (SEQ ID NO 8), a PR0344 polypeptide shown in Figure 10 (SEQ ID NO 10), a PR0343 polypeptide shown in Figure 12 (SEQ ID NO 12), a PR0347 polypeptide shown in Figure 14 (SEQ
  • a PR0715 polypeptide shown in Figure 18 (SEQ ID NO 18).
  • a PROl 017 polypeptide shown in Figure 20 (SEQ ID NO 20)
  • a PROl 1 12 polypeptide shown in Figure 22 (SEQ ID NO 22).
  • a PRO509 polypept ⁇ deshown ⁇ n F ⁇ gure24 (SEQ ID NO 24)
  • a PR0853 polypeptide shown in Figure 26 (SEQ IDNO 26).
  • a R0882 polypept ⁇ deshown ⁇ n F ⁇ gure28 (SEQ ID O 28). respectively Said another way, the "UNQ" designation refers to the specific native sequence while the "PRO” designation refers to the native sequence and active variants thereof
  • nucleic acid molecule encoding a PRO201, PR0292. PR0327, PRO 1265. PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509.
  • PR0853 or PR0882 polypeptide 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 PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-. PR0343-, PR0347-, PR0357-, PR0715-. PRO 1017-.
  • PRO 1 1 12-, PRO509-, PR0853- or PR0882-encodmg nucleic acid An isolated PRO201 -, PR0292-, PR0327-. PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from the PRO201-, PRO292-, PRO327-, PROl 265-. PR0344-, PR0343-.
  • nucleic acid molecule encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide includes nucleic acid molecules contained in cells that ordinarily express PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding se ⁇ .ience in a particular host organism
  • the control sequences that are suitable for prokaryot include a promoter, optionally an operator sequence, and a ribosome binding site
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotem that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • "operably linked” means that the DNA sequences being linked are contiguous, and.
  • Hybndization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hyb ⁇ dizable sequence the higher the relative temperature which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so
  • “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/00015 M sodium c ⁇ trate/0 1% sodium dodecyl sulfate at 50° C , (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1% bovine serum album ⁇ n/0 1% F ⁇ coll/0 1% polyv ⁇ nylpyrrol ⁇ done/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 ⁇ g/m
  • Modely stringent conditions may be identified as described by Sambrook et al Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press, 1989, and include the use of washing solutior and hybridization conditions (e g , temperature, ionic strength and % SDS) less st ⁇ ngen' than those described above
  • washing solutior and hybridization conditions e g , temperature, ionic strength and % SDS
  • An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl. 15 M t ⁇ sodium citrate).
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO201 ,
  • tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 ammo acid residues (preferably, between about 10 and 20 am o acid residues)
  • “Active” or “activity” m the context of molecules identified based upon the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptides (or their coding sequences) refers to polypeptides (e g antibodies) or organic or inorganic small molecules, peptides, e e which retain the biological and/or lmmunologicalactivities/properties of a native or naturally-occurring PRO201.
  • Bioactivity in the context of an antibody or another molecule that can be identified by the screening assays disclosed herein is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins
  • a preferred biological activity is growth inhibition of a target tumor cell
  • Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell
  • immunologicalcross-reactivity means immunologicalcross-reactivity with at least one epitope of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112.
  • PRO509, PR0853 or PR0882 polypeptide "Immunolog ⁇ caicross-react ⁇ v ⁇ ty"as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having this activity with polyclonal antisera raised against the known active PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357. PR0715, PRO 1017, PRO 1 1 12. PRO509.
  • PR0853 or PR0882 polypeptide Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intrape ⁇ toneal or subcutaneous injection in incomplete Freunds
  • the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (o g antibody) identified, to the corresponding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide is significantly higher (preferablyat least about 2-t ⁇ mes, more preferably at least about 4-t ⁇ mes, even more preferably at least about 8-t ⁇ mes, most preferably at least about 8-t ⁇ mes higher) than the binding affinity of that molecule to any other known native polypeptide
  • antagonist is used in the broadest sense, and includes any molecule that p rtially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343. PR0347.
  • agonist is used in the broadest sense and inch des any molecule that mimics a biological activity of a native PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide disclosed herein
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc
  • a "small molecule” is defined herein to have a molecular weight below about 500 Dalton
  • Antibodies (Abs) and “immunoglobulins” (Igs) are glycoprotems having the same structural characteristics While antibodiesexhibit bindingspecificityto a specific antigen, immunoglobulins include both antibodies and other antibody-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
  • antibody is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity
  • “Native antibodies” and “native immunoglobulins” are usually heterotetrame ⁇ c glycoproteins of about 150.000 Dalton, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain 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 and light chain also has regularly spaced lntracham disulfide bridges Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains Each light chain has a variable domain at one end (V L ) and a constant domain at its other end.
  • V H variable domain
  • V L variable domain at one 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 of the heavy chain
  • Particular ammo acid residues are believed to form an interface between the light- and heavy- chain variable domains
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
  • CDRs complementarity-determining regions
  • hyperva ⁇ able regions both in the light-chain and the heavy-chain variable domains
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuia ⁇ on.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, cont ⁇ buteto the formation ofthe antigen-bindingsite of antibodies (see Kabat et al MH Pub/ No 91-3242, Vol I, pages 647-669 ( 1991 ))
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody u rntibody-dependent cellular toxicity
  • hyperva ⁇ ableregion when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding
  • the hyperva ⁇ able region comprises amino acid residues from a ' complementa ⁇ tydete ⁇ iin ⁇ ngreg ⁇ on" to "CDR" (i e residues 24-34 (L I ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H 1 ), 50-65 (H2) and 95- 102 (H3) in the heavy chain variable domain, Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institute of Health, Bethesda.
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody
  • antibody fragments include Fab. Fab', F(ab'),, and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng 8(10) 1057-1062 [ 1995]), single-chain antibodv molecules, and multispecific antibodies formed from antibody fragments
  • Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab” fragments, each with a single antigen-bindingsite, and a residual "Fc” fragment whose name reflects its ability to crystallize readily
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen
  • Fv is the minimum antibody fragment which contains a complete antigen-recognitionand -binding site This region consists ofa dimer ofone heavy- and one light-chain variable domain in tight non-covalent association It is in this configuration that the three
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) 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 CH I domain including one or more cystemes from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(s) of the constant domains bear a free thiol group F(ab').
  • antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the ammo acid sequences of their constant domains
  • immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG l , IgG2, IgG3, IgG4, IgA. and IgA2
  • the heavy-chain constant domains that corres ⁇ -nd to the different classes of immunoglobulins are called ⁇ , ⁇ , e, ⁇ , and ⁇ , 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 occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore in contrast to conventional (polyclonal)ant ⁇ body preparationswhich 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 specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hyb ⁇ doma culture, uncontammatedby other immunoglobulins The modifier "monoclonal" indicates the character of the antibody as being 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
  • the "monoclonal antibodies” may also be isolated from phage antibodv libraries using the techniques described in Clackson et al nature. 352 624-628 [ 1991 ] and Marks et al J Mol Biol , 222 581 -597 ( 1991 ). for example
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cha ⁇ n(s) is identical with or homologous to correspondingsequencesin 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 Proc Nati Acad Sci -/S ⁇ , 81 6851 -6855 [ 1984]) "Humanized" forms of non-human (e g , mu ⁇ ne) antibodies are chimeric immunoglobulins, lmmunogiobulin chains or fragments thereof (such as Fv.
  • humanized antibodies are human ⁇ mmunoglobul ⁇ ns(rec ⁇ p ⁇ entant ⁇ body) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse. rat or rabbit having the desired specificity, affinity, and capacity In some instances.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modificationsare made to further refine and maximize antibody performance
  • the humanized antibody will comprise substantially all of a ⁇ 'east one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulinand all or substantiallyall of the FR regions are those of a human lmmunoglobulinsequence
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobu nconstant region (Fc), typically that of a human lmmunogiobulin
  • Fc immunoglobu nconstant region
  • the humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L )
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites
  • Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Hollmger e/ ⁇ / , Proc Nati Acad Sci US4. 90 6444-6448 (1993)
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and av include enzymes, hormones, and other proteinaceousor nonproteinaceoussolu.es
  • the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferablymore than 99% by weight.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody
  • the label may be detectable by itself (e g radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable Radionuclides that can serve as detectable labels include, for example. I- 131 , 1- 123, 1- 125, Y-90 Re-
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysacchandes (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149
  • a "l ⁇ posome” ⁇ s a small vesicle composed of various types of lipids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as an PRO201, PRO292, PRO327, PR01265. PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017. PRO 1 1 12, PRO509. P ⁇ 0853 or PR0882, or an antibody thereto and. optionally, a chemotherapeuticagent) to a mammal
  • a drug such as an PRO201, PRO292, PRO327, PR01265.
  • PRO 1 1, PRO509.
  • PRO882 PRO509.
  • an antibody thereto and. optionally, a chemotherapeuticagent to a mammal
  • the components of the hposomeare commonly arranged in a bilayer formation, similar to the hpid arrangement of biological membranes
  • the term "lmmunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of lmmunogiobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (l e , is "heterologous"),and an lmmunoglobulinconstantdomain sequence
  • the adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand
  • the lmmunogiobulin constant domain sequence in the lmmunoadhesin may be obtained from any lmmunogiobulin, such as IgG- 1 , lgG-2, IgG-3. or IgG-4 subtypes, IgA (including I
  • PR0357 PRQ715.
  • PRO 1017 PRO 1 1 12.
  • PRO509. PR0853 or PRQ882 polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO20 I (UNQ 175), PR0292 (UNQ255).
  • PR0327 LTNQ288), PRO 1265 (UNQ636), PR0344 (UNQ303), PR0343 (UNQ302), PR0347 (UNQ306), PR0357 (UNQ314), PRO715 (UNQ383),PRO1017 (UNQ500), PROl 1 12 (UNQ555), PRO509 (UNQ329), PR0853 (UNQ419) or PR0882 (UNQ448)
  • PRO509, PR0853 or PR0882 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below In the present specification the proteins encoded by nucleic acid referred to as "DNA30676”. "DNA35617” “DNA381 13", “DNA60764", “DNA40592”, “DNA43318". "DNA44176”, “DNA44804", “DNA52722”, “DNA561 12", “DNA57702”, “DNA50148”, “DNA48227”, “DNA58125” are referred to as UNQ175. UNQ255. UNQ288, UNQ636, UNQ303, UNQ302, UNQ306, UNQ314, UNQ383. UNQ500, UNQ555.
  • PRO201 PRO201, PR0292, PR0327, PR01265.
  • PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides by culturing cells transformed or transfected with a vector containing PRO201 -, PR0292-.
  • alternative methods which are well known in the art, may be employed to prepare PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343.
  • PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides For instance, the PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343, PR0347 PR0357, PR07I 5.
  • PROl 017, PROl 1 12, PRO509, PR0853 or PR08P polypeptide 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, C A ( 1969), Merrifield, J Am Chem Soc , 85 2149- 2154 (1963)]
  • In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions Various portions of the PRO201 , PR0292, PR0327.
  • PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO201, PR0292, PR0327, PR01265, PR0344. PR0343, PR0347. PR0357, PR0715. PRO1017, PROl 1 12, PRO509, PR0853 or PR0882
  • PR0853 or PR0882 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples PRO201-. PR0292-, PR0327-, PRO 1265-. PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-. PRO 1 1 12-. PRO509-. PR0853- or PR0882-encod ⁇ ng gene may also be obtained from a genomic library or by oligonucleotide synthesis
  • Probes such as antibodies to the PRO20 I , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12.
  • PRO509, PR0853 or PR0882 polypeptide, or ohgonucleotides of at least about 20-80 bases designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989) An alternative means to isolate the gene encoding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357.
  • PR0853 or PR0882 is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]
  • the oligonucleotide sequences selected as probes should be oi sufficient length and sufficiently unambiguous that false positives are minimized
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened
  • Methods of labeling are well known in the art. and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
  • Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private 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 determined through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT which employ various algorithms to measure homology
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and. if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA b Selection and Transformation of Host Cells
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO201, PRO292,PRO327, PRO1265,PRO344, PRO343, PRO347,PRO357, PRO715, PRO1017.PRO11 12, PRO509, PR0853 or PR0882 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants. or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation In general, principles, protocols, and practical techniques for maximizing the productivity ofcell culturescan be found m Mammalian Cell Biotechnology 4 Practical Approach. M Butler, ed (IRL Press. 1991 ) and Sambrook et al . supra
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram- negative or Gram-positiveorganis ⁇ is, for example, Enterobacte ⁇ aceaesuch as E coli
  • E coli strains are publicly available, such as £ coli K 12 strain MM294 (ATCC 31 ,446), £ coli X I 776 (ATCC 31 ,537), £ coli strain W31 10 (ATCC 27,325) and K5 772 (ATCC 53,635)
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-.
  • PROl ! 12-, PR 509-, PR0853- or PR0882-encod ⁇ ng vectors Saccharomvces cerevisiae is a commonly used lower eukaryotic host microorganism
  • PR0853 or PR0882 are derived from multicellularorganisms
  • invertebrate cells include insect cells such as Drosophila S2 and SpodopteraSf9, as well as plant cells
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol , 36 59 (1977)), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasm, Proc Nati Acad Sci USA, 77 4216 ( 1980)), mouse sertoli cells (TM4, Mather, Biol
  • the nucleic acid (e g , cDNA or genomic DNA) encoding PRO201 , PR0292. PR0327, PRO 1265. PR0344, PR0343, PR0347. PR0357. PR0715, PRO1017, PRO l 1 12, PRO509. PR0853 or PR0882 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression Various vectors are publicly available
  • the vector may, for example, be in the form of a plasmid.
  • vector components generally include, but are not limited to. one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
  • PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
  • the signal sequence may be a component of the vector, or it may be a part of the PRO201-, PR0292-. PR0327-. PRO 1265-, PR0344-. PR0343- PR0347- PR0357-, PR0715-.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptidesof 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 !• ,st cells Such sequences are well known for a variety of bacteria, yeast, and viruses
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for clonirg vectors in mammalian cells
  • Selection genes will typically contain a selection gene, also termed a selectable marker
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicilhn, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply c ⁇ tical nutrients not available from complex media, e g , the gene encoding D-alanine racemase for Bacilli
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO201-.
  • An appropnatehost cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaube/ ⁇ / Proc Nail Acad Sci USA, 77.4216 ( 1980)
  • a suitable selection gene for use in yeast is the trp I gene present in the yeast plasm id 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 in tryptophan. for example. ATCC No 44076 or PEP4-1 [Jones. Genetics, 85
  • Expression and cloning vectors usually contain a promoter operably linked to the PRO201 -, PR0292- , PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0 15-, PRO 1017-.
  • PRO 1 1 12-, PRO509- , PR0853- or PR0882-encod ⁇ ng nucleic acid sequence to direct mRNA synthesis
  • Promoters recognized by a variety of potential host cells are well known Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al Nature, 275 615 ( 1978), Goeddel et al .
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate k ⁇ nase [H ⁇ tzemane ⁇ / J Biol Chem , 255 2073 ( 1980)] or other glycolytic enzymes [Hess et al , J Adv Enzvme Reg , 7 149 (1968), Holland, Biochemistry, 17 4900 (1978)], such as enolase.
  • glyceraldehyde-3- ⁇ hosphate dehydrogenase hexokinase
  • pyruvate decarboxylase phosphofructokinase glucose-6-phosphate isomerase
  • 3-phosphoglycerate mutase pyruvate kinase
  • t ⁇ osephosphate isomerase
  • phosphoglucose isomerase and glucokinase
  • yeast promoters which are inducible promotershaving the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase2. isocytochromeC, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73.657
  • PROl 1 12, PRO509, PR0853 or PR0882 transcription from vectors in mammalian host cells is controlled, for example, by Dromoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 l,504pub'!3hed5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarccma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Vims 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an lmmunogiobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
  • viruses such as polyoma virus
  • Enhancers are cis-actmg elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription
  • Many enhancer sequences are now known from mammalian genes (globin, elastase. albumin, ⁇ -fetoprotein.
  • an enhancer from a eukaryotic cell vims examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PRO201 -, PR0292-. PR0327-, PRO 1265-. PR0344- PR0343- PR0347-.PR0357-,PR0715- PRO10I 7- PROl 1 12-. PRO509-. PRO853-or PR0882- coding sequence, but is preferably located at a site 5' from the promoter
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3'. untranslatedregions of eukaryotic or viral DN As or cDNAs These regions contain nucleotide segments transcribed as polyadenylatedfragments in the untranslated portion of the mRNA encoding PRO201, PR0292, PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO201 ,
  • PR0292, PR0327. PR01265, PR0344, PR0343, PR0347. PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in recombinant vertebrate cell culture are described in Gethmg et al Nature, 293 620-625 ( 1981 ). Mantel et al Nature, 281 40-46 (1979), EP 1 17,060, and EP 1 17,058 d Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting.
  • Gene expression may be measured by immunological methods, such as immunohis. :chem ⁇ calsta ⁇ n ⁇ ng of cells or tissue sections and assay of cell culture or body fluids, to qtr ititate directly the expression of gene product
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PRO! 265, PR0344, PR0343, PR0347, PR0357, PR0715.
  • PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO201, PR0292, PR0327, PR01265, PR0344, PR0343.
  • PROl 1 12, PRO509, PR0853 or PR0882 polypeptides may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (eg Tnton-X 100) or by enzymatic cleavage Cells employed in expression of PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 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 purify PRO20 I PR0292.
  • PR0344 PR0343, PR0347, PR0357.
  • PR0853 or PR0882 from recombinant cell proteins or polypeptides
  • the following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation reverse phase HPLC chromatography on silica or on a cation-exchange resin such as DEAE. chromatofocusing. SDS-PAGE. ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of the PRO201 , PR0292.
  • PR0327, PRO 1265, PR0344. PR0343. PR0347. PR0357. PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides Various methods of protein purification may be employed and such methods are known in the art and described for example in Irishr, Methods in Ercvmology, 182 ( 1990), Scopes, Protein Purification Principles and Practice, Springer- Verlag, New York ( 1982) The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO201. PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptide produced 2 Amplification of Genes Encoding the PRQ201. PRQ292. PRQ327. PRQ1265. PRQ344.
  • PROl 017, PRO 1 1 12. PRO509. PRQ853 or PRQ882 Polypeptides in Tumor Tissues and Cell Lines The present invention is based on the identification and characte ⁇ zationof genes which are amplified in certain cancer cells
  • the genome of prokaryotic and eukaryotic organisms is subjected to two seemingly conflicting requirements One is the preservation and propagation of DNA as the genetic information in its o ⁇ ginal form, to guarantee stable inheritance through multiple generations
  • cells or organisms must be able to adapt to lasting environmental changes
  • the adaptive mechanisms can include qualitative or
  • Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, / e a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene
  • the phenomenon of gene amplificationand its underlying mechanisms have been ⁇ nvest ⁇ gated/ « vf/r ⁇ in several prokaryotic and eukaryotic culture systems
  • the best-characterized example of gene amplification involves the culture of eukaryotic cells in medium containing variable concentrations of the cytotoxic dmg methotrexate (MTX)
  • MTX a folic acid analogue and interferes with DNA synthesis by blocking the enzyme d ⁇ hydrofolatereductase(DHFR)
  • genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation This is illustrated by the amplification of the bcl-I and ras genes in various epithelial and lymphoid neoplasms
  • PCR polymerase chain reaction
  • PCR-based assays are most suitable for the final identification of coding sequences, i e. genes in amplified regions
  • such genes have been identified by quantitative PCR (S Gelmini et al Clin Chem 43-752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus. testis, ovary, uterus, etc tumor, or tumor cell lines, with pooled DNA from healthy donors Quantitative PCR was performed using a TaqMan instmment (ABI) Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs
  • Human lung carcinomacell lines include A549 (SRC768),Calu-l (SRC769),Calu-6 (SRC770),H157 (SRC771 ), H441 (SRC772), H460 (SRC773). H522 (SRC832), H810 (SRC833), SKMES- 1 (SRC774) and SW900 (SRC775), all available from ATCC
  • Primary human ⁇ "ng tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRC724 (squamous cell carcinoma abbreviated as "SqCCa”)(LTl),SRC725 (non-small cell carcinoma, abbreviated as "NSCCa”)(LTla),SRC726 (adenocarcinoma, abbreviated as "AdenoCa”)(LT2), SRC727 (adenoc
  • SRC728 (squamous cell carc ⁇ noma)(LT4), SRC729 (adenocarc ⁇ noma)(LT6), SRC730 (ade ⁇ 'squamous cell carc ⁇ noma)(LT7), SRCC731 (adenocarc ⁇ noma)(LT9), SRC732 (squamous cell carc ⁇ noma)(LT10) SRC733 (adenocarc ⁇ noma)(LTl 1), SRC734 (adenocarc ⁇ noma)(LT12), SRC735 (broncho alveolar carcinoma, abbreviated as "BAC")(LT13), SRC736 (squamous cell carcmoma)(LT15), SRC737 (squamous cell carc ⁇ noma)(LT16),SRC738 (squamous cell carc ⁇ noma)(LT17), SRC739 (squamous cell carc ⁇ noma)(LT18), SRC740 (squamous cell carc ⁇ noma)
  • Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma. SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRC777), Colo320 (carcinoma, SRCC778), Colo205 (carcinoma, SRC828), HCC2998 (carcinoma, SRC830), HT29 (adenocarcinoma, SRC779), HM7 (carcinoma. SRC780).
  • KM 12 (carcinoma, SRC831 ), CaWiDr (adenocarc ⁇ noma,SRC781 ).
  • HCT 15 (carcinoma, SRC829), HCTI 16 (carcinoma, SRC782), SKCOl (adenocarcinoma, SRC783), SW403 (adenocarcinoma, SRC784), LS174T (carcinoma, SRC785), and HM7 (a high mucin producing variant of ATCC colon adenocarcinomacell line LS 174T, obtained from Dr Robert Warren, UCSF)
  • Primary colon tumors include colon adenoocarcinomas designated CT1 (SRC751 ), CT2 (SRC742), CT3 (SRC743).
  • CT4 (SRC752), CT5 (SRC753), CT6 (SRC754), CT7 (SRC755), CT8 (SRC744), CT9 (SRC756), CT10 (SRC745), CT1 1 (SRC757), CT12 (SRC746) CT14 (SRC747). CT15 (SRC748). CT 16 (SRC749). CT17 (SRC750). CT18 (SRCC758). and DcR3. BACrev, BACfwd. T160. and T159
  • Human breast carcinoma cell lines include, for example, HBL 100 (SRCC759), MB435s (SRCC760), T47D (SRCC761 ), MB468(SRCC762).MB 175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3 (SRCC767)
  • gene amplification and/or gene expression in various tissues may be measured by conventional Southem blotting.
  • Northern blotting to quantitate the transcription of mRNA Thomas, Proc Nati Acad Sci USA, 77 5201 -5205 [ 1980]
  • dot blotting DNA analysis
  • in situ hybridization using an appropriately labeled probe, based on the sequences provided herein
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes Gene expression in various tissues, alternatively, may be measured by immunologica!
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509.
  • PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO l 1 12, PRO509, PR0853 or PR0882 NA and encoding a specific antibody epitope
  • General techniques for generating antibodies, and special protocols for Northern b'-tting and in situ hybridization are provided hereinbelow
  • the gene can be mapped to a particular chromosome, e g by radiation-hybrid analysis
  • the amplification level is then determined at the location identified, and at neighboring genomic region Selective or preferential amplificationat the genomic region to which to gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival
  • Chromosome mapping includes both framework and epicenter mapping For further details see e g , Stewart et al , Genome Research 7, 422-433 ( 1997) 5 Antibody Binding Studies
  • the results of the gene amplification study can be further verified by antibody binding studies, in which the ability of ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, ant ⁇ -PR01265, ant ⁇ -PR0344. ant ⁇ -PR0343, ant ⁇ -PR0347, ant ⁇ -PR0357. ant ⁇ -PR0715. anti-PRO 1017, anti-PRO 1 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or anti- PR0882 to inhibit the expression of the PRO201 , PR0292. PR0327, PRO 1265. PR0344, PR0343, PR0347, PR0357 PR0715 PRO 1017 PROl 1 12. PRO509. PRO853 or PR0882 polypeptideson tumor (cancer) cells is tested Exemplary antibodies include polyclonal. monoclonal, humanized, bispecific. and heteroconjugate antibodies, the preparation of which will be described hereinbelow
  • Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola. Monoclonal Antibodies A Manual of Techniques, pp 147- 158 (CRC Press. Inc . 1987)
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope. of the protein to be detected
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay)
  • sandwich assay is an ELIS ⁇ assay, in which case the detectable moiety is an enzyme For ⁇ mmunoh ⁇ stochem ⁇ stry,the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
  • Cell-basedassays and animal models for tumors can be used to verify the findings of the gene amplification assay, and further understand tb ⁇ • elationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth
  • the role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above
  • Suitable cells include, for example, stable tumor cells lines such as, the B104-1- 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene)and ras'-transfectedNIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth
  • stable tumor cells lines such as, the B104-1- 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene)and ras'-transfectedNIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth
  • Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorogenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody- dependent cellular cytotox ⁇ c ⁇ ty(ADCC)
  • ADCC antibody- dependent cellular cytotox ⁇ c ⁇ ty
  • Animal models of tumors and cancers include both non-recombinant and recombinant (transgenic) animals
  • Non-recombmant animal models include, for example, rodent, e g , mu ⁇ ne models
  • Such models can be generated by introducingtumor cells into syngeneic mice using standard techniques, e g subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e g colon cancer cells implanted in colonic tissue (See, e g PCT publication No WO 97/33551, published September 18, 1997)
  • the cells introduced into such anirr tls can be derived from known tumor/cancer cell lines, such as. any of the above-listed tumor cell lines, and for example, the B 104- 1 - 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), Caco-2 (ATCC HTB-37). a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or »» . um ⁇ virgin-. _ canon.
  • Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al Br J Cancer 48 689-696 [1983]) Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures
  • the subcutaneous (s c ) space in mice is very suitable for tumor implantation
  • Tumors can be transplanteds c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions
  • tumor tissue fragments of suitable size are introduced into the s c space
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneouslv Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue Boven and Winograd (1991 ), supra
  • Animal models of breast cancer can be generated, for example, by implanting rat neuroblastomacells (from which the neu oncogen was initially isolated), or neu -transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al PNAS USA 83 9129-9133 (1986)
  • animal models of colon cancer can be generated by passaging colon cancer cells in animals, e g nude mice, leading to the appearance of tumors in these animals
  • An orthotopic transplant model of human colon cancer in nude mice has been described, for example by Wang ef ⁇ / Cancer Research 54 4726-
  • Tumors that arise in animals can be removed and cultured in vitro Cells from the in vitro cultures can then be passaged to animals Such tumors can serve as targets for further testing or dmg screening Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest Such passaging techniques can be performed with any known tumor or cancer cell lines
  • Meth A, CMS4, CMS5, CMS21 , and WEHI- 164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al , J Exp Med 146 720 [ 1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Pal ladinoe/ fl/ J Immunol J_38 4023- 4032 [ 1987]) Briefly, tumor cells are propagated in vitro in cell culture Prior to miection into the animals the cell lines are washed and suspended in buffer, at a cell density of about 10x10" to 10x l0 7 cells/ml The animals are then infected subcutaneously with 10 to 100 ⁇ l of the cell suspension, allowing one to three weeks for a tumor to appear
  • the Lewis lung (3LL) carcinoma of mice which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL)
  • SCCL small cell carcinoma of the lung
  • One way of evaluating the efficacy of a test compound in an animal model is implanted tumor is to measure the size of the tumo ⁇ - before and after treatment Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate
  • the therapeutic effects of a d g candidate can be better described as treatment-induced growth delay and specific growth delay
  • Another important variable in the description of tumor growth is the tumor volume doubling time
  • Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals, Wu and Sheng eds .
  • necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially Therefore, these changes need to be carefully monitored, by a combination of a morphomet ⁇ c method and flow cytomet ⁇ c analysis
  • Recomb ⁇ nant(transgen ⁇ c)an ⁇ mal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals
  • Ammaisthat can serve as a target for transgenic manipulation include, 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 m ⁇ cro ⁇ nject ⁇ on(Hoppe and Wanger, U S Patent No 4,873, 191 ), retrovirus-mediated gene transfer into germ lines (e g . Van der Putten et al .
  • 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 particularcell type is also possible by following, for example, the technique of Lasko . ⁇ / , Proc Nati Acad Sci USA S9 6232-636 (1992)
  • transgene expression in transgenic animals can be monitored by standard techniques For example. Southem blot analysis 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 hybridization. Northern blot analysis, PCR, or lmmunocytochemistry The animals are further examined for signs of tumor or cancer development
  • "knock out" animals can be constmcted which have a defective or altered gene encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 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 into an embryonic cell of the animal For example. cDNA encoding a particular PRO201. PR0292, P * :0327, PRO 1265, PR0344, PR0343.
  • PRO509, PR0853 or PR0882 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with establishedtechniques
  • a portion of the genomic DNA encoding a particular PR0201, PR0292,PR0327,PR01265,PR0344,PR0343,PR0347,PR0357,PR0715,PR01017,PR01 1 12, PRO509, PR0853 or PR0882 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
  • a selectable marker which can be used to monitor integration
  • several kilobases of unaltered flanking DNA are included in the vector [see e g , Thomas and Capecchi, Cell.
  • the vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e g , Li et al Cell, 69 915 ( 1992)]
  • the selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e.g , Bradley, in Teratocarcinomas and Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 1 13-152]
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified bv standard techniques and used
  • Screening assays for dmg candidates are designed to identify compounds that bind or complex with the polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule dmg candidates
  • Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides.
  • polypept ⁇ de- ⁇ mmunoglobul ⁇ n fusions and, in particular, antibodies including, without limitation, poly- and monoclonal antibodiesand antibody fragments, single-chain antibodies, anti-idiotypicantibodies, 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 variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art
  • the polypeptide encoded by the gene identified herein or the dmg candidate is immobilized on a solid phase, e on a microtiter plate, by covalent or non-covalent attachments
  • Non-covalentattachment generally is accomplished by coating the solid surface with a solution ofthe polypeptideand drying
  • 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, which may be labeled by a detectable label, to the immobilized component, e g the coated surface containing the anchored component When the reaction is complete, the non-reacted
  • GAL l-/ ⁇ rZ reporter gene under control of a GAL4-act ⁇ vated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ - galactosidase
  • MATCHMAKERTM 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 domains involved in specific protein interactions as well as to pinpoint amino acid residues that are cmcial for these interactions
  • PRO 1 1 12-, PRO509-, PR0853- or PR0882- encodmg gene identified herein and other intra- or extracellular components can be tested as follow s usually a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products To test the ability of a test compound to inhibit binding, the reaction is mn in the absence and in the presence of the test compound In addition, a placebo may be added to a third reaction mixture to serve as positive control The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove The formation of a complex in the control react ⁇ on(s) but not in the reaction mixture containing the test compound
  • compositions useful in the treatment of tumors associated with the amplification of the genes identified herein include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides. antisense and ⁇ bozyme molecules, triple helix molecules, etc that inhibit the expression and/or activity of the target gene product
  • antisense RNA and RNA molecule act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation
  • oligodeoxynbonucleotides derived from the translation initiation site e g between about -10 and +10 positions of the target gene nucleotide sequence
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA
  • Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ⁇ bozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biolo y' 4 469-471 (1994), and PCT ⁇ ubhcation 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 o gonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex For further details see, e g PCT publication No WO 97 "3551, supra These molecules can be identified by any or any combination of the screening assays discussed hereinabove and/or by any other screening techniques well
  • Some of the most promising dmg candidates according to the present invention are antibodies and antibody fragments which may inhibit the production or the gene product of the amplified genes identified herein and/or reduce the activity of the gene products l Polyclonal Antibodies
  • polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or mtraperitoneal injections
  • the immunizing agent may include the PRO201, PR0292,
  • PRO509, PR0853 or PR0882 polypeptideor a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenicin the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thvroglobulin.
  • adjuvants examples 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 11 Monoclonal Antibodies The ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265, ant ⁇ -PR0344, ant ⁇ -PR0343. anti-
  • ant ⁇ -PRO509, ant ⁇ -PR0853 or anti- PR0882 antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybndoma methods, such as those described 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 bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro
  • the immunizing agent will typically include the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof
  • peripheral blood lymphocytes ("PBLs")are used ifcells ofhuman 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 [Goding, Monoclonal Antibodies Principles and Practice, Academic Press, ( 1986) pp 59- 103]
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, oovine and human origin Usually, rat or mouse
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium
  • More preferred immortalized cell lines are munne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection (ATCC), 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, Inc , New York, (1987) pp 51-63]
  • the culture medium in which the hyb ⁇ doma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12.
  • the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibodv can for example be determined bv the Scatchard analysis of Munson and Pollard. Anal Biochem . 107 220 ( 1980)
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra! Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional lmmunoglobulinpu ⁇ fication procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of mu ⁇ ne antibodies)
  • the hybndoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce lmmunogiobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous mun
  • the antibodies may be monovalentantibodies
  • Methods for preparing m -ovalentantibodies are well known in the art For example, one method involves recombinant expression of lmmunogiobulin light chain and modified heavy chain
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross nking
  • the relevant cysteine r e sidues are substituted with another ammo acid residue or are deleted so as to prevent crosshnking
  • ant ⁇ -PRO201 The ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265.
  • ant ⁇ -PR0853 or anti- PR0882 antibodies may further comprise humanized antibodies or human antibodies
  • Humanized forms of non-human (e , munne) antibodies are chimeric immunoglobulins, lmmunogiobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human lmmunogiobulin
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • Fv framework residues of the human lmmunogiobulin are replaced by corresponding non-human residues
  • Humanized antibodies
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an “import” variable domain Humanization can be essentially performed following the method of Winter and co-workers [Jones et al . Nature.
  • humanized antibodies are chimeric ant ⁇ bod ⁇ es(U S Patent No 4.816.567), wherein substantially le ⁇ s than an intact human variable domain has been substituted by the corresponding sequence from a non-human species
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter,-/ Mol Biol , 221 381 ( 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(1) 86-95 (1991)] Similarly, human antibodies can be made by introducing of human lmmunogiobulin loci into transgenic animals, e g , mice in which the endogenous lmmunogiobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example,
  • the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzvme which converts a prodrug (e g a peptidyl chemotherapeutic agent, see WO 81/01 145) to an active anti-cancer dmg See. for example. WO 88/07378 and U S Patent No 4,975,278
  • a prodrug e g a peptidyl chemotherapeutic agent, see WO 81/01 14
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form
  • Enzymes that are useful in the method of this invention include, but are not limited to. glycosidase, glucose oxidase. human lysosyme, human giucuronidase. alkaline phosphatase usefulf or converting phosphate-contain mgprodmgs into free dmgs, arylsulfataseuseful for converting sulfate-containing prodmgs into free dmgs, cytosine deaminase useful for converting non-toxic 5-fluorocytos ⁇ ne into the anti-cancer dmg 5-fluorourac ⁇ l, proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases (e g , carboxypeptidase G2 and carboxypeptidase A) and cathepsms (such as cathepsins B and L), that are useful for converting peptide-containingprodrugs into free dmg
  • carbohydrate-cleavmgenzymes such as ⁇ -galactosidase and neuram in idase useful for converting glycosylated prodmgs into free dmgs, ⁇ -lactamase useful for converting drugs de ⁇ vatized with ⁇ -lactams into free dmgs, and penicillin amidases, such as penicillin Vamidase or penicillin G am idase, useful for converting dmgs de ⁇ vatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free dmgs
  • antibodies with enzymatic activity also known in the art as "abzymes" can be used to convert the prodmgs of the invention into free active dmgs (see, e g , Massev. Nature 328 457-458(1987)) Antibody -ab
  • the enzymes of this invention can be covalently bound to the ant ⁇ -PRO201, ant ⁇ -PR0292. anti- PR0327, anti-PRO 1265, ant ⁇ -PR0344, ant ⁇ -PR03 ⁇ 3, ant ⁇ -PR0347, ant ⁇ -PR0357, ant ⁇ -PR0715, anti- PRO 1017, anti-PROl 1 12,ant ⁇ -PRO509.ant ⁇ -PRO853 or ant ⁇ -PR0882 antibodies by techniques well known in the art such as the use of the heterobifunctionalcross-linkmgagents discussed above Alternatively, fusion proteins comprising at least the antigen binding region of the antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e g Neuberger / .' Nature 312 604-608 ( 1984))
  • 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
  • bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two lmmunogiobulin heavy-chain/light- chain pairs, where the two heavy chains have different spec ⁇ fic ⁇ t ⁇ es(M ⁇ lste ⁇ n and Cuello, Nature, 305 537-539 [1983]) Because of the random assortment of lmmunogiobulin heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific stmcture The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J , K> 3655-3659 ( 1991 ) Antibody variable domains with the desired binding spec ⁇ fic ⁇ t ⁇ es(ant ⁇ body-ant ⁇ gen combining sites) can be fused to
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain
  • one or more small am o acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g tyrosine or tryptophan)
  • Compensatory "cavities" of identical or similar size to the large side cha ⁇ n(s) are created on the interface of the second antibody molecule by replacing large am o acid side chains with smaller ones (e g alanine or threonine)
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g F(ab'), bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecfic antibodies can be prepared can be prepared using chemical linkeage Brennan et al Science 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives One of the Fab -TNB derivatives is then reconverted to the Fab -thiol by reduction with mercaptoethvlamine 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
  • Fab' fragments may be directly recovered from £ coli and chemically coupled to form bispecific antibodies
  • Shalaby e/ ⁇ / J Exp Med 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab'), molecule
  • Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in vitro to form the bispecific antibody
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets
  • Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described
  • bispecific antibodies have been produced using leucine zippers Kostelnye/ / J Immunol 148(5) 1547- 1553 (1992)
  • the leucine zipper peptides from the Fos and Jun proteins were iinked to the Fab' portions of two different antibodies by gene
  • t ⁇ specific antibodies can be prepared Tutt e/ er/ J Immw ' 147 60 ( 1991 )
  • Exemplary bispecific antibodies may bind to two different epitopes on a given "Pro protein herein
  • an ant ⁇ -"PRO” protein arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecuoe (e g CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular "PRO" protein
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular "PRO" polypeptide
  • These antibodies possess a "PRO"-bindmgarm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA
  • Another bispecific antibody of interest binds the "PRO” polypeptide and further binds tissue factor (TF) vi Heteroconiugate Antibodies Heteroconju
  • the antibody of the invention may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectivenessof the antibody in treating cancer, for example
  • cysteine res ⁇ due(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region
  • the homodime ⁇ c 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 ,
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (; e , a radioconjugate)
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (; e , a radioconjugate)
  • Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbindmg active fragments of diphtheria toxin, cholera toxin, botuhnus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleurttes fordu proteins, dianthm proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crot ⁇ n, sapav.na ⁇ aoff ⁇ c ⁇ nal ⁇ s inhibitor, gelon ⁇ n,sapo ⁇ n, m ⁇ togell ⁇ ,rest ⁇ ctoc ⁇ n,phenomyc ⁇ n, enomycinand the tncothecenes
  • Small molecule toxins include for example, ca
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succ ⁇ n ⁇ m ⁇ dyI-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccmimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)- ethylened ⁇ am ⁇ ne),d ⁇ socyanates(such as tolyene 2,6-dnsocyanate), and bis-active fluorine compounds (such as l ,5-d ⁇ fluoro-2,4-d ⁇ n ⁇ troben
  • the antibody may be conjugated to a "receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient. followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e g avidin) which is conjugated to a cytotoxic agent (e g a radionucleotide) ix Immunoliposomes
  • the antibodiesdisclosedherein may also be formulatedas immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al Proc Nati Acad Sci USA,M 3688 ( 1985), Hwang et al Proc Nati Acad Sci USA, 77 4030 (1980), and U S Pat Nos 4.485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholme, cholesterol and PEG-denvatized phosphatidvlethanolamine (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 described in Martin et al . J Biol Chem 257 286-288 (1982) via a disulfide interchange reaction A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome See Gabizon et al . J National Cancer Inst 81(19)1484 (1989) 10 Pharmaceutical Compositions
  • Antibodies specifically binding the product of an amplified gene identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred For example, based uj * , ⁇ the variable region sequences of an antibody, peptide molecules can be designed whic 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 Marascoe. ⁇ / Proc Nati Acad Sci USA 90 7889-7893 [1993])
  • Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed [1980]), in the form of lyophilized formulations or aqueous solutions
  • Acceptable earners, 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 chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3- pentanol, and m-cresol
  • chelating agents such as EDTA, sugars such as sucrose. mannitol, trehalose or sorb ⁇ tol, salt-form ⁇ ngcounter- ⁇ ons such as sodium, metal complexes ( g Zn- protein complexes), and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG)
  • Non-antibody compounds identified by the screening assays of the present invention can be formulated in an analogous manner, using standard techniques well known in the art
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokme or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatm- microcapsulesand poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres. microemulsions. nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed ( 1980)
  • the formulationsto be used for in V ⁇ administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
  • Sustained-releasepreparations may be prepared Suitable include semipermeablematnces of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyi-methacrylate), or poly(v ⁇ nylalcohol)), polylactides (U.S Pat No 3,773,919),copolymersof L-glutamic acid and ⁇ ethyl-L-glutamate.
  • non-degradable ethylene- vinyl ac ⁇ .ate degradable lactic acid-glyco c acid copolymers such as the LUPRON DEPOT TM njectable microspheres composed of lactic acid-glycohc acid copolymer and leuprolide acetate), and poly-D-(-)-3- hvdroxybuty ⁇ c acid
  • polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days
  • certain hydrogels release proteins for shorter time periods
  • encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may
  • the antibodies and other anti-tumor compounds of the present invention may be used to treat various conditions, including those characterized by overexpression and/or activation of the amplified genes identified herein Exemplary conditions or disorders to be treated with such antibodies and other compounds, including, but not limited to, small organic and inorganic molecules, peptides. antisense molecules, etc include benign or malignanttumors (e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, ling, vulval.
  • benign or malignanttumors e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, ling, vulval.
  • thyroid thyroid, hepatic carcinomas, sarcomas, glioblastomas.and various head and neck tumors
  • leukemias and lymphoid malignancies other disorders such as neuronal, glial, astrocytal.hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoe c disorders, and inflammatory, angiogenic and immunologic disorders
  • the anti-tumor agents of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, mtraperitoneal. mtracerobrospinal.subcutaneous, lntra-articular.intrasynovial.intrathecal, oral, topical, or inhalation routes Intravenous administration of the antibody is preferred
  • chemotherapeutic agents may be administered to the patient Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instmctions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapv Service Ed .
  • the chemotherapeutic agent may precede, or follow administration of the anti-tumor agent, e g antibody, or may be given simultaneously therewith
  • the antibody may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such ⁇ . x.nap ⁇ stone (see, EP 616812) in dosages known for such molecules
  • the antibodies herein are co-administered with a growth inhibitory agent
  • the growth inhibitory agent may be administered first, followed by an antibody of the present invention
  • simultaneous administration or administration of the antibody of the present invention 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 antibody herein
  • an anti-tumor agent e g an antibody herein
  • the approp ⁇ ate dosage of an anti-tumor agent 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 agent, and the discretion of the attending physician
  • the agent is suitably administered to the patient at one time or over a series of treatments
  • ⁇ g/kgto 15 mg/kg (e g 0 1- 20mg/kg) of 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 ⁇ g/kgto 100 mg/kg or more, depending on the factors mentioned above
  • the treatment is sustained until a desired suppression of disease symptoms occurs
  • other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays
  • an article of manufacturecontainingmate ⁇ als useful for the diagnosis or treatment of the disorders described above comprises a containerand a label Suitable containers 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 container 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 an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e g an antibody
  • the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
  • the article of manufacture may further comprise a second container comprising a pharmaceutical ly- 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, including other buffers, diluents, filters, needles, syringes, and package inserts with instmctions for use
  • a pharmaceutical ly- 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, including other buffers, diluents, filters, needles, syringes, and package inserts with instmctions for use
  • tumor diagnostics or prognostics While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for dmg candidates or tumor (e g cancer) treatment, the same proteins along with secreted proteins encoded by the genes amplified in tumor cells find additional use in the diagnosis and prognosis of tumors.
  • antibodies directed against the proteins products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics
  • antibodies can be used to qu ⁇ .
  • the antibody preferably is equipped with a detectable, e g fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluo ⁇ metry, or other techniques known u the art These techniques are particularly suitable, if the amplified gene encodes a cell surface protein, e g a growth factor
  • binding assays are performed essentially as described in section 5 above
  • In situ detection of antibody binding to the marker gene products can be performed, for example, by lmmunofluorescence or immunoelectron microscopy
  • a histological specimen is removed from the patient, and a labeled antibody 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
  • the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks Sambrook et al , Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press N Y , 1989, Ausubel et al Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N Y , 1989, Inms et al PCR Protocols A Guide to Methods and Applications, Academic Press, inc , N Y , 1990, Harlow et al , Antibodies A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor 1988, Gait, M J , Oligonucleotide Synthesis, IRL Press, Oxford, 1984, R I Freshney, Animal Cell Culture ' 987, Co gan et al , Current Protocols in Immunology, 1991
  • PRQ347 PRQ357. PRQ715.
  • PROl 017 PROl 112. PRO509. PRQ853 or PRQ882
  • DNA30676 has a single long open reading frame which encodes a 576 amino acids protein ( Figure 2, SEQ ID NO 2)
  • Figure 2 A vector containing DNA30676 (DNA30676-1223) was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard , Manassas, VA 201 10-2209, on December 23, 1998 and assigned ATCC Deposit No 209567
  • EST expressed sequence tag DNA database
  • LIFESEQ ® Incyte Pharmaceuticals.Palo Alto. CA
  • various EST sequences were identified that showed certain homology to human prolactin recetor protein Based upon a consensus sequence extended using repeated cycles of BLAST and the program "phrapJPhil Green, University of Washington, Seattle.
  • DNA381 13 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard , Manassas, VA z ⁇ ' 10-2209, on December 10, 1997 and assigned ATCC Deposit No 209530
  • DNA60764 Isolation of cDNA clones encoding human PRO 1265 (UNQ636) DNA60764 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc (South San Francisco. CA) upon ESTs as well as clustered and assembled EST fragments from public (e g , GenBank) and/or private (LIFESEQ ® , Incv : Pharmaceuticals. Inc .
  • the signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration
  • the nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined If neither meets the requirement, the candidate sequence is not scored
  • the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals
  • EST Cluster No 86995 an EST cluster sequence from the LIFESEQ® database, designated EST Cluster No 86995 that was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies
  • the homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enrvmology 266 460-480 ( 1996)) 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 a consensus DNA sequence w ith the program phrap"
  • One or more of the ESTs used in the assembly was derived from a cDNA library prepared from RNA isolated from inflamed human adenoid tissue A consensus sequence was assembled, and.
  • the full length clone shown in Figure 7 contained a single open reading frame with an apparent translational initiation site at nucleotide positions 79-81 and ending at the stop codon found at nucleotide positions 1780- 1782 ( Figure 7, SEQ ID NO 7)
  • the pred ⁇ ctedpolypept ⁇ deprecursor(F ⁇ gure 8, SEQ ID NO 8 ) is 567 amino acids long PRO 1265 has a calculated molecular weight of approximately 62,881 daltons and an estimated pi of approximately 8 97
  • Additional features include a signal peptide sequence at about ammo acids 1-21 , potential N-glycosylation sites at about ammo acids 54-57, 134-137, 220-223, and 559-562, and a region having ammo acid sequence identity with D-ammo acid oxidase proteins at about amino acids 61 -80
  • ECD extracellulardomain sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases
  • the EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto.
  • DNA40592 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 sequence as far as possible using the sources of EST sequences discussed above Using probes s> nthesized based upon the extended consensus sequence, a cDNA clone designated DNA40592 was isolated from a human fetal kidney tissue library
  • Clone UNQ302 (DNA43318) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 53-55 and ending at the stop codon at nucleotide positions 1004- 1006 ( Figure 1 1 ) The predicted polypeptide precursor is 317 ammo acids long ( Figure 12) Various unique aspects of the PR0343 protein are shown in Figure 12 Clone UNQ302 (DNA43318-1217) was deposited with ATCC on November 21 , 1997 and is assigned ATCC deposit no 209481
  • a consensus sequence was determined Based on the 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 of the full-length coding sequence for PR0347 RNA for constmction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228) The cDNA libraries used to isolate the cDNA clones were constmcted by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
  • sequence expression tag "2452972" ( Figure 30, SEQ ID NO 30) by Incyte Pharmaceut ⁇ cals,Palo Alto, CA, was used to begin a data base search
  • ECD extracellular domain
  • sequences including the secretion signal, if any
  • EST search expressed sequence tag
  • the EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ ® , Incvte Pharmaceuticals Palo Alto, CA) The search was performed as described above for ECD homology searches
  • DNA44804 A consensus DNA sequence was assembled relative to other EST sequences using phrap Using probes synthesized based upon the consensus sequence, a cDNA clone designated DNA44804 was isolated from a human fetal liver library, and sequenced in its entirety The entire nucleotide sequence of DN A44804 is shown in Figure 15 (SEQ ID NO 15)
  • DNA44804 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 137-139 and ending at the stop codon at nucleotide positions 1931-1933
  • the predicted polypeptide precursor is 598 ammo acids long ( Figure 16 SEQ ID NO 16) Clone DNA44804- 1248 was deposited with ATCC on December 10, 1997 and is assigned ATCC deposit No 209527
  • Figure 18 has an estimated molecular weight of about 27,433 daltons and a pi of about 9 85
  • PR0715 is a novel tumor necrosis factor protein
  • a vector containing DNA52722 (DNA52722-1229) was deposited with ATCC on January 7, 1998 and was assigned ATCC deposit no 209570
  • the extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins ii om the Swiss-Protpublic protein database were used to search expressed sequence tag (EST) databases, following the ECD homology search described above
  • the EST databases included public EST databases (e g , GenBank, Merck/Wash U) and a proprietary EST DNA database (LIFESEQ ® ,
  • a bacte ⁇ ophage library of human retinal cDNA (commercially available from Clontech) was screened by hybridization with a synthetic oligonucleotide probe based on an EST sequence (GenBank locus AA021617), which showed some degree of homology to members of the TNFR family
  • Five positive clones (containing cDNA inserts of 1 8-1 9kb) were identified in the cDNA library, and the positive clones were confirmed to be specific by PCR using the above hybridization probe as a PCR primer
  • Single phage plaques containing each of the five positive clones were isolated by limiting dilution and the DNA was purified using a Wizard Lambda Prep DNA purification kit (commercially available from Promega)
  • the cDNA inserts from three of the five bacte ⁇ ophage clones were excised from the vector arms by digestion with EcoRI, gel-pu ⁇ fied,and subcloned into pRK5 and sequenced on both strands
  • the three clones contained an identical open reading frame (with the exception of an intron found in one of the clones)
  • the pasicted amino acid sequence of the full length HVEM contains 283 amino ai-Hs (See Fig 24, SEQ ID NO 24)
  • a putative transmembrane region of the HVEM comprises amino acids 201- 225 of Fig 24 and a putative cytoplasmic region of the HVEM comprises amino acids 226-283 of Fig 24
  • the sequence differs from the HVEM sequence reported in Montgomery et al , supra, in at least two amino acids as shown in Fig 26, codon 108 encodes a se ⁇ ne and codon 140 encodes an alanine
  • An alignment using the AlignTM -.mputer program) of a 58 amino acid long cytoplasmic region of HVEM with other known members of the human TNF receptor family showed some sequence similarity, in particular to CD40 ( 12 identities) and LT-beta receptor ( 1 1 identities)
  • RNA for constmction ofthe cDNA libraries was isolated from human fetal kidney tissue (LIB228) The cDNA libraries used to isolate the cDNA clones were contmcted by standard methods using commercially available reagents such as those from Invitrogen. San Diego, CA
  • glycosaminoglycanattachmentsite corresponding to amino acids from about 46 to about 49 of SEQ ID NO 26, and two sequences typical of the short-chain alcohol dehydrogenase family, corresponding to amino acids from about 37 to about 49 and about 1 14 to about 124 of SEQ ID NO 26, respectively Clone DNA48227- 1350 has been deposited with the ATCC and is assigned ATCC deposit no 209812
  • PR0882 (UNQ448) is identical with cardiotrophin- 1
  • the amino acid sequence of this 201 amino acid protein is present in the public Dayhoff database under Accession Nos P R83967, P W29238 and CTF1 HUMAN. among others, and is shown in Figure 28
  • the nucleotide of the DNA encoding the PR0882 (UNQ448) is shown in Figure 29 (SEQ ID NO 29)
  • Cardiotrophin- 1 has also been disclosed in WO9730146, published on 21 August 1997 and W09529237, published on 2 November 1995
  • EXAMPLE 2 Gene Amplification This example shows that the PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-.
  • PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng genes are amplified in the genome of certain human lung, colon and/or breast cancers and/or cell lines
  • Amplification is associated with overexpression of the gene product, indicating that the binding specificities for at least two different • nigens In the present case, one of the binding specificities is for the PRO201.
  • PR0292 PR0327, PR01265.
  • PR0344, PR0343. PR0347, PR0357, PR0715 is for the PRO201.
  • PRO10 I 7, PROl 1 12, PRO509, PR0853 or PR0882 proteins are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast z r ⁇ other cancers
  • Therapeutic agent may take the form of antagonists of binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 -, PR0292-, PR0327- , PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-.
  • PRO 1 1 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng genes for example, mu ⁇ ne-human chimeric, humanized or human antibodies against a binding specificities for at least two different antigens
  • one of the binding specificities is for the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347.
  • the starting material for the screen was genomic DNA isolated from a variety cancers
  • the DNA is quantitated precisely, eg fluoromet ⁇ cally
  • DNA was isolated from the cells often normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown)
  • the 5' nuclease assay for example, TaqManTM
  • real-time quantitative PCR for example, ABI P ⁇ zm 7700 Sequence Detection SystemTM (Perkin Elmer, Applied Biosystems Division, Foster City, CA)
  • the results were used to determine whetherthe DNA encoding binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327, PR01265 PR0344, PR0343, PR0347.
  • PRO1 1 12 PRO509, PR0853 or PR0882 is over-represented m any of the primary lung or colon cancers or cancer cell lines or breast cancer cell lines that were screened
  • the primary lung cancers were obtained from individualswith tumors of the type and stage as indicated in Table 1
  • An explanation of the abbreviations used for the designation of the primary tumors listed in Table 1 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore
  • the results of the TaqmanTM are reported in delta ( ⁇ ) CT units One unit corresponds 1 PCR cycle or approximately a 2-fold amplification relative to normal, two units corresponds to 4-fold. 3 units to 8-fold amplification and so on Quantitation was obtained using p ⁇ mers and a TaqmanTM fluorescent prove derived from the binding specificities for at least two different antigens In the present case, one of the binding specificitiesis for the PRO201-, PR0292-, PR0327-, PR01265-, PR0344-.
  • PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng gene Regions of binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327. PRO 1265. PR0344.
  • PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 which are most likely to contain unique nucleic acid se ⁇ uences and which are least likely to have spliced out introns are preferred for the primer and probe derivation, e g 3-untranslated region
  • the sequences for the primers and probes (forward, reverse and probe) used for the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 . ' 2, PRO509, PR0853 or PR0882 gene amplification were as follows PRO201 (DNA30676) 30676 tm f
  • PRQ292 (DNA35617) 35617 tm f 5'-GATCCTGGG CGACGTCTTC-3' (SEQ ID NO 35)
  • PRQ327 (DNA38 I 13) 381 13 tm f 5'-CTCAAGAAGCACG CGTACTGC-3' (SEQ ID NO 38) 381 13.tm.p
  • PRO1265 (DNA60764): 60764.tm.fl
  • PRQ344 (DNA40592): 40592.tm.fl
  • PRQ343 (DNA43318): 43318.tm.fl
  • PRQ357 (DNA44804): 44804.tm.fl 5'-CCTCGGTCTCCTCATCTGTGA-3' (SEQ ID NO: 53)
  • PRO 1017 (DNA561 12 : 561 12.tm.fl
  • PRO 1 1 12 (DNA57702): 57702.tm.fl
  • PRO509 (DNA50148): 50148.tm.fi 5'-GGAGGAGACAATACCCTCATTCA-3' (SEQ ID NO: 65)
  • PRQ853 (DNA48227) 48227 tm fl
  • the 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor amplification in real time
  • Two oligonucleotidep ⁇ mers are used to generate an amplicon typical of a PCR reaction
  • a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers
  • the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe
  • the TAQ DNA polymerase enzyme cleaves the probe in a template-dependent manner
  • the resultant probe fragments disassociate in solution, and signal from 'he released reporter dye is free from the quenching effect of the second fiuorophore
  • the system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer
  • the system amplifies samples in a 96-well format on a thermocycler During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD
  • the system includes software for mnning the instmment and for analyzing the data
  • Nuclease assay data are initial ly expressed as Ct. or the threshold cycle Th is is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence
  • the ⁇ Ct values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer DNA results to normal human DNA results
  • Table 1 describes the stage. T stage and N stage ofva ⁇ ous primary tumors which were used to screen the PRO201, PR0292. PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715.
  • PRO1017, PROl 112, PRO509, PR0853 or PR0882 compounds of the invention Table 1 Primary Lung and Colon Tumor Profiles
  • DNA was prepared from cultured cell lines, primary tumors, normal human blood The isolation was performed using purification kit, buffer set and protease and all from Quiagen, according to the manufacturer's instmctions and the description below Cell culture Ivsis
  • Buffer C 1 10 mL, 4°C
  • ddH20 40 mL.4°C
  • the cell nuclei were pelleted by cent ⁇ fuging in a Beckman swinging bucket rotor at 2500 rpm at 4°C for 15 minutes The supernatant was discarded and the nuclei were suspended with a vortex into 2 mL Buffer C 1 (at 4°C) and 6 mL ddH-O, followed by a second 4°C centrifugation at 2500 rpm for 15 minutes
  • the nuclei were then resuspended into the residual buffer using 200 ⁇ l per tip G2 buffer ( 10 ml) was added to the suspended nuclei while gentle vortexing w as applied Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds Quiagen protease (200 ⁇ l, prepared as indicated above) was added and incubated at
  • DNA levels in each tube were quantified by standard A260, A280 spectrophotometry on a 1 20 dilution (5 ⁇ l DNA + 95 ⁇ l ddH 2 0) using the 0 1 ml quartz cuvetts in the Beckman DU640 spectrophotometer A260/A280 ratios were in the range of 1 8-1 9
  • Each DNA samples was then diluted further to approximately 200 ng/ml in TE (pH 8 5) If the original material was highly concentrated (about 700 ng/ ⁇ l), the material was placed at 50°C for several hours until resuspended
  • Fluoromet ⁇ cDNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelinesas modified below This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes The Hoechst dye working solution (#H33258. 10 ⁇ l.
  • PRO201 (UNQ175) (DNA30676), PRO-327 (UNQ288)(DNA381 13) and PR01265 (UNQ636)(DNA60764) were also reexamined along with selected tumors irom the above initial screen with framework mapping Figure 32 and Table 3 indicate the chromosomal mapping ot the framework markers that were used in the present example The tramework markers are located approximately every 20 megabases and were used to control aneuploidy
  • PRO201 (UNQ 175XDNA30676), PR0327 (UNQ288)(DNA381 13) and PR01265 (UNQ636)(DNA60764) were also reexamined with epicenter mapping
  • the markers indicated in Tables 4A, 4B and 4Care located in close proximity (in the genome) to DNA30676.
  • the distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers One cR is very roughly equivalent to 20 kilobases.
  • the marker SHGC-35441 is the marker found to be the closest to the location on chromosome 19 where DNA30676 maps, is closest to DNA381 13 and SHGC-33698 is closest to DNA60764.
  • Table 6 6 land 6 2 indicate the Ct values for results of epicenier mapping relative to DNA30676 DNA381 13 and DNA60764 respectivelv indicating the relative amplification in the region more immediate to the actual location of DNA30676 DNA381 1 and DNA670764 along chromosome 19
  • Table 6 2 indicates the Ct ⁇ alues for the results of epicenter mapping relative to DNA60764 indicating relative amplification in the region more immediate to the actual location of DNA60764 along chromosome 19
  • DNA34353 DNA40620 and DNA54002 are other independently identified molecules which have been observed to map to to the same region of chromosome 19 as DNA60764
  • PR0292 (UNQ266) (DNA35617) was also examined with framework mapping Figure 33 and Table 7 indicate the chromosomal mapping of the framework markers that were used in this analysis T e framework markers are located approximately every 20 megabases and were used to control aneuploidy
  • PR0343 (UNQ302) (DNA433 I 8) and PR0882 (UNQ448) (DNA58125) were also examined with both framework and epicenter mapping Figure 34 and Table 9 indicate the chromosomal mapping of the framework markers that were used in this analysis
  • the framework markers are located approximately every 20 megabases and were used to control aneuploidy
  • Tables 9 1 and 9 2 in indicate the epicenter markers used for the mapping of DNA43318 and DNA58125
  • the markers shown in Tables 9 1 and 92 are located in close proximity (in the genome) to DNAs DNA43318 and DNA58125, respectivelv, and are used to assess the relative amplification in the immediate vicinitv of Chromosome 16 wherein the respective molecules map
  • the distance between individual markers in measured in centiravs (cR) which is a radiation breakage unit roughly equal to a roughly equiv alent to 20 kilobases
  • the markers AFMa061 ⁇ b5 and SHGC 36123 are located the closest to the location on
  • Table 10 1 and 10 2 indicate the ⁇ Ct values from epicenter mapping relative to DNA43318 and DNA58125, respectively indicating the relative amplification in the region more immediate to the actual location of the respective molecules along chromosome 16
  • PRO 1017 (UNQ500) (DNA561 12) was also examined with framework mapping.
  • Figure 35 and Table 1 1 indicate the chromosomal mapping of the framework markers that were used in this analysis The framework markers are located approximately every 20 megabases and were used to control aneuploidy.
  • PROI017 (U Q500)(DNA561 12) was also examined with epicenter mapping.
  • Table 1 1.1 indicates the epicenter markers which are located in close proximity to DNA561 12 which were employed to assess the relative amplification in the immediate vicinity of chromosome 7 wherein DNA561 12 is located.
  • the distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers.
  • cR centirays
  • SHGC-22698 is the marker found to be the closest to the location on chromosome 7 where DNA561 12 maps.
  • Table 1 1 Framework Marker used on Chromosome 7 for DNA 561 12
  • Table 1 1 2 indicated the DCt values for the results of epicenter mapping relative to DNA561 12, indicating the relative amplification in the region more immediate to the actual location of DNA561 12 along chromosome 7
  • PR0715 (UNQ383) (DNA52722) and PR0853 (UNQ419) (DNA48227) were also reexamined with both framework and epicenter mapping
  • Figures 36A and 36B and Table 13 indicate the chromosomal localizations of the framework markers that were used for the procedure
  • the framework markers are located approximately every 20 bases and were used to control aneuploidy
  • Tables 14A and 14B indicate the epicenter mapping markers that were used in the procedure
  • the epicenter markers were located in close proximity to DNA52722 and DNA48226, respectively and are used to determine the relative DNA amplification in the immediate vicinity of DNA52722 and DNA48226
  • the distance between individual markers is measured in centirays, which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers
  • One cR is very roughly equivalent to about 20 kilobases
  • "BAC" means bacterial artificial chromosome The ends of a BAC clone which contained the gene of interest were sequenced Ta
  • Table 16 indicates the ⁇ Ct values of the above described framework markers along chromosome 17 relative to DNA52722 and DNA48227 for selected tumors. While not shown, the similar ⁇ Ct values for the framework markers in the analysis of DNA48227 were reported.
  • Table 18 indicates the ⁇ Ct values for the indicated epicenter markers indicating the relative amplification along chromosome 17 in the immediate vicinity of DNA52722
  • Tables 18A and 18B indicate the ⁇ Ct values for the indicated epicenter markers, indicating the relative amplification of selected lung and colon tumors, respectively, along chromosome 17 in the immediate vicinity of DNA48227
  • PR0357 (UNQ324 DNA44804) was reexamined with selected tumors from the above initial screen with framework mapping Figure 40 and Table 19 indicate the chromosomal mapping of the framework markers that were used in the present example
  • the framework markers are located approximately every 20 megabases and were used to control aneuploidv
  • PR0357 (UNQ324 DNA44804) was also examined with epicenter mapping
  • the markers indicated in Table 20 are located in close proximitv (in the genome) to DNA44804 and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein DNA44804 is located
  • the distance between individual markers is measured in centirays (cR) which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between the two markers
  • cR centirays
  • SHGC-6154 is the marker tound to be the closest to the location on chromosome 16 where DNA44804 maps
  • Table 22 indicates the ⁇ Ct values for the results oi epicenter mapping relative to DNA44804 indicating the relative amplification in the region more immediate to the actual location of DNA44804 along chromosome 16
  • DNA30676 is the gene responsible for the amplification of the particular region on Chromosome 19 Because amplification of DNA30676 occurs in various lung and colon tumors and cell lines (especially colon), it is highly probable to play a significant role in tumor formation or growth As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA30676 (PRO201 ) would be expected to have utility in cancer therapy.
  • antagonists e.g., antibodies directed against the protein encoded by DNA30676 (PRO201 ) would be expected to have utility in cancer therapy.
  • ⁇ Ct values for DNA35617 PR0292, UNQ266
  • a ⁇ Ct value of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
  • Table 2 indicates that significant amplification of DNA35617 occurred in primary lung tumors: LT12, LT13, LT15, LT19, LT21 , in primary colon tumors CT2, CT8. CT10, CT14; in lung tumor cell lines H441 and H810, in colon tumor cell lines SW620, Colo320, HT29 and LS 174T.
  • the ⁇ Ct and average ⁇ Ct values for these hits are: ( 1 ) primary lung tumors: 1 35, 1 93, 2.15, 2.33, 1.42; (2) primary colon tumors: 3.54, 1.52, 1.63, 1.58; (3) lung tumor cell lines: 1.13, 1.08; and (4) colon tumor cell lines: 1.41 , 1.34, 1.10, 1.50. Relative to normal tissue, this represents approximately a 2.55, 3.81 , 4.44, 5.03, 2.67-fold increase, respectively, in gene copy for the primary lung tumor hits, a 1 1.63, 2.87, 3.09, 2.99-fold increase, respectively, in gene copy for the primary colon tumors, a 2 19 and 2.1 1 -fold increase in gene copy for the lung tumor cell lines, and a 2.66. 3.53, 2.14 and 2.83-foId increase in gene copy for the colon tumor cell lines.
  • Amplification of DNA35617 was also confirmed by framework mapping.
  • Table 8 indicates that significant amplification of DNA35617 was confirmed in LT12, LT13, LT15, LT16 and CT2.
  • CT8, CT10 and CT14 The reported ⁇ Ct values for the primary tumors were 1.61 , 1.95, 2.86 and 1.68, while for the primary colon tumors the values were 3.75, 1.76, 1.68 and 1.75 These represent a 3.05, 3.86, 7.26, 3.20- fold increase in gene copy for the lung tumors and a 13.45, 3 39, 3.20 and 3.36-fold increase in gene copy for the colon tumors
  • the amplification of the closest known framework markers does not occur to a greater extent that that of DNA35617.
  • DNA356 I 7 is the gene responsible for the amplification of the particular region on chromosome 17 Because amplification of DNA35617 occurs in various tumor tissues, especially colon tumors, it is highly probably to plav a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g . antibodies) directed against the protein encoded by DNA35617 would be expected to have utility in cancer therapy
  • Table 2 indicates that significant amplification of DNA381 13 occurred in primary lung tumors: SRC734 (LT12), SRC735 (LT13), SRC736 (LT15), SRC737 (LT16), SRC738 (LT17), SRC740 (LT19); primary colon tumors: SRC742 (CT2), SRC743 (CT3), SRC744 (CT8), SRC745 (CTIO), SRC746 (CT12), SRC747 (CT14), SRC748 (CT15), SRC749 (CT16), SRC750 (CT17), SRC751 (CTI), SRC752 (CT4), SRC753 (CT5), SRC754 (CT6), SRC756 (CT9), SRC757 (CTI 1), SRC758
  • the ⁇ Ct values and average ⁇ Ct values for the primary lung tumor hits are: 2.57, 2.09, 3 44, 1.91 , 1.43, 3.58; primary colon tumors are: 3 64, 1.12, 1.64, 2.53, 1.88, 2.51, 1.30, 1.47, 1.71, 1.37, 2.22, 2.50, • 2.28, 1.58, 3.33 and 1.07; lung tumor cell lines 1.65, 2.23, 1.12, 1.18, 1.02 and 1.1 ; and colon tumor cell lines 2.24, 1.01, 1.23 and 1.61.
  • Amplification has been confirmed by framework mapping for DNA381 13 in primary lung tumors LT10 and in primary colon tumors CT2, CT3, CT8, CT10, CT12, CT14, CT16.
  • the reported ⁇ Ct values for the p ⁇ mary lung tumor is 1 45; the primary colon tumors are 2.94, 1.23, 1.45, 1.72, 1.60, 2.03 and 1.07. Relative to normal tissue, this represents approximately a 2.73-fold increase for the lung tumor and a 7.67, 2.34, 2.73, 3.29, 3.03, 4 08 and 2.10-fold increase for the colon tumors.
  • Epicenter mapping resulted in confirmation of significant amplification in: primary lung tumors LT12, LT13, LT15, LT16, LT17 and in p ⁇ mary colon tumors CTI , CT2, CT3, CT4, CT5, CT6, CT8, CT9, CT10, CTI 1 , CT12, CT14, CT16.
  • the reported ⁇ Ct values for the primary lung tumors are. 1.57, 1.22, 2.47, 2.23, 1.02 and for the primary colon tumors are 1.38, 2 94, 1.23, 2.23, 2 51, 2.29, 1 45. 1.59, 1.72, 3.34, 1 6, 2.03, 1 07 and 1 08. Relative to normal tissue, this represents a 2.97, 2.33, 5 54, 4 69.
  • DNA381 13 is the gene which is driving the amplification of this particular region of Chromosome 19
  • the amplification of marker S41 (which does not map closely to DNA381 13) could be an independent amplification event or even an error in the ordering of the markers
  • antagonists e g., antibodies directed against the protein encoded by DNA381 13 (PR0327) would be expected to have utility in cancer therapy
  • PRO 1265 The ⁇ Ct values for DNA60764 (PR01265)(UNQ636) in a variety of lung tumors are reported in
  • Table 2 A ⁇ Ct value of > l was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA60765 occurred in primary lung tumors LT3, LT12, LT13. LT15, LT16 and LT17 The ⁇ Ct values of these hits are 1.03, 2.17, 2.24, 3.51 , 3.32 and 1.02. This represents an increase in gene copy of approximately 2.04, 4.50, 4.72, 11.39, 9.99 and 2.03
  • Amplification has also been confirmed framework mapping for DNA60764 in LT16.
  • the reported ⁇ Ct value was 1.37, which represents a 2 58 fold increase in gene copy relative to normal tissue.
  • Epicenter mapping has also confirmed amplification of DNA60764 in LT12, LT13, LT15, LT16, CTI , CT4, CT5, CT7 and CTI 1.
  • These tumors report ⁇ Ct values of 2.35, 2.37, 3.88, 3.32 in the lung tumors and 1.74, 1.86, 3.28, 1.29 and 2.32 in the colon tumors. Relative to normal tissue, this represents an increase in gene copy of approximately 5.10, 5.17, 14.72 and 9.98 in the lung tumors and 3.34, 3.63, 9.71 , 2.45 and 4.99 in the colon tumors.
  • DNA60764 is ghe gene responsible for the amplification of the particular region in Chromosome 19 Because amplification of DNA60764 occurs in various lung and colon tumors, it is highly probably to play a significant role in tumor formation or growth. As a result, antagonists (e g., antibodies) directed against the protein encoded by DNA60764 would be expected to have utility in cancer therapy.
  • PRQ344 The ⁇ Ct values for DNA40592 (PR0344, UNQ303) in a variety of lung and colon tumors are reported in Table 2. A ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA40592 occurred in primary lung tumors LT13 and LT19, in primary colon tumors CT2. CT14, CT15. CTI, CT4, CT5, CTI 1. The ⁇ Ct values and average ⁇ Ct of these hits are 1.34, 1.52 in the lung tumors, and 1.84, 1.16, 1.17, 1.24, 1.46, 1.51 and 1 35. This represents approximately a 2 53, 2.87 and 3.58, 2.23, 2.25, 2.36,
  • the ⁇ Ct values for DNA43318 (PRO343)(UNQ302) in a variety of lung and colon tumors are reported in Table 2.
  • a ⁇ Ct of >1 was typicallv used as the threshold value for amplification scoring, as this represents a doubling of gene copy
  • Table 2 indicates that significant amplification of DNA43318 occurred in primary lung tumor: LT13, LT15, LT19, CT2, CT8, CTIO. CT12, CT14, CT15, CT16. CT17 and CT5.
  • the ⁇ Ct and average ⁇ Ct values for the primary lung tumor hits are 1 67, 1.47 and 1.66. while the colon tumor hits are 2.72, 1.43, 1.68, 1.33, 2.02, 1.73, 1 16, 1.5, 1.34. This represents an increase in gene copy of approximately 3.18. 2.77 and 3.16 -fold for the lung tumors and 6.59, 2.69, 3.20, 2.51 , 4.06, 3.31 , 2.23, 2.83 and 2.53 -fold for the colon tumors.
  • Amplification has been confirmed by framework mapping for DNA43318: in primary lung tumors LT12, LT13, LT15, LT16 and LT18: and in primary colon tumors CT2, CT4, CT5, CT8.
  • CTIO, CT14, CT15 and CT16 The reported ⁇ Ct values are 1 16. 1.72, 2.73, 1 46 and 1.06 for the lung tumors and 3.54, 1 17, 2.63, 1.44, 1.36, 1.68, 1.07 and 1.01 for the colon tumors.
  • Relative to normal tissue this represents about a 2.23, 3.29, 6.63, 2.75 and 2.08-fold increase in gene copy for the lung tumors, and about a 11.63, 2.25, 6.19, 2.71 , 2.57, 3.20, 2.10 and 2.01-fold increase for the colon tumors.
  • Epicenter mapping for DNA43318 confirmed significant amplification in LT12, LT13, LT15, LT16, CT4, CT5, CT6, CTI 1 and CT2.
  • the reported ⁇ Ct values are 1.32, 1.94, 3.07, 1.83, 1.02, 2.40, 3.78, 1.51 and 2.48.
  • Relative to normal tissue this represents a 2.50, 3.84, 8.40, 3.56-fold increase in the lung tumors and a 2.03, 5.28, 13.74, 2.85, 5.58-fold increase in gene copy for the colon tumors.
  • DNA43318 is the gene responsible for the amplification of the particular region of Chromosome 16 Because amplification of DNA43318 occurs in various lung and colon tumors, it is highly probably to play a significant role in tumor formation or growth As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA43318 would be expected to have utility in cancer therapy.
  • the ⁇ Ct values for DNA44176 (PR0347, UNQ306) in a variety of lung and colon tumors are reported in Table 2.
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
  • Table 2 indicates that significant amplification of DNA44176 occurred in primary lung tumors LT15 and primary colon tumors CT2, CT8, CT14, CT15, CT16, CT5 and CTI 1
  • the ⁇ Ct values and average ⁇ Ct of these hits are 1.76 in the lung tumors, and 1.85, 1.12, 1.27, 1.07, 1.19, 1.80. 1.42 in the colon tumors. This represents approximately a 3.39. 3.61. 2 17. 2 41. 2.10. 2.28. 3 48. 2.67-fold increase, respectively, in gene copy relative to normal tissue.
  • DNA44176 Because amplification of DNA44176 occurs in various tumors, it is likely associated with tumor formation or growth. As a result, antagonists, (e.g., antibodies) directed against the protein encoded by DNA44176 (PR0347) would be expected to be useful in cancer therapy
  • ⁇ Ct values for DNA44804 (PR0357)(UNQ314) in a variety of lung and colon tumor and cell lines are reported in Table 2.
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
  • Table 2 indicates that significant amplification of DNA44804 occurred in primary lung tumors: LT9, LT10, LTI 1, LT12. LT13, LT15, LT16, LT17, LT19, LT21 ; in primary colon tumors CT2, CT8, CTIO, CT14, CT15, CT16, CTI , CT4, CT5, CT6, CT7 and CT11.
  • the ⁇ Ct and average ⁇ Ct values of the lung tumor hits are: 1.42, 1.63, 1.47, 1.42, 1.52, 2.18, 1.23, 1.71, 2.47. 1.66: and the colon tumor hits are 2.01 , 1.1 , 1.42, 1.5, 1.25, 1.05, 1.04, 1.27, 1.69, 1.08, 1.73, 1.83.
  • DNA44804 is the gene responsible for the amplification of the particular region on chromosome 16. Because amplification of DNA44804 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA44804 would be expected to have utility in cancer therapy.
  • the ⁇ Ct values for DNA52722 (PR0715)(UNQ383) in a variety of lung and colon tumor and cell lines are reported in Table 2.
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
  • Table 2 indicates that significant amplification of DNA52722 occurred in LT12, LT13.
  • the ⁇ Ct and average ⁇ Ct values for the p ⁇ mary lung tumors are 1.29, 1.51.
  • DNA52722 is the gene responsible for the amplification of the particular region on Chromosome 17 Because amplification of DNA52722 occurs in various lung and colon tumors and cell lines, it is highly probably to play a significant role in tumor formation or growth As a result, antagonists (e g , antibodies) directed against the protein encoded by DNA52722 (PR0715) would be expected to have utility in cancer therapy
  • PRO1017 The ⁇ Ct values for DNA561 12 (PRO 1017, UNQ500) in a variety of lung and colon tumors are reported in Table 2 A ⁇ Ct value of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA56112 occurred in primary lung tumors LTla, LT3, LT6, LT7, LT9, LT10, LTI 1, LT12, LT13, LT15, LT16, LT17.
  • the ⁇ Ct and average ⁇ Ct values for (1 ) the primary lung tumor hits are 1 78, 2 37, 2 79, 1 11 , 2 73, 3 51 , 1 96, 2 20, 1 90 2 24, 1 78, 1 76, 1 24 and 2 24, and (2) p ⁇ mary colon tumor hits are 3 13, 2 74, 1 78, 2 67, 2 29, 2 87, 2 30, 1 75, 1 82, 1 10 2 20, 1 1 1 , 1 28 and 1 47
  • LTI 1. LT12. LT13. LT15. LT16, LT18 and LT22
  • the reported ⁇ Ct values are 1.21 , 1.59. 1 40, 1 07, 1 98. 1.15. 2.31 , 1 83, 2.79. 2 22, 1.06 and 1 18. These values represent a 2 31 , 3 01 , 2 64, 2 10, 3 95, 2 22. 4 96. 3 56. 6 92. 4 66. 2 08 and 2 27-fold increase, respectively, in gene copy for the indicated lung tumors
  • the ⁇ Ct values for the primary lung tumors are. 1.47, 1.51 , 2 27, 1.62 and 1.03; and the primary colon tumors are 1.29, 1.46, 2.28, 1.84. 2.56. 1 22 and 1.52 These values represent a 2.77, 2.85, 4.82, 3 07, 2 04-fold increase, respectively, in the lung tumors and a 2 45. 2 75, 4.86, 3.58, 5.90, 2.33 and 2 87-fold increase, respectively, in the colon tumors
  • DNA561 12 is the gene responsible for the amplification of the particular region on chromosome 17. Because amplification of DNA561 12 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g.. antibodies) directed against the protein encoded by DNA56112 would be expected to have utility in cancer therapy.
  • ⁇ Ct values for DNA57702 (PROl 1 12, UNQ555) in a variety of lung and colon tumors are reported in Table 2.
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
  • Table 2 indicates that significant amplification of DNA57702 occurred in lung tumors LT10, LTI 1, LT12, LT13, LT15, LT17, LT18 and in colon tumors CT2, CT8,
  • the ⁇ Ct values and average ⁇ Ct of these hits are 1 13, 1.52, 1.19, 1.63, 1.77. 1.45 and 1.25 for the lung tumor and 2.26, 1.06, 1.57, 1.31 , 1 89, 1.46, 1.25, 1 24. 1.53. 1.97, 1.57 and 2.28 for the colon tumors. This represents approximately a 2.19, 2.87, 2.28, 3 09, 3.41, 2.73, 2.38-fold amplification, respectively, for the lung tumor, and
  • DNA57702 (PROl 1 12) would be expected to be useful in cancer therapy.
  • the ⁇ Ct values for DNA50148 (PRO509, UNQ329) in a variety of lung and colon tumors are reported in Table 2.
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
  • Table 2 indicates that significant amplification of DNA50148 occurred in primary colon tumors CT15.
  • the ⁇ Ct values and average ⁇ Ct ot these hits are I 16 1 02 1 01 1 34 1 35 which represents a 2 23 2 03 2 01. 2 53, 2 55-fold increase respectively in gene copv relative to normal tissue
  • DNA50148 Because amplification of DNA50148 occurs in various tumors it is likely associated with tumor formation or growth as a result, antagonists, (e g , antibodies) directed against the protein encoded by DNA50148 (PRO509 would be expected to be useful in cancer therapv
  • the ⁇ Ct values for DNA48227 (PR0853)(UNQ419) in a variety of lung and colon tumors and cell lines are reported in Table 2
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy
  • Table 2 indicates that significant amplification of DNA48227 occurred in p ⁇ mary lung tumors LT12 and lung cell line H522, as well as in primary colon tumors CT2, CT3, CT8 CT10, CT12, CT14, CT15, CT16, CT17, CTI , CT4, CT5, CT7 and CT1 1
  • the ⁇ Ct and average ⁇ Ct values for these hits are 1 03, 1 02, 2 4, 1 52, 1 55, 1 97, 1 36, 1 75, 1 75, 1 1 1 , 1 11 ,
  • DNA48227 is the gene responsible for the amplification of the particular region on chromosome 17 Because amplification of DNA47227 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth As a result, antagonist (e g , antibodies) directed against the protein encoded by DNA48227 would be expected to have utility in cancer therapy
  • PRQ882 The ⁇ Ct value for DNA58125 (DNA882, UNQ448) in a variety of lung and colon tumors and cell lines is reported in Table 2 A ⁇ Ct value of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA58125 occurred in primary lung tumors LTla LT3, LT6, LT9, LT10, LTI 1 , LT12, LT13, LT15.
  • ⁇ Ct and average ⁇ Ct values for the primary lung tumor hits are 1 18, 1 26, 1 10, 1 81 , I 95, 1 29, 1 07, 1 36, 1 58, 1 45, 1 87, the colon tumor hits report 2 18, 1 64, 1 10. 1 64. 1 05. 1 62. 1 25. 1 29. 1 17. 1 1. 2.03, 2 13. lung tumor cell lines
  • Amplification has been confirmed by framework mapping for DNA58125 in primary lung tumors LT3, LT13, LT15, LT17, LT18, CTI .
  • the reported ⁇ Ct values for the primary lung tumors are 1.02, 1.33, 1.83, 1.03 and 1 08.
  • the p ⁇ mary colon tumors values are 1.08, 2.27, 1 34, 1.13, 2.17, 1.41 , 1.23, 1.74, 1.13, 1.74. 1.30 and 1.04. Relative to normal tissue, this represents approximately a 2.03, 2.51, 3.56, 2.04. 2.1 1 -fold increase, respectively, for the lung tumors, and a 2.1 1. 4.82, 2.53, 2.19, 4.50, 2.66, 2.34, 3.34, 2.19, 3 34, 2.46 and 2.06-fold increase, respectively, for the colon tumors.
  • Epicenter mapping further confirmed significant amplification in primary colon tumors LTI 2,
  • LT13, LT15, LT16, LT17 and in primary colon tumors CTI, CT4, CT6, CT7, CT9, CTI 1, CT2, CT8, CT10 and CT16.
  • the reported ⁇ Ct values in the primary lung tumors are 1.02, 1.52, 2.04, 1.09 and 1.32, while the colon tumor values are 2.29, 1.49, 1.83, 1.20, 1.67, 1.02, 1.07, 2.27, 1.50 and 2.24 Relative to normal tissue, this represents approximately a 2.03, 2.87, 4.1 1 , 2.13, 2.50-fold increase, respectively, in gene copy for the lung tumors and a 4.89, 2 81 , 3.56, 2.30, 3.18, 2.03, 2.10, 4.82, 2.83, 4 72-fold increase, respectively, for the colon tumors.
  • DNA58125 is the gene responsible for the amplification of the particular region on chromosome 16. Because amplification of DNA58125 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA58125 would be expected to have utility in cancer therapy.
  • In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping In situ hybridization was performed following an optimized version of the protocol by
  • the tubes were incubated at 37°C for one hour. 1 0 ⁇ l RQ1 DNase were added, followed by incubation at 37°C for 15 minutes. 90 ⁇ l TE ( 10 mM T ⁇ s pH 7.6/l mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon- 50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 ⁇ l TE were added.
  • the sections were deproteinated in 20 ⁇ g/ml proteinase K (500 ⁇ l of 10 mg/ml in 250 ml RNase-free RNase buffer; 37°C, 15 minutes ) - human embryo, or 8 x proteinase K (100 ⁇ l 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.
  • Hybridization 1 0 x 10' cpm probe and 1 0 ⁇ l tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes The slides were cooled on ice, and 48 ⁇ l hybridization buffer were added per slide After vortexing, 50 ⁇ I ''P mix were added to 50 ⁇ l prehyb ⁇ dization on slide The slides were incubated overnight at 55 °C
  • DNA381 13 (Prolactin Rceptor Homlog)
  • DNA52722 (TNF Homolog) dna52722p1 GGA TTC TAA TAC GAC TCA CTA TAG GGC CGC CCC GCC ACC TCC T dna52722p2 CTA TGA AAT TAA CCC TCA CTA AAG GGA CTC GAG ACA CCA CCT GAC CCA
  • TCT 52722 p4 CTA TGA AAT TAA CCC TCA CTA AAG GGA CTA GGG GGT GGG AAT GAA
  • AAG High expression levels were observed in many tissues, including placenta, osteoblasts. injured renal tubules, injured liver colorectal liver matastasis and gall bladder. Tested sample had acetominophen induced liver injury and hepatic cirrhosis
  • DNA48227 (Reductase Homolog) 607.p 1 : GGA TTC TAA TAC GAC TCA CTA TAG GGC CCA ACA GCG GCA TCG GAA AGA 607.p2. CTA TGA AAT TAA CCC TCA CTA AAG GGA GGA GCA CCA GCC AAG CCA ATG
  • Elevated expression was observed in the mucosa of the chimp stomach.
  • the following method describes use of a nucleotide sequence encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide as a hybridization probe.
  • SEQ ID NO: 23 SEQ ID NO: 23
  • PR0853 Fig. 25, SEQ ID NO: 25
  • PR0882 Fig. 27, SEQ ID NO: 27
  • homologous DNAs such as those encoding naturally-occurring variants of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in human tissue cDNA libraries or human tissue genomic libraries
  • Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions.
  • Hybridization of radiolabeled PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882-de ⁇ ved 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 0.1 x SSC and 0 l % SDS at 42"C.
  • PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can then be identified using standard techniques known in the art.
  • PRO201 PR0292. PR0327, PR01265. PR0344, PR0343. PR0347 PR0357. PR0715.
  • the vector is digested with restriction enzyme and dephosphorylated
  • the PCR amplified sequences are then hgated into the vector
  • the vector will preferably include sequences which encode for an antibiotic resistance gene, a tip promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO201, PR0292, PR0327. PRO 1265, PR0344, PR0343, PR0347, PR0357. PR0715. PRO1017. PROl 1 12, PRO509, PR0853 or PR0882 coding region, lambda transcriptional terminator, and an argU gene
  • the hgation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, du ⁇ ng which the expression promoter is turned on
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation
  • the cell pellet obtained by the centrifugation can be solubihzed using various agents known in the art. and the solubihzed PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein
  • PRO201 Expression of PRO201. PRQ292. PRQ327, PRO 1265, PRQ344 PRQ343 PRQ347, PRQ357, PRQ715. PRO1017. PROl 1 12. PRO509. PRQ853 or PRQ882 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PRO201 , PR0292, PR0327, PR01265, PR0344, PRO-343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 by recombinant expression in mammalian cells
  • the vector, pRK5 (see EP 307.247, published March 15, 1989), is employed as the expression vector
  • the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA is hgated into pRK5 with selected restriction enzymes to allow insertion of the PRO20I , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715.
  • PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA using hgation methods such as described in Sambrook et al .
  • the selected host cells may be 293 cells Human 293 cells (ATCC
  • CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 ⁇ g pRK5-PRO201.
  • pRK5-PR0357 pRK5-PR0715, pRK- 1017, pRK5-PR01 1 12.
  • pRK5-PRO509, pRK5-PR0853 are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 ⁇ g pRK5-PRO201.
  • pRK5- PR0882 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al , Cell, 31 543 ( 1982)] and dissolved in 500 ⁇ l of 1 mM T ⁇ s-HCl, 0.1 mM EDTA, 0.227 M CaCl- To this mixture is added, dropwise, 500 ⁇ l of 50 mM HEPES (pH 7.35), 280 mM NaCl. 1.5 mM NaP0 4 , and a precipitate is allowed to form for 10 minutes at 25"C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37"C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ O/ml 3, S-cyste ⁇ ne and 200 ⁇ Ci/ml "S-meth ⁇ on ⁇ ne.
  • the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel.
  • the processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 , or EBAF-2 polypeptide.
  • the cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays In an alternative technique.
  • PR0715, PRO 1017, PRO l 1 12, PRO509, PR0853 or PR0882 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Nati. Acad. Sci., 12:7575 ( 1981 ).
  • 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-PRO201 , pRK5-PR0292, pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5-PR0357, pRK5-PR0715, pRK- 1017, pRK5-PR01 1 12., pRK5-PRO509, pRK5- PR0853, pRK5-PR0882 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- mtroduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin and 0.1 ⁇ g/ml bovine transfemn. After about four days, the conditioned media is cent ⁇ fuged and filtered to remove cells and debris.
  • the sample containing expressed PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343, PR0347, PR0357, PR0715, PROl 017, PRO l 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
  • PRO 1 1 12, PRO509. PR0853 or PR0882 can be expressed in CHO cells The pRK5-PRO201 , pRK5-PR0292. pRK5-PR0327. pRK5-PR01265. pRK5-PR0344 pRK5-PR0343. P RK5-PR0347 pRK5-PR0357, pRK5-PR0715. pRK- 1017.
  • pRK5-PR01 1 12 pRK5- PRO509, pRK5-PR0853.
  • pRK5-PR0882 vector can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as "S-methionine After determining the presence of PRO201. PR0292. PR0327. PRO 1265. PR0344, PR0343. PR0347, PR0357, PR0715, PRO1017. PRO l 1 12. PRO509, PR0853 or PR0882 polypeptide.
  • the culture medium may be replaced with serum tree medium
  • the cultures are incubated for about 6 days, and then the conditioned medium is harvested.
  • PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 can then be concentrated and purified by any selected method.
  • PROl 1 12, PRO509, PR0853 or PR0882 may also be expressed in host CHO cells.
  • PRO509, PR0853 or PR0882 may be subcloned out of the pRK5 vector.
  • the subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector.
  • the poly-His tagged PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones.
  • the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression.
  • PROl 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as by N ⁇ 2+ -chelate affinity chromatography.
  • PR0292 (UNQ266) and PRO509 (UNQ329) were expressed in CHO cells by a transient expression procedure.
  • PRO1017 (UNQ500) and PRO509 (UNQ329) were stably expressed in CHO cells.
  • the proteins were expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins were fused to an IgGI constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
  • IgG construct immunoadhesin
  • CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's
  • the vector used expression in CHO cells is as described in Lucas et al , Nucl. Acids Res. 24- 9 (1774-1779 ( 1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • DHFR expression permits selection for stable maintenance of the plasmid following transfection Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect ® (Quiagen) DospeJ or Fugene J (Boeh ⁇ nger Mannheim) The ceils were grown and described in Lucas et al , supra Approximately 3 x 10 7 cells are frozen in an ampule for further growth and production as described below
  • the ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing The contents were pipetted into a centrifuge tube containing 10 mLs of media and cent ⁇ fuged at 1000 rpm for 5 minutes The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0 2 ⁇ m filtered PS20 with 5% 0 2 ⁇ m diafiltered fetal bovine serum) The cells were then aliquoted into a 100 L spinner containing 90 mL of selective media Alter 1-2 days, the cells were transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37 °C After another 2-3 days, a 250 mL, 500 mL and 2000 L spinners were seeded with 3 x 10 s cells/mL The cell media was exchanged with fresh media by centrifugation and resuspension in production medium Although any suitable CHO media may be employed, a
  • the proteins were purified using a Ni NTA column (Qiagen) Before purification, lmidazole was added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4 C C After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) constructs
  • PRO201 PRQ292.
  • PRQ327 PROl 265.
  • PRQ344 PRQ343 PRQ347 PRQ357.
  • PRQ715 PRO1017.
  • PRO l 1 12.
  • PRQ853 or PRQ882 in Yeast The following method describes recombinant expression of PRO201.
  • yeast expression vectors are constructed for intracellular production or secretion of
  • PRO509, PR0853 or PR0882 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO201 , PR0292, PR0327, PRO 1265, PRO-344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882
  • DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO.347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 signal peptide or other mammalian
  • PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 Yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% t ⁇ chloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO l 1 12, PRO509, PR0853 or PR0882 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • the concentrate containing PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 may further be purified using selected column chromatography resins.
  • PRQ201 PRQ292.
  • PRQ327 PROl 265.
  • PRQ344 PRQ343 PRQ347.
  • PRQ357 PRQ71 .
  • PRO1017 PROl 1 PRO509. PRQ853 or PRQ882 in Baculovirus-Infected Insect Cells
  • the following method describes recombinant expression in Baculovirus-infected insect cells.
  • PRO201 The sequence coding for PRO201. PR0292. PR0327, PR01265, PR0344. PR0343. PR0347, PR0357. PR0715, PRO10I 7.
  • PROl 1 12, PRO509. PR0853 or PR0882 is fused upstream of an epitope tag contained within a baculovirus expression vector
  • epitope tags include poly-His tags and lmmunogiobulin tags (like Fc regions of IgG).
  • Plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 or the desired portion of the coding sequence of PRO201 , PR0292.
  • pVL1393 Novagen
  • PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9") cells (ATCC CRL 171 1 ) using hpofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28 ' C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors- A Laboratory Manual. Oxford: Oxford University Press (1994). Expressed poly-His tagged PRO201. PR0292, PR0327, PR01265, PR0344. PR0343,
  • PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 can then be purified, for example, by N ⁇ 2+ -chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature. 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9, 12.5 mM MgCl 2 ; 0.1 mM EDTA, 10% glycerol, 0.1 % NP-40; 0.4 M KCI), and sonicated twice for 20 seconds on ice.
  • sonication buffer 25 mL Hepes, pH 7.9, 12.5 mM MgCl 2 ; 0.1 mM EDTA, 10% glycerol, 0.1 % NP-40; 0.4 M KCI
  • the sonicates are cleared by centrifugation. and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 ⁇ m filter.
  • loading buffer 50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8
  • a N ⁇ 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
  • the filtered cell extract is loaded onto the column at 0.5 mL per minute.
  • the column is washed to baseline A 280 with loading buffer, at which point fraction collection is started.
  • the column is washed with a secondary wash buffer (50 mM phosphate: 300 mM NaCl. 10% glycerol, pH 6 0), which elutes nonspecifically bound protein
  • a secondary wash buffer 50 mM phosphate: 300 mM NaCl. 10% glycerol, pH 6 0
  • the column is developed with a 0 to 500 M Imidazole gradient in the secondary wash buffer.
  • One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with to alkaline phosphatase (Qiagen) Fractions containing the eluted H ⁇ s l ⁇ -tagged PRO201.
  • PRO292 PR0327 PR01265 PR0344. PR0343. PR0347 PR0357.
  • PRO509. PR0853 or PR0882. respectively are pooled and dialyzed against loading buffer
  • purification ot the IgG tagged (or Fc tagged) PRO201.
  • PR0292. PR0327. PR01265, PR0344. PR0343. PR0347. PR0357. PR0715, PRO1017.
  • PR0853 or PR0882 can be performed using known chromatography techniques, including for instance. Protein A or protein G column chromatography
  • PR0327 (UNQ288). PR0344 (UNQ303).
  • PRO509 (UNQ329) were expressed in baculovirus infected Sf9 insect cells While the expression was actually performed in a 0 5-2 L scale, it can be readily scaled up for larger (e g 8 L) preparations
  • the proteins were expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region was fused to an IgGI constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms
  • baculovirus expression vector pb PH IgG for IgG fusions and pb PH.His.c for poly-His tagged proteins
  • vector and Baculogold® baculovirus DNA were co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectin (Gibco BRL)
  • pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences
  • the cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated for 5 days at 28°C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf
  • the first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown m ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were incubated for 3 days at 28°C. The supernatant was harvested and filtered Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed.
  • the conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters
  • the protein construct were purified using a Ni-NTA column (Qiagen) Before purification, imidazole was added to the conditioned media to a concentration of 5 mM
  • the conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 03 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4°C
  • the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole
  • the highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol.
  • lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid.
  • the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5 '-of) of an epitope tag contained with a baculovirus expression vector
  • suitable systems such as Pfu (Stratagene)
  • Pfu Stratagene
  • epitope tags include poly-his tags and lmmunogiobulin tags (like Fc regions of IgG).
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl-1 (Novagen)
  • the pIEl-1 and pIEl-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells (1)
  • the plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lel-mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIEl-1 and pIEl-2 include the te translation initiation site and can be used to produce fusion proteins
  • the desired sequence or the desired portion of the sequence is amplified by PCR with primers complementary to the 5' and 3' regions
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites
  • the product was then digested with those selected restriction enzymes and
  • H ⁇ 5 cells are grown to a confluency of 50% under the conditions of, 27°C, no CO : , NO pen/strep
  • 30 ug of pIE based vector containing the sequence was mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH B ⁇ osc ⁇ ences # 14401 -78P (note this media is light sensitive)), and in a separate tube, 100 ul of CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium
  • the two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2ml of DNA/CellFECTIN mix and this is layered on H ⁇ 5 cells that have been washed once with Ex-Cell media The plate is then incubated in darkness for 1 hour at room temperature
  • Immunogens that may be employed include purified PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, fusion proteins containing PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, and cells expressing recombinant PRO201 , PR0292, PR0327, PR01265, PR0344, PRO-343, PR0347, PR0357, PR0715, PROI017, PROl 112, PRO509, PR0853 or PR0882 on the cell surface Selection of the immunogen can be made by the skilled artisan without undue experimentation
  • mice such as Balb/c are immunized with the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or lntrape ⁇ toneally 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 hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected ad
  • mice are sacrificed and the spleen cells are harvested
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected munne myeloma cell line 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, aminopte ⁇ n, 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 Determination of "positive" hybndoma cells secreting the desired monoclonal antibodies against PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 is within the skill in the art
  • the positive hybndoma cells can be injected lntrape ⁇ toneally into syngeneic Balb/c mice to produce ascites containing the anti- PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 monoclonal antibodies.
  • the hyb ⁇ doma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

Abstract

The invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The invention is based on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including prevention) of certain cancers, and may act of predictors of the prognosis of tumor treatment.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR
Related Applications
This is a non-provisional application filed under 37 C F R 1 53(b)( 1 ), claiming priority under 35 U S C 1 19(e) to provisional application nos serial no 60 088 742,filed 10 June. 1998. serial no 60/086.414 filed 22 May 1998. serial no 60,070.440, filed 05 January 1998, serial no 60/109,304 filed 20 November
1998, serial no 60/083,500 filed 29 April 1998. and serial no "to be assigned" (Attorney Docket No
PR 1533), filed 10 November 1998 the contents of which are incorporated herein by reference
Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of rumor
Background of the Invention Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al , CA Cancel J Clin 43, 7 [ 1993])
Cancer is characterized by the increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis) In a cancerous state a cell proliferates under conditions in which normal cells would not grow Cancer manifests itself in a wide vdnety of forms, characterized by different degrees of invasiveness and aggressiveness Alteration of gene expression is intimately related to the uncontrolled cell growth and de- differentiationwhich are a common feature of all cancers The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which v ^uld normally function to prevent malignant cell growth, and/or overexpression of certair dominant genes, such as oncogenes, that act to promote malignant growth Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype (Hunter,
CeU 64, 1 12 [1991], Bishop. Cell 64. 235-248 [1991])
A well known mechanism of gene (e g oncogene) overexpression in cancer cells is gene amplification This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome ( Alitalo et al , Adv Cancer Res 47, 235-281 [1986]) It is believed that the overexpression of the gene parallels gene amplification, I e is proportionate to the number of copies made
Proto-oncogenesthat encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer For example, it has been found that the human ErbB2 gene (erbB2. also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoprote receptor (pl 85HER2, HER2) related to the epidermal growth factor receptor (EGFR), is overexpressedin about 25% to 30% of human breast cancer (Slamon et al . Science 235 1 7-182 [1987], Slamon et al , Science 244 707-712 [ 1989]) It has been reported that gene amplification of a proto-oncogeneis an event typically involved in the more malignant forms of cancer and could act as a predictor of clinical outcome (Schwab et al Genes Chromosomes Cancer 1. 181- 193 [1990], Ahtalo et al supra) Thus. erbB2 overexpression is commonly regarded as a predictorof a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al . [ 1987] and [ 1989], supra, Ravύm and Chamness. G we 159 19-27 [1995]. and Hynes and Stern, Biochim Biophvs Ada 1 198 165- 184 [ 1994]). and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines (Baselga et al , Oncology 1 1 (3 Suppl 1 ) 43-48 [1997]) However, despite the association of erbB2 overexpression with poor prognosis, the odds of HER2-posιtιve patients responding clinically to treatment with taxanes were greater than three times those of HER2-negatιve patients (Ibid) A recombιnanthumanιzedantι-ErbB2(antι-HER2)monoclonal antibody (a humanized version of the muπneantι-ErbB2 antibody 4D5. referred to as rhuMAb HER2 or Herceptin®) has been clinically active in patients with ErbB2-overexpressιng metastatic breast cancers that had received extensive prior anticancer therapy (Baselga et al , -/ Clin Oncol 14 737-744 [1996]) Summary of the Invention
The present invention concerns compositions and methods for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans The present invention is based on the identification of genes that are amplified in the genome of tumor cells Such gene amplification is expected to be associated with the overexpression of the gene product and concπbute to tumoπgenesis Accordingly, the proteins encoded by the amplified genes are believed to be useful targets for the diagnosis and/or treatment (including preventιon)of certain cancers, and may act of predictors of the prognosis of tumor treatment
In one embodiment, the present invention concerns an isolated antibody which binds a polypeptide which is designated PRO201 (UNQ175), PR0292 (UNQ255), PR0327 (UNQ288). PR01265 (UNQ636), PR0344 (UNQ303), PR0343 (UNQ302), PR0347 (UNQ306), PR0357 (UNQ314). PR0715 (UNQ383), PRO 1017 (UNQ500), PRO 1 1 12 (UNQ555), PRO509 (UNQ329), PR0853 (UNQ419) or PR0882 (UNQ448), wherein "PRO" stands for "protein" and "UNQ" stands for "unique" Throughout this specification, the "PRO" and "UNQ" numbers are used interchangeably to designate the respective proteins, wherein UNQ designates the native human sequence only and PRO designates the native sequence and active variants thereof In one aspect, the antibody induces death of a cell overexpressing a PRO20I , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357. PR0715, PRO1017, PR01 1 12, PRO509, PR0853 or PR0882 polypeptide In another aspect, the antibody is a monoclonal antibody, which preferably has nonhuman compiementaπtydetermιnιngregιon (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 single-chain antibody, or an anti-idiotypic antibody
In another embodiment, the invention concerns a composition comprising an antibody which binds a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509 , PR0853 or PR0882 polypeptide m admixture with a pharmaceutically acceptable carrier In one aspect, the composition comprises a therapeutically effective amount of the antibody In another aspect the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent Preferably, the composition is sterile
In a further embodiment, the invention concerns nucleic acid encoding an antι-PRO201 , anti- PR0292. antι-PR0327.antι-PR01265,antι-PR0344.antι-PR0343,antι-PR0347.antι-PR0357,antι-PR0715, antι-PRO1017, anti-PROl 1 12, antι-PRO509, antι-PR0853 or antι-PR0882 antibody, and vectors and recombinant host cells comprising such nucleic acid
In a still further embodiment, the invention concerns a method for producing an antι-PRO201 , anti- PR0292. antι-PR0327,antι-PR01265,antι-PR0344.antι-PR0343,antι-PR0347.antι-PR0357 antι-PR0715, antι-PRO1017,antι-PROl 1 12,antι-PRO509, antι-PR0853 or antι-PR0882 antibody by culturing a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the antibody from the cell culture
The invention further concerns antagonists and agonists of a PRO201 , PR0292, PR0327. PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide that inhibit one or more of the functions or activities of the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide
In a further embodiment, the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides The nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions Furthermore, such sequences can be used as part of πbozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes In another embodiment, the invention concerns a method for determimngthe presence of a PRO201,
PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509 , PR0853 or PR0882 polypeptidecompπsingexposinga cell suspected of containing the PRO201, PR0292, PRO327, PRO1265,PRO344,PRO343,PRO347,PRO357,PRO715,PRO1017,PROl 112, PRO509, PR0853 or PR0882 polypeptide to an antι-PRO201, antι-PR0292, antι-PR0327, antι-PR01265, antι-PR0344, anti- PR0343, antι-PR0347, antι-PR0357, antι-PR0715, antι-PRO1017, anti-PROl 1 12, antι-PRO509, anti- PR0853 or antι-PR0882 antibody and determining binding of the antibody to the cell
In yet another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptιde(a) in 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 expression ievel in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained
In another embodiment, the present invention concerns a method of diagnosing tumor in a mammal, comprising (a) contacting an antι-PRO201 , antι-PR0292, antι-PR0327, antι-PR01265, antι-PR0344, anti- PR0343. antι-PR0347, antι-PR0357. antι-PR0715 antι-PRO1017, anti-PROl 1 12, antι-PRO509, anti- PR0853 or antι-PR0882 antibody with a test sample of tissue cells obtained from the mammal , and (b) detecting the formation of a complex between the antι-PRO201 , antι-PR0292. antι-PR0327, anti-PRO 1265, antι-PR0344,antι-PR0343,antι-PR0347, antι-PR0357, antι-PR0715, antι-PRO1017, anti-PROl 1 12, anti- PRO509. antι-PR0853 or antι-PR0882 antibody and the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343. PR0347, PR0357. PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in the test sample 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 sampie indicates the presence of tumor in the mammal from which the test tissue cells were obtained The antibody preferably carries a detectable label Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluoπmetry, or other techniques known in the art
The test sample is usually obtained from an individual suspected to have neoplastic cell growth or proliferation (e g cancerous cells) In another embodiment, the present invention concerns a cancer diagnostic kit, comprising an anti-
PRO201, antι-PRO292,antι-PRO327.antι-PRO1265,antι-PRO344,antι-PRO343,antι-PRO347,antι-PRO357, antι-PR0715, antι-PRO1017, anti-PROl 1 12, antι-PRO509, antι-PR0853 or antι-PR0882 antibody and a carrier (e g a buffer) in suitable packaging The kit preferably contains instructions for using the antibody to detect the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide
In yet another embodiment, the invention concerns a method for inhibitingthe growth of tumor cells comprising exposing a cell which overexpressesa PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017. PROl 1 12, PRO509, PR0853 or PR0882 polypeptideto an effective amount of an agent inhibiting the expression and/or activity of the PR 201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347. PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide The agent preferably is an antι-PRO201, antι-PR0292, antι-PR0327. anti-PROl 265, anti- PR0344, antι-PR0343, antι-PR0347, antι-PR0357, antι-PR0715, antι-PRO1017, anti-PROl 1 12, anti- PRO509, antι-PR0853 or antι-PR0882 antibody, a small organic and inorganic molecule, peptide, phosphopeptide, antisense or πbozyme molecule, or a triple helix molecule In a specific aspect, the agent, e g antι-PRO201,antι-PRO292,antι-PRO327,antι-PRO1265,antι-PRO344.antι-PRO343,antι-PRO347,antι- PRO357,antι-PRO715,antι-PRO1017,antι-PROl 1 12,antι-PRO509,antι-PRO853 or antι-PR0882 antibody induces cell death In a further aspect, the tumor cells are further exposed to radiation treatment and/or a cytotoxic or chemotherapeutic agent
In a further embodiment, the invention concerns an article of manufacture, comprising a container, a label on the container, and a composition comprising an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327, PR01265. PR0344 PR0343 PR0347, PR0357, PR0715 PRO1017, PRO l 1 12 PRO509 PR0853 or
PR0882 polypeptide, and the active agent in the composition is an agent inhibiting the expression and/or activity of the PRO201 PR0292, PR0327, PR01265 PR0344, PR0343, PR0347 PR0357. PR0715
PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide In a preferred aspect, the active agent is an antι-PRO201. antι-PR0292, antι-PR0327. anti-PRO 1265,antι-PR0344, antι-PR0343. antι-PR0347 anti-
PR0357 antι-PR0715, anti-PROl 017, anti-PRO l 1 12,antι-PRO509, antι-PRO853 or antι-PRO882 antibody
A method for identifying a compound capable of inhibiting the expression and/or activity of a
PRO201 , PR0292, PR0327. PRO 1265. PR0344, PR0343, PR0347, PR0357, PR0715. PRO 1017, PRO 1 1 12,
PRO509, PR0853 or PR0882 polypeptide, comprising contacting a candidate compound with a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12,
PRO509, PR0853 or PR0882 polypeptide under conditions and for a time sufficient to allow these two components to interact In a specific aspect, either the candidate compound or the PRO201 , PR0292,
PR0327, PR01265,PR0344,PR0343. PR0347, PR0357 PR0715, PROl 017, PROl 1 12. PRO509 PR0853 or PR0882 polypeptide is immobilized on a solid support Brief Description of the Figures
Figure 1 (SEQ ID NO 1 ) is the nucleotide sequence of DNA30676 encoding a PRO201 (UNQ 175) Figure 2 (SEQ ID NO 2) is the ammo acid sequence of the native human PRO201 (LTNQ175) polypeptide
Figure 3 (SEQ ID NO 3) is the nucleotide sequence of DNA35617 encoding a PR0292 Figure 4 (SEQ ID NO 4) is the amino acid sequence of the native human PR0292 (UNQ266) polypeptide
Figure 5 (SEQ ID NO 5) is the nucleotide sequence of DNA381 13 encoding a PR0327 Figure 6 (SEQ ID NO 6) is the ammo acid sequence of the native human PR0327 (UNQ288) polypeptide Figure 7 (SEQ ID NO 7) is the nucleotide sequence of DNA60764 encoding a PRO 1265
Figure 8 (SEQ ID NO 8) is the ammo acid sequence of the native human PRO 1265 (UNQ636) polypeptide
Figure 9 (SEQ ID NO 9) is the nucleotide sequence of DNA40592 encoding a PR0344 Figure 10 (SEQ ID NO 10) is the ammo acid sequence of the native human PR0344 (UNQ303) polypeptide
Figure 1 1 (SEQ ID NO 1 1 ) is the nucleotide sequence of DN A43318 encoding a PR0343 Figure 12 (SEQ ID NO 12) is the ammo acid sequence of the native human PR0343 (UNQ302) polypeptide
Figure 13 (SEQ ID NO 13) is the nucleotide sequence of DNA44176 encoding a PR0347 Figure 14 (SEQ ID NO 14) is the ammo acid sequence of the native human PR0347 (UNQ306) polypeptide
Figure 15 (SEQ ID NO 15) is the nucleotide sequence of DNA44804 encoding a PR0357 Figure 16 (SEQ ID NO 16) is the ammo acid sequence of the native human PR0357 (UNQ314) polypeptide Figure 17 (SEQ ID NO 17) is the nucleotide sequence of DNA52722 encoding a PR0715 Figure 18 (SEQ ID NO 18) is the amino acid sequence of the native human PR0715 (UNQ383) polypeptide
Figure 19 (SEQ ID NO 19) is the nucleotide sequence of DN A561 12 encoding a PRO 1017 Figure 20 (SEQ ID NO 20) is the amino acid sequence of the native human PRO 1017 (UNQ500) polypeptide
Figure 21 (SEQ ID NO 21 ) is the nucleotide sequence of DNA57702 encoding a PROl 1 12 Figure 22 (SEQ ID NO 22) is the ammo acid sequence of the native human PROl 1 12 (UNQ555) Figure 23 (SEQ ID NO 23) is the nucleotide sequence of DNA50148 encoding a PRO509 Figure 24 (SEQ ID NO 24) is the amino acid sequence of the native human PRO509 polypeptide
(UNQ329)
Figure 25 (SEQ ID NO 25) is the nucleotide sequence of DNA48227 encoding a PRO1350 Figure 26 (SEQ ID NO 26) is the ammo acid sequence of the native human PRO 1350 polypeptide (UNQ419) Figure 27 (SEQ ID NO 27) is the nucleotide sequence of DNA58125 encoding a PR0882
Figure 28 (SEQ ID NO 28) is the amino acid sequence of the native human PR0882 polypeptide (UNQ448)
Figure 29 (SEQ ID NO 29) is the nucleotide sequence of DNA28710 used in the cloning of DNA 30676 encoding PRO201 Figure 30 (SEQ ID NO 30) is the nucleotide sequence encoding EST 2452972
Figure 31 (SEQ ID NO 31 ) is the nucleotide sequence encoding EST2099855 Figure 32 is a map of chromosome 19 showing the mapping regions of DNA30676, DNA381 13 and DNA60764
Figure 33 is a map of chromosome 1 1 show ing the mapping region of DNA354617 Figure 34 is a map of chromosome 16 showing the mapping region of DNA58125 and DNA43318
Figure 35 is a map of chromosome 7 showing the mapping region of DNA561 12 Figure 36A is map of chromosome ' 7 showing the mapping region of DNA52722 Figure 36B is a map of chromosome 17 showing the mapping region of DNA48227 Figures 37-39 are in situ hybridizations as described in the example section Figure 40 is a map of chromosome 16 showing the mapping region of DNA44804
Detailed Description of the Invention I Definitions
The phrases "gene amplification" and "gene duplication ' are used interchangeably and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon " Usually. the amount of the messenger RNA (mRNA) produced, I e the level of gene expression, also increases in the proportion of the number of copies made of the particular gene expressed "Tumor as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized bv unregulated cell growth Examples of cancer include but are not limited to, carcinoma, lymphoma blastoma.sarcoma, and leukemia More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastromtestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma. colorectal cancer, endometπal carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented In tumor (e g cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e g radiation and/or chemotherapy
The "pathology" of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc "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, horses, cats, cows, etc Preferably, the mammal is human
"Carriers" as used herein include pharmaceuticallyacceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being e) ;θsed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin, or ιmmunoglobulιns,hydrophιlιc polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagme, arginine or lysine, monosacchaπdes,dιsacchaπdes,and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counteπons such as sodium, and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is intended to include radioactive isotopes (e g I13', I125, Yw and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer Examples of chemotherapeuticagents include adπamycin doxorubicin.epirubicin, 5-fluorouracιl, cytosine arabinoside ("Ara-C") cyclophosphamide. thiotepa, busulfan. cytoxin, taxoids, e g paclitaxel (Taxol Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate cisplatin.melphalan, vinblastine.bleomycin.etoposide. ifosfamide.mitomycinC, itoxantrone, vincristine. vinorelbine, carboplatm, teniposide, daunomycin, carminomycin, ammopteπn. dactinomycin, mitomycms, esperamιcιns(see U S Pat No 4,675, 187), 5-FU, 6-thιoguanιne.6-mercaptopuπne actinomycm D, VP- 16. chlorambucil.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 inhibitory 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 m 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 arrest and M-phase arrest Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubιcιn,epιrubιcιn,daunorubιcιn,etoposιde and bleomycin Those agents that arrest G 1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, predmsone, dacarbazme, mechlorethamιne,cιsplatιn, methotrexate, 5-fluorouracιl,and ara-C Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds . Chapter 1, entitled "Cell cycle regulation oncogens, and antineoplastic drugs" by Murakami et al (WB Saunders Philadelphia, 1995), especially p 13
"Doxorubicin" is an athracycline antibiotic The full chemical name of doxorubicin is (8S-cιs)- 10- [(3-amιno-2,3,6-tπdeoxy-o.-L-lyxo-hexapyranosyI)oxy]-7,8,9, 10-tetrahydro-6,8.1 l -tπhydroxy-8- (hydroxyacety 1)- 1 -methoxy-5, 12-naphthacenedιone
The term "cytokme" is . generic 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 cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroidhormone, thyroxine, insulin, proinsulin, relaxm, prorelaxin, glycoprote hormones such as follicle stimulating hormone (FSH), thyroid stιmulatιnghormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mullerian-inhibiting substance, mouse gonadotropm-associatedpeptide, inhibin, activin. vascular endothelial growth factor, ιntegπn,thrombopoιetιn (TPO), nerve growth factors such as NGF-β, platelet-growth factor, transforming growth factors (TGFs) such as TGF-α and TGF-β, insulin-like growth factor-I and -II, erythropoietin (EPO), osteomductive factors, interferons such as mterferon -α, -β, and -γ, colonv 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-1 1, 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 herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines The term prodrug ' as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is iess cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form See, e g Wilman, "Prodrugs in Cancer Chemotherapy \ Biochemical Society Transactions. 14, pp 375-382, 615th Meeting, Belfast (1986), and Stella et al "Prodrugs A Chemical Approach to Targeted Drug Delivery", Directed Drug delivery, Borchardte/ / (ed ), pp 147-267. Humana Press (1985) The prodrugs of this invention include, but are not limited to, phosphate-contaimngprodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glysocyiated prodrugs, β-lactam- containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-contaming prodrugs, 5-fluorocytosιne and other 5-fluorouπdιne prodrugs which can be converted into the more active cytotoxic free drug Examples of cytotoxic drugs that can be deπvatized into a prodrugs form for use in this invention include, but are not limited to, those chemotherapeutic agents described above
As used herein, the terms "PRO201 J "PR0292," "PR0327," "PRO 1265," "PR0344," "PR0343," "PR0347," "PR0357," "PR0715." "PRO1017J "PROl 1 12," "PRO509," "PR0853" or "PR0882" or
"PRO201 polypeptide". "PR0292 polypeptιde","PR0327 polypeptιde","PR01265 polypeptide", "PR0344 polypeptide", "PR0343 polypeptide", "PR0347 polypeptide", "PR0357 polypeptide", "PR0715 polypeptide", "PROl 017 polypeptide", "PROl 1 12 polypeptide", "PRO509 polypeptide", "PR0853 polypeptide" or "PP0882 polypeptide) encompass both native sequence and active variants thereof Th PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic means The term specifically encompasses naturally-occurringtruncated or secreted forms (e g , an extracellular domain sequence), naturally-occurring variant forms (e g ,
Figure imgf000011_0001
spliced forms) and naturally-occurring allelic variants of the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptide Fragments of the respective native polypeptides herein include, but are not limited, to polypeptide variants from which the native N-terminal signal sequence has been fully or partially deleted or replaced by another sequence, and extracellular domains of the respective native sequences, regardless whether such truncated (secreted) forms occur in nature Fragments are preferably sufficient in length for the production of an antibody specifically binding the corresponding native "PRO" polypeptide
As used herein, the terms "UNQ 175", "UNQ255", "UNQ288", "UNQ636", "UNQ303", "UNQ302", "UNQ306", "UNQ314", "UNQ383", "UNQ500", "UNQ555", "LTNQ329", "UNQ419" and "LTNQ448" are used to refer to the native sequence polypeptide corresponding to the PRO designated sequences above appearing as a full-length native presequence or a mature form of a PRO201 polypeptide shown in Figure 2 (SEQ ID NO 2), a PR0292 polypeptide shown in Figure 4 (SEQ ID NO 4), a PR0327 polypeptide shown in Figure 6 (SEQ ID NO 6), a PRO 1265 polypeptide shown in Figure 8 (SEQ ID NO 8), a PR0344 polypeptide shown in Figure 10 (SEQ ID NO 10), a PR0343 polypeptide shown in Figure 12 (SEQ ID NO 12), a PR0347 polypeptide shown in Figure 14 (SEQ ID NO 14), a PR0357 polypeptide shown in Figure 16 (SEQ ID NO 16). a PR0715 polypeptide shown in Figure 18 (SEQ ID NO 18). a PROl 017 polypeptide shown in Figure 20 (SEQ ID NO 20), a PROl 1 12 polypeptide shown in Figure 22 (SEQ ID NO 22). a PRO509 polypeptιdeshown ιn Fιgure24 (SEQ ID NO 24) a PR0853 polypeptide shown in Figure 26 (SEQ IDNO 26). and a R0882 polypeptιdeshown ιn Fιgure28 (SEQ ID O 28). respectively Said another way, the "UNQ" designation refers to the specific native sequence while the "PRO" designation refers to the native sequence and active variants thereof
An "isolated" nucleic acid molecule encoding a PRO201, PR0292. PR0327, PRO 1265. PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 polypeptide 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 PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-. PR0343-, PR0347-, PR0357-, PR0715-. PRO 1017-. PRO 1 1 12-, PRO509-, PR0853- or PR0882- encodmg nucleic acid An isolated PRO201 -, PR0292-, PR0327-. PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encodιng nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from the PRO201-, PRO292-, PRO327-, PROl 265-. PR0344-, PR0343-. PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encodιng nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide includes nucleic acid molecules contained in cells that ordinarily express PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding seς.ience in a particular host organism The control sequences that are suitable for prokaryot ., for example, include a promoter, optionally an operator sequence, and a ribosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotem that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous, and. in the case of a secretory leader, contiguous and in reading phase However, enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oiigonucleotide adaptors or linkers are used in accordance with conventional practice
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybndizationgenerally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybπdizable sequence the higher the relative temperature which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions, see Ausubel et al Current Protocols in Molecular Biology, Wiley Interscience Publishers. (1995)
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that (1) employ low ionic strength and high temperature for washing, for example 0 015 M sodium chloride/00015 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% bovine 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 stringent conditions" may be identified as described by Sambrook et al Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press, 1989, and include the use of washing solutior and hybridization conditions (e g , temperature, ionic strength and % SDS) less stπngen' than those described above An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising 20% formamide, 5 x SSC (150 mM NaCl. 15 M tπsodium 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 in 1 x SSC at about 37-50°C The skilled artisan will recognize how to adjust the tempe. -ure, ionic strength, etc as necessary to accommodate factors such as probe length and .ιe like
The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO201 ,
PP0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 polypeptide fused to a "tag polypeptide" The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 ammo acid residues (preferably, between about 10 and 20 am o acid residues)
"Active" or "activity" m the context of molecules identified based upon the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptides (or their coding sequences) refers to polypeptides (e g antibodies) or organic or inorganic small molecules, peptides, e e which retain the biological and/or lmmunologicalactivities/properties of a native or naturally-occurring PRO201. PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 "Biological activity in 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 ) is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins A preferred biological activity is growth inhibition of a target tumor cell Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell
The phrase "lmmunologicalproperty" means immunologicalcross-reactivity with at least one epitope of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112. PRO509, PR0853 or PR0882 polypeptide "Immunologιcaicross-reactιvιty"as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having this activity with polyclonal antisera raised against the known active PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357. PR0715, PRO 1017, PRO 1 1 12. PRO509. PR0853 or PR0882 polypeptide Such antisera are prepared in conventional fashion by injecting goats or rabbits, for example, subcutaneously with the known active analogue in complete Freund's adjuvant, followed by booster intrapeπtoneal or subcutaneous injection in incomplete Freunds The immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologically cross-reactive molecule (o g antibody) identified, to the corresponding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide is significantly higher (preferablyat least about 2-tιmes, more preferably at least about 4-tιmes, even more preferably at least about 8-tιmes, most preferably at least about 8-tιmes higher) than the binding affinity of that molecule to any other known native polypeptide
The term "antagonist" is used in the broadest sense, and includes any molecule that p rtially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343. PR0347. PR0357, PR0715, PRO1017, PROl 1 12, or PRO509 polypeptide disclosed herein In a similarmanner, the term "agonist" is used in the broadest sense and inch des any molecule that mimics a biological activity of a native PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide disclosed herein Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc
A "small molecule" is defined herein to have a molecular weight below about 500 Dalton "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoprotems having the same structural characteristics While antibodiesexhibit bindingspecificityto a specific antigen, immunoglobulins include both antibodies and other antibody-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 in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity
"Native antibodies" and "native immunoglobulins" are usually heterotetrameπc glycoproteins of about 150.000 Dalton, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain 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
Figure imgf000015_0001
and light chain also has regularly spaced lntracham disulfide bridges Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains Each light chain has a variable domain at one end (VL) and a constant domain 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 of the heavy chain Particular ammo acid residues are believed to form an interface between the light- and heavy- chain variable domains
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 binding and specificity of each particular antibody for its particular antigen However, the variability is not evenly distributed throughout the variable domains of antibodies It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervaπable regions both in the light-chain and the heavy-chain variable domains The more highly conserved portions of variable domains are called the framework (FR) The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuiaπon. connected by three 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 chain, contπbuteto the formation ofthe antigen-bindingsite of antibodies (see Kabat et al MH Pub/ No 91-3242, Vol I, pages 647-669 ( 1991 )) The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody u rntibody-dependent cellular toxicity
The term 'hypervanableregion" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding The hypervaπable region comprises amino acid residues from a ' complementaπtydeteπτiinιngregιon" to "CDR" (i e residues 24-34 (L I ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H 1 ), 50-65 (H2) and 95- 102 (H3) in the heavy chain variable domain, Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institute of Health, Bethesda. MD [ 1991]) and/or those residues from a "hypervaπable loop (I e residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain , Clothia and Lesk, J Mol Biol 196 901 -917 [ 1987]) "Framework" or "FR" residues are those variable domain residues other than the hypervaπable region residues as herein defined
"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody Examples of antibody fragments include Fab. Fab', F(ab'),, and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng 8(10) 1057-1062 [ 1995]), single-chain antibodv molecules, and multispecific antibodies formed from antibody fragments Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-bindingsite, and a residual "Fc" fragment whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen "Fv" is the minimum antibody fragment which contains a complete antigen-recognitionand -binding site This region consists ofa dimer ofone heavy- and one light-chain variable domain in tight non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V -VL dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) 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 CH I domain including one or more cystemes from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group F(ab'). antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the ammo acid sequences of their constant domains
Depending on the amino acid sequence of the constant domain of their heavy chains, lmmunoglobulinscan be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG l , IgG2, IgG3, IgG4, IgA. and IgA2 The heavy-chain constant domains that corres^-nd to the different classes of immunoglobulins are called α, δ, e, 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 occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore in contrast to conventional (polyclonal)antιbody preparationswhich 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 specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybπdoma culture, uncontammatedby other immunoglobulins The modifier "monoclonal" indicates the character of the antibody as being 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 hybπdoma method first described by Ko leret al Nature. 256 495 [1975], or may be made b\ recombinant DNA methods (see, e g , U S Patent No 4.816,567) The "monoclonal antibodies" may also be isolated from phage antibodv libraries using the techniques described in Clackson et al nature. 352 624-628 [ 1991 ] and Marks et al J Mol Biol , 222 581 -597 ( 1991 ). for example
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding 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 correspondingsequencesin 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 Proc Nati Acad Sci -/SΛ, 81 6851 -6855 [ 1984]) "Humanized" forms of non-human (e g , muπne) antibodies are chimeric immunoglobulins, lmmunogiobulin chains or fragments thereof (such as Fv. Fab. Fab', F(ab'): or other antigen-binding subsequencesof antibodies) which contain minimal sequence derived from non-human lmmunogiobulin For the most part, humanized antibodies are human ιmmunoglobulιns(recιpιentantιbody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse. rat or rabbit having the desired specificity, affinity, and capacity In some instances. Fv FR residues of the human lmmunogiobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modificationsare made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of aι 'east one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulinand all or substantiallyall of the FR regions are those of a human lmmunoglobulinsequence The humanized antibody optimally also will comprise at least a portion of an immunoglobu nconstant region (Fc), typically that of a human lmmunogiobulin For further details, see Jones et al Nature. 321 522-525 ( 1986). Reιchmanne/ α/ Nature. 332 323-329 [1988]. and Presta. Cκ/τ C Struct Biol , 2 593-596( 1992) The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 1 13, Rosenburg and Moore eds , Springer- Verlag, New York, pp 269-315 ( 1994)
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Hollmger e/ α/ , Proc Nati Acad Sci US4. 90 6444-6448 (1993)
An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and av include enzymes, hormones, and other proteinaceousor nonproteinaceoussolu.es In preferred embodiments, the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferablymore than 99% by weight. (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or intemal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody The label may be detectable by itself (e g radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable Radionuclides that can serve as detectable labels include, for example. I- 131 , 1- 123, 1- 125, Y-90 Re- 188, Re- 186, At-21 1 , Cu-67, Bι-212. and Pd- 109
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), polysacchandes (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 "lιposome" ιs a small vesicle composed of various types of lipids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as an PRO201, PRO292, PRO327, PR01265. PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017. PRO 1 1 12, PRO509. P^ 0853 or PR0882, or an antibody thereto and. optionally, a chemotherapeuticagent) to a mammal The components of the hposomeare commonly arranged in a bilayer formation, similar to the hpid arrangement of biological membranes
As used herein, the term "lmmunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of lmmunogiobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (l e , is "heterologous"),and an lmmunoglobulinconstantdomain sequence The adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The lmmunogiobulin constant domain sequence in the lmmunoadhesin may be obtained from any lmmunogiobulin, such as IgG- 1 , lgG-2, IgG-3. or IgG-4 subtypes, IgA (including IgA- 1 and IgA-2), IgE, IgD or IgM
II Compositions and Methods of the Invention
I Preparation of the PRO201. PRQ292. PRQ327. PRO 1265. PR0344. PR0343. PRQ347.
PR0357. PRQ715. PRO 1017. PRO 1 1 12. PRO509. PR0853 or PRQ882 polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO20 I (UNQ 175), PR0292 (UNQ255). PR0327 (LTNQ288), PRO 1265 (UNQ636), PR0344 (UNQ303), PR0343 (UNQ302), PR0347 (UNQ306), PR0357 (UNQ314), PRO715 (UNQ383),PRO1017 (UNQ500), PROl 1 12 (UNQ555), PRO509 (UNQ329), PR0853 (UNQ419) or PR0882 (UNQ448) In particular, cDNAs encoding certain PRO201. PR0292, PR0327 PRO 1265, PR0344 PR0343, PR0347, PR0357, PR07 I 5. PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptides have been identified and isolated, as disclosed in further detail in the Examples below In the present specification the proteins encoded by nucleic acid referred to as "DNA30676". "DNA35617" "DNA381 13", "DNA60764", "DNA40592", "DNA43318". "DNA44176", "DNA44804", "DNA52722", "DNA561 12", "DNA57702", "DNA50148", "DNA48227", "DNA58125" are referred to as UNQ175. UNQ255. UNQ288, UNQ636, UNQ303, UNQ302, UNQ306, UNQ314, UNQ383. UNQ500, UNQ555. UNQ329, UNQ419 or UNQ448, respectively However, the above sequences including further native homologues and variants are included m the definition of a PRO201 , PR0292, PR0327, PRO 1265, PR0344. PR0343, PR0347. PR0357, PR0715. PRO1017, PROl 1 12, PRO509 PR0853 or PR0882 polypeptide. regardless of their origin or mode of expression
The description below relates primarily to production of PRO201, PR0292, PR0327, PR01265. PR0344, PR0343, PR0347, PR0357. PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides by culturing cells transformed or transfected with a vector containing PRO201 -, PR0292-. PR0327-, PRO 1265-, PR0344-, PR0343-, PRO 347-, PR0357-, PR0715-, PRO 1017-, PRO 1 112-, PRO509-, PR0853- or PR0882-encodιng nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343. PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides For instance, the PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343, PR0347 PR0357, PR07I 5. PROl 017, PROl 1 12, PRO509, PR0853 or PR08P polypeptide 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, C A ( 1969), Merrifield, J Am Chem Soc , 85 2149- 2154 (1963)] In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions Various portions of the PRO201 , PR0292, PR0327. PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO201, PR0292, PR0327, PR01265, PR0344. PR0343, PR0347. PR0357, PR0715. PRO1017, PROl 1 12, PRO509, PR0853 or PR0882
a Isolation of DNA Encoding a PRO201 , PRQ292. PRQ327. PRQ1265. PRQ344.
PRQ343. PRQ347. PRQ357. PRQ715. PRO1017. PROl 1 12. PRO509. PRQ853 or PRQ882 polypeptide DNA encoding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PRO715. PRO1017.PRO1 1 12, PRO509, PR0853 or PR0882 may be obtained from a cDNA library prepared from tissue believed to possess the PRO20I PR0292. PR0327, PRO 1265, PR0344 PR0343. PR0347, PR0357, PR0715, PROl 01 , PROl 1 12. PRO509. PR0853 or PR0882 mRNA and to express it at a detectable level Accordingly.human PRO201. PR0292, PR0327, PR01265, PR0344. PR0343. PR0347, PR0 57, PR0715. PRO 1017, PRO 1 1 12, PRO509. PR0853 or PR0882 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples PRO201-. PR0292-, PR0327-, PRO 1265-. PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-. PRO 1 1 12-. PRO509-. PR0853- or PR0882-encodιng gene may also be obtained from a genomic library or by oligonucleotide synthesis
Libraries can be screened with probes (such as antibodies to the PRO20 I , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptide, or ohgonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989) An alternative means to isolate the gene encoding PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357. PR0715, PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]
The Examples below describe techniques for screening a cDNA library The oligonucleotide sequences selected as probes should be oi sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods of labeling are well known in the art. and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private 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 determined through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT which employ various algorithms to measure homology
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and. if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA b Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PRO201, PRO292,PRO327, PRO1265,PRO344, PRO343, PRO347,PRO357, PRO715, PRO1017.PRO11 12, PRO509, PR0853 or PR0882 production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants. or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation In general, principles, protocols, and practical techniques for maximizing the productivity ofcell culturescan be found m Mammalian Cell Biotechnology 4 Practical Approach. M Butler, ed (IRL Press. 1991 ) and Sambrook et al . supra
Methods of transfection are known to the ordinarily skilled artisan, for example. CaPOj and electroporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such ceils The calcium treatment employing calcium chloride, as described in Sambrook et al , supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al Gene. 22 315 ( 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 transformations have been described in U S Patent No 4,399,216 Transformations into yeast are typically carried out according to the method of Van Solingen et al J Bact , J_30 946 ( 1977) and Hsiao et al Proc Nati Acad Sci (USA), 76 3829 ( 1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e g , polybrene, polyornιthιne,may also be used For various techniques for transforming mammalian cells, see Keown et al , Methods in Ercvmology, 185 527-537 ( 1990) and Mansour et al , Nature, 336 348-352 ( 1988)
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include but are not limited to eubacteπa, such as Gram- negative or Gram-positiveorganisπis, for example, Enterobacteπaceaesuch as E coli Various E coli strains are publicly available, such as £ coli K 12 strain MM294 (ATCC 31 ,446), £ coli X I 776 (ATCC 31 ,537), £ coli strain W31 10 (ATCC 27,325) and K5 772 (ATCC 53,635)
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-. PRO1017-. PROl ! 12-, PR 509-, PR0853- or PR0882-encodιng vectors Saccharomvces cerevisiae is a commonly used lower eukaryotic host microorganism
Suitable host cells for the expression of glycosylated PRO201. PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509. PR0853 or PR0882 are derived from multicellularorganisms Examples of invertebrate cells include insect cells such as Drosophila S2 and SpodopteraSf9, 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 CVl line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol , 36 59 (1977)), Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasm, Proc Nati Acad Sci USA, 77 4216 ( 1980)), mouse sertoli cells (TM4, Mather, Biol Reprod , 21 243-251 ( 1980)), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), and mouse mammary tumor (MMT 060562, ATCC CCL51 ) The selection of the appropriate host cell is deemed to be within the skill in the art c Selection and Use of a Replicable Vector
The nucleic acid (e g , cDNA or genomic DNA) encoding PRO201 , PR0292. PR0327, PRO 1265. PR0344, PR0343, PR0347. PR0357. PR0715, PRO1017, PRO l 1 12, PRO509. PR0853 or PR0882 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression Various vectors are publicly available The vector may, for example, be in the form of a plasmid. cosmid viral particle, or phage The appropriate nucleic acid sequence mav be inserted into the vector by a variety of procedures In general, DNA is inserted into an appropriate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to. one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide In general, the signal sequence may be a component of the vector, or it may be a part of the PRO201-, PR0292-. PR0327-. PRO 1265-, PR0344-. PR0343- PR0347- PR0357-, PR0715-. PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encodιng DNA that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be. e g , the yeast invertase leader, alpha factor leader
Figure imgf000022_0001
Kluweromvce α-factor leaders, the latter described in U S Patent No 5,010, 182), or acid phosphatase leader, the C αlbicαns glucoamylase leader (EP 362, 179 published 4 April 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 polypeptidesof 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 !• ,st cells Such sequences are well known for a variety of bacteria, yeast, and viruses The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for clonirg vectors in mammalian cells
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicilhn, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply cπtical nutrients not available from complex media, e g , the gene encoding D-alanine racemase for Bacilli
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO201-. PRO292-, PR0327-, PR01265-, PR0344-, PR0343-, PR0347-,
PRO357-, PRO715-, PRO1017-, PROl 1 12-, PRO509- PR0853- or PR0882-encodmg nucleic acid, such as DHFRorthymidmekinase An appropnatehost cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaube/ α/ Proc Nail Acad Sci USA, 77.4216 ( 1980) A suitable selection gene for use in yeast is the trp I gene present in the yeast plasm id 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 in tryptophan. for example. ATCC No 44076 or PEP4-1 [Jones. Genetics, 85 12 ( 1977)]
Expression and cloning vectors usually contain a promoter operably linked to the PRO201 -, PR0292- , PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0 15-, PRO 1017-. PRO 1 1 12-, PRO509- , PR0853- or PR0882-encodιng nucleic acid sequence to direct mRNA synthesis Promoters recognized by a variety of potential host cells are well known Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al Nature, 275 615 ( 1978), Goeddel et al . Nature, 281 544 (1979)], alkaline phosphatase. a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res . 8 4057 ( 1980), EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al Proc Nati Acad Sci USA, 80 21-25 (1983)] Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding PRO201, PR0292, PR0327, PRO 1265. PR0344, PR0343. PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kιnase [Hιtzemane α/ J Biol Chem , 255 2073 ( 1980)] or other glycolytic enzymes [Hess et al , J Adv Enzvme Reg , 7 149 (1968), Holland, Biochemistry, 17 4900 (1978)], such as enolase. glyceraldehyde-3-ρhosphate dehydrogenase, hexokinase, pyruvate decarboxylase phosphofructokinase glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tπosephosphate isomerase, phosphoglucose isomerase, and glucokinase
Other yeast promoters, which are inducible promotershaving the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase2. isocytochromeC, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73.657
PRO201, PRC292, PR0327. PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017. PROl 1 12, PRO509, PR0853 or PR0882 transcription from vectors in mammalian host cells is controlled, for example, by Dromoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 l,504pub'!3hed5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarccma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Vims 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an lmmunogiobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
Transcription of a DNA encoding a PRO201 , PR0292, PR0327, PR01265. PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector Enhancers are cis-actmg elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription Many enhancer sequences are now known from mammalian genes (globin, elastase. albumin, α-fetoprotein. and insulin) Typically, however, one will use an enhancer from a eukaryotic cell vims Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers The enhancer may be spliced into the vector at a position 5' or 3' to the PRO201 -, PR0292-. PR0327-, PRO 1265-. PR0344- PR0343- PR0347-.PR0357-,PR0715- PRO10I 7- PROl 1 12-. PRO509-. PRO853-or PR0882- coding sequence, but is preferably located at a site 5' from the promoter
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellularorganisms) 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'. untranslatedregions of eukaryotic or viral DN As or cDNAs These regions contain nucleotide segments transcribed as polyadenylatedfragments in the untranslated portion of the mRNA encoding PRO201, PR0292, PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO201 ,
PR0292, PR0327. PR01265, PR0344, PR0343, PR0347. PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in recombinant vertebrate cell culture are described in Gethmg et al Nature, 293 620-625 ( 1981 ). Mantel et al Nature, 281 40-46 (1979), EP 1 17,060, and EP 1 17,058 d Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting. Northern blotting to quantitate the transcription of mRNA [Thomas, Proc Nati Acad Sci USA, 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybπd duplexes or DN A-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 immunohis. :chemιcalstaιnιng of cells or tissue sections and assay of cell culture or body fluids, to qtr ititate directly the expression of gene product Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PRO! 265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO201, PR0292, PR0327, PR01265, PR0344, PR0343. PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA and encoding a specific antibody epitope e Purification of Polypeptide
Forms of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO10I7. PROl 1 12, PRO509, PR0853 or PR0882 polypeptides may be recovered from culture medium or from host cell lysates If membrane-bound, it can be released from the membrane using a suitable detergent solution (eg Tnton-X 100) or by enzymatic cleavage Cells employed in expression of PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 112, PRO509, PR0853 or PR0882 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 purify PRO20 I PR0292. PR0327, PR01265. PR0344 PR0343, PR0347, PR0357. PR0715. PRO1017 PRO l 1 12 PRO509. PR0853 or PR0882 from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation reverse phase HPLC chromatography on silica or on a cation-exchange resin such as DEAE. chromatofocusing. SDS-PAGE. ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of the PRO201 , PR0292. PR0327, PRO 1265, PR0344. PR0343. PR0347. PR0357. PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Ercvmology, 182 ( 1990), Scopes, Protein Purification Principles and Practice, Springer- Verlag, New York ( 1982) The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO201. PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptide produced 2 Amplification of Genes Encoding the PRQ201. PRQ292. PRQ327. PRQ1265. PRQ344.
PRQ343, PRQ347, PRQ357. PRQ715. PROl 017, PRO 1 1 12. PRO509. PRQ853 or PRQ882 Polypeptides in Tumor Tissues and Cell Lines The present invention is based on the identification and characteπzationof genes which are amplified in certain cancer cells The genome of prokaryotic and eukaryotic organisms is subjected to two seemingly conflicting requirements One is the preservation and propagation of DNA as the genetic information in its oπginal form, to guarantee stable inheritance through multiple generations On the other hand, cells or organisms must be able to adapt to lasting environmental changes The adaptive mechanisms can include qualitative or
.antitative modificationsof the genetic material Qualitative modifications include DNA r . tations, in which coding sequences are altered resulting in a stmcturally and/or functionally different protein Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, / e a gene, increases, leading to an increased number of available templates for transcription, an increased number of translatable transcripts, and, ultimately, to an increased abundance of the protein encoded by the amplified gene The phenomenon of gene amplificationand its underlying mechanismshave been ιnvestιgated/« vf/rø in several prokaryotic and eukaryotic culture systems The best-characterized example of gene amplification involves the culture of eukaryotic cells in medium containing variable concentrations of the cytotoxic dmg methotrexate (MTX) MTX is a folic acid analogue and interferes with DNA synthesis by blocking the enzyme dιhydrofolatereductase(DHFR) During the initial exposure to low concentrationsof MTX most cells (>99 9%)wιll die A small number of cells survive, and are capable of growing in increasing concentrations of MTX by producing large amounts of DHFR-RNA and protein The basis of this overproduction is the amplification of the single DHFR gene The additional copies of the gene are found as extrachromosomal copies in the form of small, supernumerary chromosomes (double minutes) or as integrated chromosomal copies Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs (antibiotics for bacteria and chemotherapeuticagents for eukaryotic cells) and neoplastic transformation Transformation of a eukaryotic cell as a spontaneous event or due to a viral or chemical/environmental insult is typically associated with changes in the genetic material of that cell One of the most common genetic changes observed in human malignanciesare mutations of the p53 protein p53 controls the transition of cells from the stationary (G l) to the replicative (S) phase and prevents this transition the presence of DNA damage In other words, one of the main consequences of disabling p53 mutations is the accumulation and propagation of DNA damage, ; e genetic changes Common types of genetic changes in neoplastic cells are, in addition to point mutations, amplifications and gross, stmctural alterations, such as translocations The amplification of DNA sequences may indicate specific functional requirement as illustrated in the DHFR experimental system Therefore, the amplification of certain oncogenes in malignancies points toward a causative role of these genes in the process of malignant transformation and maintenance of the transformed phenotype This hypothesis has gained support in recent studies For example, the bcl-2 protein was found to be amplified in certain types of non-Hodgkin s lymphoma This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells Members of the gene family of growth factor receptors have been found to be amplified in various types of cancers suggesting that overexpression of these receptors may make neoplastic cells less susceptible to limiting amounts of available growth factor Examples include the amplification of the androgen receptor in recurrent prostate cancer during androgen deprivation therapyand the amplification of the growth factor receptor homologue ERG? in breast cancer Lastly, genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation This is illustrated by the amplification of the bcl-I and ras genes in various epithelial and lymphoid neoplasms
These earlier studies illustrate the feasibility of identifying amplified DNA sequences in neoplasms, because this approach can identify genes important for malignant transformat n The case of ERB2 also demonstrates the feasibility from a therapeutic standpoint, since transforming proteins may represent novel and specific targets for tumor therapy
Several different techniques can be used to demonstrate amp'-fied genomic sequences Classical cytogenetic analysis of chromosome spreads prepared from cancer cells is adequate to identify gross stmctural alterations, such as translocations, deletions and inversions Amplified genomic regions can only be visualized, if they involve large regions with high copy numbers or are present as extrachromosomal material While cytogeneticswas the first technique to demonstrate the consistent association of specific chromosomal changes with particular neoplasms, it is inadequate for the identification and isolation of manageable DNA sequences The more recently developed technique of comparative genomic hybridization (CGH) has lllustratedthe widespread phenomenon of genomic amplification in neoplasms Tumor and normal DNA are hybridizedsimultaneouslyonto metaphases of normal cells and the entire genome can be screened by image analysis for DNA sequences that are present in the tumor at an increased frequency (WO 93/18,186, Gray et al , Radiation Res 137. 275-289 [1994]) As a screening method, this type of analysis has revealed a large number of recumngamplicons (a stretch of amplified DNA) in a variety of human neoplasms Although CGH is more sensitive than classical cytogenetic analysis in identifying amplified stretches of DNA, it does not allow a rapid identification and isolation of coding sequences within the amplicon by standard molecular genetic techniques
The most sensitive methods to detect gene amplification are polymerase chain reaction (PCR)-based assays These assays utilize very small amount of tumor DNA as starting material, are exquisitely sensitive, provide DNA that is amenable to further analysis, such as sequencing and are suitable for high-volume throughput analysis
The above-mentioned assays are not mutually exclusive, but are frequently used in combination to identify amplifications in neoplasms While cytogenetic analysis and CGH represent screening methods to survey the entire genome for amplified regions. PCR-based assays are most suitable for the final identification of coding sequences, i e. genes in amplified regions
According to the present invention, such genes have been identified by quantitative PCR (S Gelmini et al Clin Chem 43-752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus. testis, ovary, uterus, etc tumor, or tumor cell lines, with pooled DNA from healthy donors Quantitative PCR was performed using a TaqMan instmment (ABI) Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs
Human lung carcinomacell lines include A549 (SRC768),Calu-l (SRC769),Calu-6 (SRC770),H157 (SRC771 ), H441 (SRC772), H460 (SRC773). H522 (SRC832), H810 (SRC833), SKMES- 1 (SRC774) and SW900 (SRC775), all available from ATCC Primary human ι"ng tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRC724 (squamous cell carcinoma abbreviated as "SqCCa")(LTl),SRC725 (non-small cell carcinoma, abbreviated as "NSCCa")(LTla),SRC726 (adenocarcinoma, abbreviated as "AdenoCa")(LT2), SRC727 (adenocarcιnoma)(LT3). SRC728 (squamous cell carcιnoma)(LT4), SRC729 (adenocarcιnoma)(LT6), SRC730 (adeπ 'squamous cell carcιnoma)(LT7), SRCC731 (adenocarcιnoma)(LT9), SRC732 (squamous cell carcιnoma)(LT10) SRC733 (adenocarcιnoma)(LTl 1), SRC734 (adenocarcιnoma)(LT12), SRC735 (broncho alveolar carcinoma, abbreviated as "BAC")(LT13), SRC736 (squamous cell carcmoma)(LT15), SRC737 (squamous cell carcιnoma)(LT16),SRC738 (squamous cell carcιnoma)(LT17), SRC739 (squamous cell carcιnoma)(LT18), SRC740 (squamous cell carcιnoma)(LT19), SRC741 (lung cell carcinoma, abbreviated as "LCCa")(LT21 ), SRC81 1 (adenocarcιnoma)(LT22)
Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma. SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRC777), Colo320 (carcinoma, SRCC778), Colo205 (carcinoma, SRC828), HCC2998 (carcinoma, SRC830), HT29 (adenocarcinoma, SRC779), HM7 (carcinoma. SRC780). KM 12 (carcinoma, SRC831 ), CaWiDr (adenocarcιnoma,SRC781 ). HCT 15 (carcinoma, SRC829), HCTI 16 (carcinoma, SRC782), SKCOl (adenocarcinoma, SRC783), SW403 (adenocarcinoma, SRC784), LS174T (carcinoma, SRC785), and HM7 (a high mucin producing variant of ATCC colon adenocarcinomacell line LS 174T, obtained from Dr Robert Warren, UCSF) Primary colon tumors include colon adenoocarcinomas designated CT1 (SRC751 ), CT2 (SRC742), CT3 (SRC743). CT4 (SRC752), CT5 (SRC753), CT6 (SRC754), CT7 (SRC755), CT8 (SRC744), CT9 (SRC756), CT10 (SRC745), CT1 1 (SRC757), CT12 (SRC746) CT14 (SRC747). CT15 (SRC748). CT 16 (SRC749). CT17 (SRC750). CT18 (SRCC758). and DcR3. BACrev, BACfwd. T160. and T159
Human breast carcinoma cell lines include, for example, HBL 100 (SRCC759), MB435s (SRCC760), T47D (SRCC761 ), MB468(SRCC762).MB 175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3 (SRCC767)
3 Tissue Distribution
The results of the gene amplification assays herein can be verified by further studies, such as, by determining mRNA expression in various human tissues
As noted before, gene amplification and/or gene expression in various tissues may be measured by conventional Southem blotting. Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Nati Acad Sci USA, 77 5201 -5205 [ 1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes Gene expression in various tissues, alternatively, may be measured by immunologica! methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO l 1 12, PRO509, PR0853 or PR0882 NA and encoding a specific antibody epitope General techniques for generating antibodies, and special protocols for Northern b'-tting and in situ hybridization are provided hereinbelow
4 Chromosome Mapping
If the amplification of a given gene is functionally relevant, then that gene should be amplified more than neighboring genomic regions which are not important for tumor survival To test this, the gene can be mapped to a particular chromosome, e g by radiation-hybrid analysis The amplification level is then determined at the location identified, and at neighboring genomic region Selective or preferential amplificationat the genomic region to which to gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival Chromosome mapping includes both framework and epicenter mapping For further details see e g , Stewart et al , Genome Research 7, 422-433 ( 1997) 5 Antibody Binding Studies
The results of the gene amplification study can be further verified by antibody binding studies, in which the ability of antι-PRO201 , antι-PR0292, antι-PR0327, antι-PR01265, antι-PR0344. antι-PR0343, antι-PR0347, antι-PR0357. antι-PR0715. anti-PRO 1017, anti-PRO 1 1 12, antι-PRO509, antι-PR0853 or anti- PR0882 to inhibit the expression of the PRO201 , PR0292. PR0327, PRO 1265. PR0344, PR0343, PR0347, PR0357 PR0715 PRO 1017 PROl 1 12. PRO509. PRO853 or PR0882 polypeptideson tumor (cancer) cells is tested Exemplary antibodies include polyclonal. monoclonal, humanized, bispecific. and heteroconjugate antibodies, the preparation of which will be described hereinbelow
Antibody binding studies may be carried out in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola. Monoclonal Antibodies A Manual of Techniques, pp 147- 158 (CRC Press. Inc . 1987)
Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody The amount of target protein (encoded by a gene amplified in a tumor cell) in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitatedeterminingthe amount of standardthat becomes bound, the antibodies preferablyare insolubilized 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 involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope. of the protein 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 binds to the analyte. thus forming an insoluble three-part complex See, eg , US Pat No 4,376,1 10 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay) For example, one type of sandwich assay is an ELISΛ assay, in which case the detectable moiety is an enzyme For ιmmunohιstochemιstry,the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
6 Cell-Based Tumor Assays
Cell-basedassays and animal models for tumors (e g cancers) can be used to verify the findings of the gene amplification assay, and further understand tb ■•elationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth The role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above
In a differentapproach, cells of a cell type known to be involved in a particular tumor are transfected with the cDN As herein, and the ability of these cDN As to induce excessive growth is analyzed Suitable cells include, for example, stable tumor cells lines such as, the B104-1- 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene)and ras'-transfectedNIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogenic growth Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumorogenic cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody- dependent cellular cytotoxιcιty(ADCC) Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of cancer
In addition, primary cultures derived from tumors in transgenic animals (as described below) can be used in the cell-basedassays herein, although stable cell lines are preferred Techniques to derive continuous cell lines from transgenic animals are well known in the art (see. e g Small et al Mol Cell Biol 5, 642-648 [1985])
7 Animal Models
A variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagonists of the native polypeptides. including small molecule antagonists The in vivo nature of such models makes them particularly predictive of responses in human patients Animal models of tumors and cancers (e g breast cancer, colon cancer, prostate cancer, lung cancer, etc ) include both non-recombinant and recombinant (transgenic) animals Non-recombmant animal models include, for example, rodent, e g , muπne models Such models can be generated by introducingtumor cells into syngeneic mice using standard techniques, e g subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, or orthopin implantation, e g colon cancer cells implanted in colonic tissue (See, e g PCT publication No WO 97/33551, published September 18, 1997) Probably the most often used animal species m oncological studies are lmmunodeficient mice and, in particular, nude mice The observation that the nude mouse with hypo/aplasia could successfully act as a host for human tumor xenografts has lead to its widespread use for this purpose The autosomal recessive nu gene has been introduced into a very large number of distinct congenic strains of nude mouse, including, for example, ASW, A/He, AKR, BALB/c, B 10 LP, C 17, C3 H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL In addition, a wide variety of other animals with inherited immunological defects other than the nude mouse have been bred and used as recipients of tumor xenografts For further details see, e g The Nude Mouse m Oncology Research. E Boven and B Winograd, eds , CRC Press, Inc , 1991
The cells introduced into such anirr tls can be derived from known tumor/cancer cell lines, such as. any of the above-listed tumor cell lines, and for example, the B 104- 1 - 1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene),
Figure imgf000030_0001
Caco-2 (ATCC HTB-37). a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or »» .um<„-. _ canon. Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al Br J Cancer 48 689-696 [1983]) Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures
The subcutaneous (s c ) space in mice is very suitable for tumor implantation Tumors can be transplanteds c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions For solid block or trochar implantation, tumor tissue fragments of suitable size are introduced into the s c space Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneouslv Tumor cells can also be injected as subdermal implants In this location, the inoculum is deposited between the lower part of the dermal connective tissue and the s c tissue Boven and Winograd (1991 ), supra
Animal models of breast cancer can be generated, for example, by implanting rat neuroblastomacells (from which the neu oncogen was initially isolated), or neu -transformed NIH-3T3 cells into nude mice, essentially as described by Drebin et al PNAS USA 83 9129-9133 (1986) Similarly, animal models of colon cancer can be generated by passaging colon cancer cells in animals, e g nude mice, leading to the appearance of tumors in these animals An orthotopic transplant model of human colon cancer in nude mice has been described, for example by Wang ef α/ Cancer Research 54 4726-
4728 ( 1994) and Too et al Cancer Research 55, 681 -684 ( 1995) This model is based on the so-called "METAMOUSE" sold by AntiCancer, Inc (San Diego, California)
Tumors that arise in animals can be removed and cultured in vitro Cells from the in vitro cultures can then be passaged to animals Such tumors can serve as targets for further testing or dmg screening Alternatively, the tumors resulting from the passage can be isolated and RNA from pre-passage cells and cells isolated after one or more rounds of passage analyzed for differential expression of genes of interest Such passaging techniques can be performed with any known tumor or cancer cell lines
For example, Meth A, CMS4, CMS5, CMS21 , and WEHI- 164 are chemically induced fibrosarcomas of BALB/c female mice (DeLeo et al , J Exp Med 146 720 [ 1977]), which provide a highly controllable model system for studying the anti-tumor activities of various agents (Pal ladinoe/ fl/ J Immunol J_38 4023- 4032 [ 1987]) Briefly, tumor cells are propagated in vitro in cell culture Prior to miection into the animals the cell lines are washed and suspended in buffer, at a cell density of about 10x10" to 10x l07 cells/ml The animals are then infected subcutaneously with 10 to 100 μl of the cell suspension, allowing one to three weeks for a tumor to appear
In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most thoroughly studied experimental tumors, can be used as an investigational tumor model Efficacy in this tumor model has been correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL) This tumor can be introduced in normal mice upon injection of tumor fragments from an affected mouse or of cells maintained in culture (Zupi et al Br J Cancer 41 suppl 4, 309 [1980]), and evidence indicates that tumors can be started from injection of even a single cell and that a very high proportion of infected tumor cells <• rvive For further information about this tumor model see Zacharski Haemostasis 6 300-320 [ 1986])
One way of evaluating the efficacy of a test compound in an animal model is implanted tumor is to measure the size of the tumo<- before and after treatment Traditionally, the size of implanted tumors has been measured with a slide caliper in two or three dimensions The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted into the corresponding volume by using a mathematical formula However, the measurement of tumor size is very inaccurate The therapeutic effects of a d g candidate can be better described as treatment-induced growth delay and specific growth delay Another important variable in the description of tumor growth is the tumor volume doubling time Computer programs for the calculation and description of tumor growth are also available, such as the program reported by Rygaard and Spang-Thomsen, Proc 6th Int Workshop on Immune-Deficient Animals, Wu and Sheng eds . Basel, 1989, 301 It is noted, however, that necrosis and inflammatory responses following treatment may actually result in an increase in tumor size, at least initially Therefore, these changes need to be carefully monitored, by a combination of a morphometπc method and flow cytometπc analysis
Recombιnant(transgenιc)anιmal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals Ammaisthat can serve as a target for transgenic manipulation include, 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 mιcroιnjectιon(Hoppe and Wanger, U S Patent No 4,873, 191 ), retrovirus-mediated gene transfer into germ lines (e g . Van der Putten et al . Proc Nati Acad Sci USA 82 6148-615 [ 1985]), gene targeting in embryonic stem cells (Thompson etal Cell 56 313-321 [1989]), electroporation of embryos (Lo, Mol Cel Biol 3 1803-1814 [ 1983]), sperm-mediated gene transfer (Lavitrano e/ a/ , 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 particularcell type is also possible by following, for example, the technique of Lasko . α/ , Proc Nati Acad Sci USA S9 6232-636 (1992)
The expression of the transgene in transgenic animals can be monitored by standard techniques For example. Southem blot analysis 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 hybridization. Northern blot analysis, PCR, or lmmunocytochemistry The animals are further examined for signs of tumor or cancer development
Alternatively, "knock out" animals can be constmcted which have a defective or altered gene encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 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 into an embryonic cell of the animal For example. cDNA encoding a particular PRO201. PR0292, P*:0327, PRO 1265, PR0344, PR0343. PR0347, PR0357, PR0715, PRO 1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with establishedtechniques A portion of the genomic DNA encoding a particular PR0201, PR0292,PR0327,PR01265,PR0344,PR0343,PR0347,PR0357,PR0715,PR01017,PR01 1 12, PRO509, PR0853 or PR0882 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e g , Thomas and Capecchi, Cell. 51 503 (1987) for a description of homologous recombination vectors] The vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e g , Li et al Cell, 69 915 ( 1992)] The selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e.g , Bradley, in Teratocarcinomas and Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 1 13-152] A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal Progeny harboring the homologously recombined DNA in their germ cells can be identified bv 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, by their ability to defend against certain pathological conditions and by their development of pathologicalconditionsdue to absence of the PRO201. PR0292, PR0327. PR01265 PR0344. PR0343, PR0347 PR0357. PR0715, PRO 1017 PROl 1 12 PRO509. PR0853 or PR0882 polypeptide The efficacy of antibodies specifically binding the polypeptides identified herein and other dmg candidates, can be tested also in the treatment of spontaneousammal tumors A suitable target for such studies is the feline oral squamous cell carcinoma (SCC) Feline oral SCC is a highly invasive, malignant tumor that is the most common oral malignancy of cats, accounting for over 60% of the oral tumors reported in this species It rarely metastasizes to distant sites, although this low incidence of metastasis may merely be a reflection of the short survival times for cats with this tumor These tumors are usually not amenable to surgery, primarily because of the anatomy of the feline oral cavity At present, there is no effective treatment for this tumor Prior to entry into the study, each cat undergoes complete clinical examination, biopsy, and is scanned by computed tomography (CT) Cats diagnosed with subhngual oral squamous cell tumors are excluded from the study The tongue can become paralyzed as a result of such tumor, and even if the treatment kills the tumor the animals may not be able to feed themselves Each cat is treated repeatedly, over a longer period of time Photographs of the tumors will be taken daily during the treatment period, and at each subsequent recheck After treatment, each cat undergoes another CT scan CT scans and thoracic radiograms are evaluated every 8 weeks thereafter The data are evaluated for differences in survival, response and toxicity as compared to control groups Positive response may require evidence of tumor regression, preferably with improvement of quality of life and/or increased life span
In addition, other spontaneous animal tumors, such as fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested Of these mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans Howeve. the use of this model is limited by the rare occurrence of this type of tumor in animals 8 Screening Assays for Dmg Candidates
Screening assays for dmg candidates are designed to identify compounds that bind or complex with the polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule dmg candidates Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides. (poly)peptιde-ιmmunoglobulιn fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodiesand antibody fragments, single-chain antibodies, anti-idiotypicantibodies, 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 variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art
All assays are common in that they call for contacting the drug candidate with 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 on a microtiter plate, by covalent or non-covalent attachments Non-covalentattachment generally is accomplished by coating the solid surface with a solution ofthe polypeptideand 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, which may be labeled by a detectable label, to the immobilized component, e g the coated surface containing the anchored component When the reaction is complete, the non-reacted components are removed, e g by washing, and complexes anchored on the solid surface are detected When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex
If the candidate compound interacts with but does not bind to a particular PRO201. PR0292. PR0327, PRO 1265, PR0344, PR0343. PR0347, PR0357. PR0715, PRO 1017, PRO 1 1 12, PRO509. PR0853 or PR0882 poiypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions Such assays include traditional approaches, such as, cross-linking, co-immunoprecipitation, and co-purification through gradients or chrom-itographiccolumns In addition, protein-protein interactions can be monitored by using a > past-based genetic system described by Fields and co-workers [Fields and Song, Nature 340 245-246 ( 1989), Chien el al , Proc Nati Acad Sci USA 88' 9578-9582 (1991)] as disclosed by Chevray and Nathans [Proc Nati Acad Sci USA 89 5789-5793 (1991 )] Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DN A-binding domain, while the other one functioning as the transf ion activation domain The yeast expression system described in the foregoing publ1 ttions (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DN A-bindmg domain of GAL4. and another, in which candidate activating proteins are fused to the activation domain The expression of a GAL l-/αrZ reporter gene under control of a GAL4-actιvated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for β- galactosidase A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are cmcial for these interactions
Compounds that interfere with the interaction of a PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-. PRO 1 1 12-, PRO509-, PR0853- or PR0882- encodmg gene identified herein and other intra- or extracellular components can be tested as follow s usually a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products To test the ability of a test compound to inhibit binding, the reaction is mn in the absence and in the presence of the test compound In addition, a placebo may be added to a third reaction mixture to serve as positive control The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove The formation of a complex in the control 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 9 Compositions and Methods for the Treatment of Tumors
The compositions useful in the treatment of tumors associated with the amplification of the genes identified herein include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides. antisense and πbozyme molecules, triple helix molecules, etc that inhibit the expression and/or activity of the target gene product
For example, antisense RNA and RNA molecule act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation When antisense DNA is used, oligodeoxynbonucleotides derived from the translation initiation site, e g between about -10 and +10 positions of the target gene nucleotide sequence, are preferred Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA
Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific πbozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biolo y' 4 469-471 (1994), and PCT πubhcation 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 o gonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of puπnes or pyπmidines on one strand of a duplex For further details see, e g PCT publication No WO 97 "3551, supra These molecules can be identified by any or any combination of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art 9 1 Antibodies
Some of the most promising dmg candidates according to the present invention are antibodies and antibody fragments which may inhibit the production or the gene product of the amplified genes identified herein and/or reduce the activity of the gene products l Polyclonal Antibodies
Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or mtraperitoneal injections The immunizing agent may include the PRO201, PR0292,
PR0327, PRO 1265, PR0344. PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12. PRO509, PR0853 or PR0882 polypeptideor a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenicin the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thvroglobulin. 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 11 Monoclonal Antibodies The antι-PRO201 , antι-PR0292, antι-PR0327, anti-PRO 1265, antι-PR0344, antι-PR0343. anti-
PR0347, antι-PR0357, antι-PR0715, antι-PRO1017, anti-PROl 1 12. antι-PRO509, antι-PR0853 or anti- PR0882 antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybndoma methods, such as those described 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 bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro
The immunizing agent will typically include the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof Generally, either peripheral blood lymphocytes("PBLs")are used ifcells ofhuman 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 [Goding, Monoclonal Antibodies Principles and Practice, Academic Press, ( 1986) pp 59- 103] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, oovine 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 guanme phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine. am lnopteπn. and thymidine ("HAT medium"), which substances prevent v.? growth of HGPRT- deficient cells
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are munne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection (ATCC), 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, Inc , New York, (1987) pp 51-63]
The culture medium in which the hybπdoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 Preferably, the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art The binding affinity of the monoclonal antibodv can for example be determined bv the Scatchard analysis of Munson and Pollard. Anal Biochem . 107 220 ( 1980)
After the desired hybndoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra! Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional lmmunoglobulinpuπfication procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography. gel electrophoresis, dialysis, or affinity chromatography The monoclonal antibodiesmay also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of muπne antibodies) The hybndoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce lmmunogiobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous munne sequences [U S Patent No 4,816,567, Morrison et at , *uprd or by covalently joining to the lmmunogiobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody
The antibodiesmay be monovalentantibodies Methods for preparing m -ovalentantibodiesare well known in the art For example, one method involves recombinant expression of lmmunogiobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross nking Alternatively, the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosshnking
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 Human and Humanized Antibodies
The antι-PRO201, antι-PR0292, antι-PR0327, anti-PRO 1265. antι-PR0344, antι-PR0343, anti- PR0347, antι-PR0357, antι-PR0715, antι-PRO1017, anti-PROl 1 12, antι-PRO509. antι-PR0853 or anti- PR0882 antibodiesmay further comprise humanized antibodies or human antibodies Humanized forms of non-human (e , munne) antibodies are chimeric immunoglobulins, lmmunogiobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human lmmunogiobulin Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances, Fv framework residues of the human lmmunogiobulin are replaced by corresponding non-human residues Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human lmmunogiobulin and all or substantially all of the FR regions are those of a human lmmunogiobulin consensus sequence The humanized antibody optimally also will comprise at least a portion of an lmmunogiobulin constant region (Fc), typically that of a human lmmunogiobulin [Jones et al , Nature 321 522-525 ( 1986), Riechmann et al Nature 332 323-329 ( 1988), and Presta, Curr Op Struct Biol , 2 593-596 (1992)]
Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers [Jones et al . Nature. 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988), Verhoeyen et al Science, 239 1534- 1536 ( 1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeric antιbodιes(U S Patent No 4.816.567), wherein substantially le^s than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter,-/ Mol Biol , 221 381 ( 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(1) 86-95 (1991)] Similarly, human antibodies can be made by introducing of human lmmunogiobulin loci into transgenic animals, e g , mice in which the endogenous lmmunogiobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example, in U S 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 K), 779-783 ( 1992), Lonberg et al , Nature 368 856-859 ( 1994), Morrison. Nature 368 812-13 (1994), Fishwild et al . Nature Biotechnology J4 845-51 (1996), Neuberger Nature Biotechnology 14 826 (1996). Lonberg and Huszar, Intern Rev Immunol 13 65-93 (1995) iv Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)
The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzvme which converts a prodrug (e g a peptidyl chemotherapeutic agent, see WO 81/01 145) to an active anti-cancer dmg See. for example. WO 88/07378 and U S Patent No 4,975,278 The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form
Enzymes that are useful in the method of this invention include, but are not limited to. glycosidase, glucose oxidase. human lysosyme, human giucuronidase. alkaline phosphatase usefulf or converting phosphate-contain mgprodmgs into free dmgs, arylsulfataseuseful for converting sulfate-containing prodmgs into free dmgs, cytosine deaminase useful for converting non-toxic 5-fluorocytosιne into the anti-cancer dmg 5-fluorouracιl, proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases (e g , carboxypeptidase G2 and carboxypeptidase A) and cathepsms (such as cathepsins B and L), that are useful for converting peptide-containingprodrugs into free dmgs, D-alanylcarboxypeptidases. useful for converting prodmgs that contain D-ammo acid substituents. carbohydrate-cleavmgenzymes such as β-galactosidase and neuram in idase useful for converting glycosylated prodmgs into free dmgs, β-lactamase useful for converting drugs deπvatized with β-lactams into free dmgs, and penicillin amidases, such as penicillin Vamidase or penicillin G am idase, useful for converting dmgs deπvatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free dmgs Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes" can be used to convert the prodmgs of the invention into free active dmgs (see, e g , Massev. Nature 328 457-458(1987)) Antibody -abzymeconjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population
The enzymes of this invention can be covalently bound to the antι-PRO201, antι-PR0292. anti- PR0327, anti-PRO 1265, antι-PR0344, antι-PR03^3, antι-PR0347, antι-PR0357, antι-PR0715, anti- PRO 1017, anti-PROl 1 12,antι-PRO509.antι-PRO853 or antι-PR0882 antibodies by techniques well known in the art such as the use of the heterobifunctionalcross-linkmgagents discussed above Alternatively, fusion proteins comprising at least the antigen binding region of the antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e g Neuberger / .' Nature 312 604-608 ( 1984))
v 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
PR0201, PR0292,PR0327,PR01265,PR0344,PR0343,PR0347,PR0357.PR0715,PR01017,PR011 1J PRO509, PR0853 or PR0882, 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 lmmunogiobulin heavy-chain/light- chain pairs, where the two heavy chains have different specιficιtιes(Mιlsteιn and Cuello, Nature, 305 537-539 [1983]) Because of the random assortment of lmmunogiobulin heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific stmcture The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J , K> 3655-3659 ( 1991 ) Antibody variable domains with the desired binding specιficιtιes(antιbody-antιgen combining sites) can be fused to lmmunogiobulin constant domain sequences The fusion preferably is with an lmmunoglobulinheavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CH 1 ) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the lmmunogiobulin heavy-chain fusions and, if desired, the lmmunogiobulin 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, \2Λ_ 210 ( 1986)
According to another approach desci bed 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 preferred interface comprises at least a part of the CH3 region of an antibody constant domain In this method, one or more small am o acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g tyrosine or tryptophan) Compensatory "cavities" of identical or similar size to the large side chaιn(s) are created on the interface of the second antibody molecule by replacing large am o acid side chains with smaller ones (e g alanine or threonine) This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g F(ab'), bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example, bispecfic antibodies can be prepared can be prepared using chemical linkeage Brennan et al Science 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives One of the Fab -TNB derivatives is then reconverted to the Fab -thiol by reduction with mercaptoethvlamine 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 £ coli and chemically coupled to form bispecific antibodies Shalaby e/ α/ J Exp Med 175 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab'), molecule Each Fab' fragment was separately secreted from £ coli and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example bispecific antibodies have been produced using leucine zippers Kostelnye/ / J Immunol 148(5) 1547- 1553 (1992) The leucine zipper peptides from the Fos and Jun proteins were iinked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody" technology described by Hollmgere/ α/ Proc Nati Acad Sci USA 90 6444-6448 ( 1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-bmdingsites Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported See, Gmber et al J Immunol 152 5368 (1994)
Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt e/ er/ J Immw ' 147 60 ( 1991 ) Exemplary bispecific antibodies may bind to two different epitopes on a given "Pro protein herein
Altematιvely,an antι-"PRO" protein arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecuoe (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" protein Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular "PRO" polypeptide These antibodies possess a "PRO"-bindmgarm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA Another bispecific antibody of interest binds the "PRO" polypeptide and further binds tissue factor (TF) vi Heteroconiugate Antibodies Heteroconjugate antibodiesare composedof two covalentlyjoined 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 crosshnking agents For example, lmmunotoxins mav be constmcted using a disulfide exchange reactionor by forming a thioetherbond Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutynmιdate and those disclosed, for example, in U S Patent No 4,676,980 vii Effector function engineering
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectivenessof the antibody in treating cancer, for example For example cysteine resιdue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeπc antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al ,
J Exp Med 176 1 191-1 195 ( 1992) and Shopes, B J Immunol 148 2918-2922 (1992) Homodimeπc antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff etal Cancer Research 53 2560-2565 (1993) Alternatιvely,an antibody can be engineered which 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) VIM Immunoconiugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (; e , a radioconjugate)
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbindmg active fragments of diphtheria toxin, cholera toxin, botuhnus toxin, exotoxin A chain (from Pseudomonas aeruginosa), πcin A chain, abπn A chain, modeccin A chain, alpha-sarcin, Aleurttes fordu proteins, dianthm proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotιn, sapav.naπaoffιcιnalιs inhibitor, gelonιn,sapoπn, mιtogellιπ,restπctocιn,phenomycιn, enomycinand the tncothecenes Small molecule toxins include for example, calicheamicins, maytansmoids, palytoxin and CC1065 A variety of radionuclides are available for the production of radioconjugated antibodies Examp'cs include 2,2Bι, 13II, l3lIn, 90Y and ,86Re
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succιnιmιdyI-3-(2-pyπdyldιthιol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccmimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bιs-(p-dιazonιumbenzoyl)- ethylenedιamιne),dιιsocyanates(such as tolyene 2,6-dnsocyanate), and bis-active fluorine compounds (such as l ,5-dιfluoro-2,4-dιnιtrobenzene) For example, a πcin immunotoxincan be prepared as described in Vitetta et al , Science 238 1098 (1987) Carbon- 14-labeled l-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 in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient. followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e g avidin) which is conjugated to a cytotoxic agent (e g a radionucleotide) ix Immunoliposomes
The antibodiesdisclosedherein may also be formulatedas immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al Proc Nati Acad Sci USA,M 3688 ( 1985), Hwang et al Proc Nati Acad Sci USA, 77 4030 (1980), and U S Pat Nos 4.485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholme, cholesterol and PEG-denvatized phosphatidvlethanolamine (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 described in Martin et al . J Biol Chem 257 286-288 (1982) via a disulfide interchange reaction A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome See Gabizon et al . J National Cancer Inst 81(19)1484 (1989) 10 Pharmaceutical Compositions
Antibodies specifically binding the product of an amplified gene identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred For example, based uj *,τ the variable region sequences of an antibody, peptide molecules can be designed whic 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 Marascoe. α/ Proc Nati Acad Sci USA 90 7889-7893 [1993])
Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed [1980]), in the form of lyophilized formulations or aqueous solutions Acceptable earners, 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 chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3- pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvιnylpyrrohdone,amιno acids such as glycme, glutamine, asparagine, histidine, arginine. or lysine, monosacchaπdes, disacchandes, and other carbohydrates including glucose, mannose, or dextrins. chelating agents such as EDTA, sugars such as sucrose. mannitol, trehalose or sorbιtol, salt-formιngcounter-ιons such as sodium, metal complexes ( g Zn- protein complexes), and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG)
Non-antibody compounds identified by the screening assays of the present invention can be formulated in an analogous manner, using standard techniques well known in the art
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokme or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatm- microcapsulesand poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres. microemulsions. nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed ( 1980)
The formulationsto be used for in VΠΌ administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
Sustained-releasepreparationsmay be prepared Suitable
Figure imgf000044_0001
include semipermeablematnces of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyi-methacrylate), or poly(vιnylalcohol)), polylactides (U.S Pat No 3,773,919),copolymersof L-glutamic acid and γ ethyl-L-glutamate. non-degradable ethylene- vinyl ac< .ate, degradable lactic acid-glyco c acid copolymers such as the LUPRON DEPOT ™ njectable microspheres composed of lactic acid-glycohc acid copolymer and leuprolide acetate), and poly-D-(-)-3- hvdroxybutyπc acid While polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modify ingsulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions 1 1 Methods of Treatment
It is contemplated that the antibodies and other anti-tumor compounds of the present invention may be used to treat various conditions, including those characterized by overexpression and/or activation of the amplified genes identified herein Exemplary conditions or disorders to be treated with such antibodies and other compounds, including, but not limited to, small organic and inorganic molecules, peptides. antisense molecules, etc include benign or malignanttumors (e g renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, ling, vulval. thyroid, hepatic carcinomas, sarcomas, glioblastomas.and various head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal.hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoe c disorders, and inflammatory, angiogenic and immunologic disorders
The anti-tumor agents of the present invention, e g antibodies, are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, mtraperitoneal. mtracerobrospinal.subcutaneous, lntra-articular.intrasynovial.intrathecal, oral, topical, or inhalation routes Intravenous administration of the antibody is preferred
Other therapeutic regimens may be combined with the administration of the anti-cancer agents, eg antibodies of the instant invention For example, the patient to be treated with such anti-cancer agents may also receive radiation therapy Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instmctions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapv Service Ed . M C Perry, Williams & Wilkins, Baltimore, MD (1992) The chemotherapeutic agent may precede, or follow administration of the anti-tumor agent, e g antibody, or may be given simultaneously therewith The antibody may be combined with an anti-oestrogen compound such as tamoxifen or an anti-progesterone such α. x.napπstone (see, EP 616812) in dosages known for such molecules
It may be desirable to also administer antibodies against other tumor associated antigens, such as antibodies which bind to theErbB2, 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 (' rclosed herein may be co-administered to the patient Sometimes, it may be beneficial -• also administer one or more cytokines to the patient In a preferred embodiment, the antibodies herein are co-administered with a growth inhibitory agent For example, the growth inhibitory agent may be administered first, followed by an antibody of the present invention However, simultaneous administration or administration of the antibody of the present invention 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 antibody herein
For the prevention or treatment of disease, the appropπate dosage of an anti-tumor agent, e g an antibody herein 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 agent, and the discretion of the attending physician The agent is suitably 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 μg/kgto 15 mg/kg (e g 0 1- 20mg/kg) of 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/kgto 100 mg/kg or more, depending on the factors mentioned above For repeated administrations over several davs 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
12 Articles of Manufacture In another embodimentof the invention. an article of manufacturecontainingmateπals useful for the diagnosis or treatment of the disorders described above is provided The article of manufacture comprises a containerand a label Suitable containers 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 container 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 an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e g an antibody The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
The article of manufacture may further comprise a second container comprising a pharmaceutical ly- 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, including other buffers, diluents, filters, needles, syringes, and package inserts with instmctions for use
13 Diagnosis and Prognosis of Tumors
While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for dmg candidates or tumor (e g cancer) treatment, the same proteins along with secreted proteins encoded by the genes amplified in tumor cells find additional use in the diagnosis and prognosis of tumors For example, antibodies directed against the proteins products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics
For example, antibodies, including antibody fragments, can be used to quά . αtively or quantitatively detect the expression of proteins encoded by the amplified genes ("marker gene products") The antibody preferably is equipped with a detectable, e g fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluoπmetry, or other techniques known u the art These techniques are particularly suitable, if the amplified gene encodes a cell surface protein, e g a growth factor Such binding assays are performed essentially as described in section 5 above In situ detection of antibody binding to the marker gene products can be performed, for example, by lmmunofluorescence or immunoelectron microscopy For this purpose, a histological specimen is removed from the patient, and a labeled antibody 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 referred to in the examples were used according to manufacturer's instmctions 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, 10801 University Blvd , Manassas, VA 201 10-2209 All original deposits referred to in the present application were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations 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, Inc and ATCC, which assures permanent and unrestnctedavailability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U S patent or upon laying open to the public of any U S or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U S Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's mles pursuant thereto (including 37 CFR § 1 14 with particular reference to 886 OG 638)
Unless otherwise roted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks Sambrook et al , Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press N Y , 1989, Ausubel et al Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N Y , 1989, Inms et al PCR Protocols A Guide to Methods and Applications, Academic Press, inc , N Y , 1990, Harlow et al , Antibodies A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor 1988, Gait, M J , Oligonucleotide Synthesis, IRL Press, Oxford, 1984, R I Freshney, Animal Cell Culture ' 987, Co gan et al , Current Protocols in Immunology, 1991
EXAMPLE 1
Isolation of cDNA clones Encoding Human PRO201 , PRQ292. PRQ327. PRO 1265. PRQ344. PRQ343.
PRQ347. PRQ357. PRQ715. PROl 017. PROl 112. PRO509. PRQ853 or PRQ882
1 Isolation of cDNA clones encoding a human PRO201 (UNO 175) (nspl) An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte PharmaceutιcaIs,Palo Alto, CA) was searched and an EST was identified (1328938, DNA28710, Figure 29, SEQ ID NO 29) which shared significant sequence identity with the adaptor protein She Based upon the EST, a full-length cDNA clone (DNA30676, Nspl, Figure 1, SEQ ID NO 1 ) was obtained from a human fetal kidney library using an in vivo cloning technique. DNA30676 has a single long open reading frame which encodes a 576 amino acids protein (Figure 2, SEQ ID NO 2) A vector containing DNA30676 (DNA30676-1223) was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd , Manassas, VA 201 10-2209, on December 23, 1998 and assigned ATCC Deposit No 209567
2 Isolation of cDNA clones encoding a human PRQ292 (UNQ266) (Cathepsm-d) PR0292 is identical with the human death associated protein DAP-7. also called cathepsin D The amino acid sequence of this 412 amino acids protein is present in the public Dayhoff database under Accession
Nos CATD Humanand P R74207, and is shown in Figure 4 (SEQ ID NO 4) The nucleotide sequence of the DNA encoding PR0292 is shown m Figure 3 (SEQ ID NO 3) DAP-7 has also been disclosed in WO 95/10630 publιshedon April 20, 1995. and in Faust e. α/ Proc Nad Acad Set (75/1 82 4910-4914 ( 1985)
3 Isolation of cDNA clones encoding a human PRQ327 (UNQ288)
An expressed sequence tag (EST) DNA database (LIFESEQ®. Incyte Pharmaceuticals.Palo Alto. CA) was searched and various EST sequences were identified that showed certain homology to human prolactin recetor protein Based upon a consensus sequence extended using repeated cycles of BLAST and the program "phrapJPhil Green, University of Washington, Seattle. Washington), oligonucleotides were synthesized and a cDNA clone designated DNA381 13 (Figure 5, SEQ ID NO 5) was isolated from a human fetal lung tissue library (LIB26) and sequenced Clone DNA381 13 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 1 19- 121, and ending at the stop codon at nucleotide positions 1385-1387 (Figure 5) The predicted polypeptideprecursor, PR0327, is 422 ammo acids long (Figure 6. SEQ ID NO 6) The full-length PR0327 protein has an estimated molecular weight of about 46.302 daltons and a pi of about 9 42
A vector containing DNA381 13 (DNA381 13-1230)was deposited with the American Type Culture Collection (ATCC), 10801 University Blvd , Manassas, VA zϋ ' 10-2209, on December 10, 1997 and assigned ATCC Deposit No 209530
4 Isolation of cDNA clones encoding human PRO 1265 (UNQ636) DNA60764 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc (South San Francisco. CA) upon ESTs as well as clustered and assembled EST fragments from public (e g , GenBank) and/or private (LIFESEQ®, Incv : Pharmaceuticals. Inc . Palo Alto, CA) databases The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration The nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined If neither meets the requirement, the candidate sequence is not scored In order to determine whether the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals
Use of the above described signal sequence algorithm allowed identification of an EST cluster sequence from the LIFESEQ® database, designated EST Cluster No 86995 that was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enrvmology 266 460-480 ( 1996)) 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 a consensus DNA sequence w ith the program phrap" One or more of the ESTs used in the assembly was derived from a cDNA library prepared from RNA isolated from inflamed human adenoid tissue A consensus sequence was assembled, and. based on homology between the consensus sequence and Incyte EST no 20965 shown in Figure 26 (SEQ ID NO 26), EST clone no 20965 was purchased and the cDNA insert was obtained and sequenced The sequence of this cDNA insert is shown in Figure 7 (SEQ ID NO 7) is herein designated as DNA60764
The full length clone shown in Figure 7 contained a single open reading frame with an apparent translational initiation site at nucleotide positions 79-81 and ending at the stop codon found at nucleotide positions 1780- 1782 (Figure 7, SEQ ID NO 7) The predιctedpolypeptιdeprecursor(Fιgure 8, SEQ ID NO 8 ) is 567 amino acids long PRO 1265 has a calculated molecular weight of approximately 62,881 daltons and an estimated pi of approximately 8 97 Additional features include a signal peptide sequence at about ammo acids 1-21 , potential N-glycosylation sites at about ammo acids 54-57, 134-137, 220-223, and 559-562, and a region having ammo acid sequence identity with D-ammo acid oxidase proteins at about amino acids 61 -80
Clone DNA60764 (UNQ636), designated as DNA60764-1533 was deposited with the ATCC on November 10, 1998, and is assigned ATCC deposit no 203452
5 Isolation of cDNA clones encoding human PRQ344 (UNO303)
The extracellulardomain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e g , GenBank) 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 sequence- vith the program "phrap" (Phil Green, University of Washington. Seattle, Washington) This method will be hereinafterreferred to as "ECD homology search"
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 sequence as far as possible using the sources of EST sequences discussed above Using probes s> nthesized based upon the extended consensus sequence, a cDNA clone designated DNA40592 was isolated from a human fetal kidney tissue library
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0344 [herein designated as UNQ303 (DNA40592)] (Figure 9, SEQ ID NO 9) and the derived protein sequence for PR0344 (Figure 10, SEQ ID NO 10) Clone UNQ303 (DNA40592) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 227-229 and ending at the stop codon at nucleotide positions 956-958 (Figure 9) The predicted polypeptide precursor is 243 ammo acids long (Figure 10) Importantregionsofthe amino acid sequence encoded by nucleotides 1 to 729 of PR0344 include the signal peptide, corresponding to am o acids 1-15, the start of the mature protein, corresponding to amino acids 16-67, and two potential N-myπstoylatιonsιtes„ correspondingto amino acids 68-215 and 216- 243, respectively Clone DNA40592 (DNA40592- 1242) was deposited with ATCC on November 21 , 1997 and assigned ATCC deposit No 209492
6 Isolation of cDNA clones encoding human PRQ343
Folhwng the ECD homology search described in section 5 above, a consensus sequence was identified Based on this sequence, oligonucleotideswere synthesized and used to isolate cDNA clones from a human fetal lung tissue (library LIB26)
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0343 [herein designated as UNQ302 (DNA43318)] (Figure 1 1 SEQ ID NO 1 1 ), and the derived protein sequence for PR0343 The entire nucleotide sequence of UNQ302 (DN A43318) is shown in Figure 1 1 (SEQ ID NO 1 1 )
Clone UNQ302 (DNA43318) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 53-55 and ending at the stop codon at nucleotide positions 1004- 1006 (Figure 1 1 ) The predicted polypeptide precursor is 317 ammo acids long (Figure 12) Various unique aspects of the PR0343 protein are shown in Figure 12 Clone UNQ302 (DNA43318-1217) was deposited with ATCC on November 21 , 1997 and is assigned ATCC deposit no 209481
7 Isolation of cDNA clones encoding human PRQ347
Using the ECD homology search described above, a consensus sequence was determined Based on the 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 of the full-length coding sequence for PR0347 RNA for constmction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228) The cDNA libraries used to isolate the cDNA clones were constmcted by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for PR0347 [herein designated as UNQ306 (DNA - " 176)] (Figure 13, SEQ ID NO 13) and the derived protein sequence for PR0347 (Figure 14. SEQ ID NO 14)
The entire nucleotide sequence of UNQ306 (DNA44176) is shown in Figure 13 (SEQ ID NO 13) Clone UNQ306 (DNA44176) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 123-125 and ending at the stop codon at nucleotide positions 1488-1490 (Figure 13) The predicted polypeptide precursor is 455 am o acids long (Figure 14, SEQ ID NO 14) The full- length PR0347 protein shown in Figure 14 has an estimated molecular weight of about 50,478 daltons and a pi of about 8 44 Clone UNQ306 (DNA44I 76) was deposited with ATCC on December 10, 1997, and is assigned ATCC deposit no 209532
8 Isolation of cDNA clones encoding human PRQ357
The sequence expression tag "2452972" (Figure 30, SEQ ID NO 30) by Incyte Pharmaceutιcals,Palo Alto, CA, was used to begin a data base search The extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases which overlapped with a portion of
"2452972" The EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ®, Incvte Pharmaceuticals Palo Alto, CA) The search was performed as described above for ECD homology searches
A consensus DNA sequence was assembled relative to other EST sequences using phrap Using probes synthesized based upon the consensus sequence, a cDNA clone designated DNA44804 was isolated from a human fetal liver library, and sequenced in its entirety The entire nucleotide sequence of DN A44804 is shown in Figure 15 (SEQ ID NO 15) DNA44804 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 137-139 and ending at the stop codon at nucleotide positions 1931-1933 The predicted polypeptide precursor is 598 ammo acids long (Figure 16 SEQ ID NO 16) Clone DNA44804- 1248 was deposited with ATCC on December 10, 1997 and is assigned ATCC deposit No 209527
9 Isolation of cDNA clones encoding PRQ715
A proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto CA) was searched for EST sequences encoding polypeptides having homology to human TNF-α This search resulted in the identificationof Incyte Expressed Sequence Tag No 2099855 (see Figure 31, SEQ ID NO 31 ) Based upon the alignment of various EST clones, a single clone (725887, Accession No AA292358) was sequenced The full-length DNA52722 sequence was then obtained from sequencing the insert DNA from clone 725887 (Accession No AA292358)
The entire nucleotide sequence of DNA52722 is shown in Figure 17 (SEQ ID NO 17) Clone DNA52722 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 1 14-1 16 and ending at the stop codon at nucleotide positions 864-866 (Figure 17) The predicted polypeptide is 250 amino acids long (Figure 18 SEQ ID NO 18) The full-length PR0715 protein shown in
Figure 18 has an estimated molecular weight of about 27,433 daltons and a pi of about 9 85
Analysis of the amino acid sequence of the full-length PR0715 polypeptide suggests that it possesses significant homology to members of the tumor necrosis factor family of proteins thereby indicating that PR0715 is a novel tumor necrosis factor protein
A vector containing DNA52722 (DNA52722-1229) was deposited with ATCC on January 7, 1998 and was assigned ATCC deposit no 209570
10 Isolation of cDNA clones encoding PROl 017 The extracellulardomain (ECD) sequences (includingthe secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search 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 (Altschul et al Methods in Enzymology 266 460-480 (1996)) as a compansonof 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) An initial consensus DNA sequence was assembled relative to other EST sequences using phrap The initial 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 An extended consensus sequence was then performed using the same procedures Further examination of the two consensus assemblies generated in the previous paragraph. EST clone
AA243086 (Merck clone 664402) was further examined DNA sequencing gave the full-length DNA sequence for PRO 1017 [herein designated as UNQ500 (DN A561 12- 1379)](SEQ ID NO 19) and the derived protein sequence for PRO1017
The entire nucleotide sequence of DNA561 12 is shown in Figure 19 (SEQ ID NO 19) Clone UNQ500 (DN A561 12- 1379)contaιns a single open reading frame with an apparent translational initiation site at nucleotide positions 128- 130 and ending at the stop codon at nucleotide positions 1370- 1372 The predicted polypeptide precursor is 414 ammo acids long Figure 20 The full-length PRO1017 protein in Figure 20 has an estimated molecular weight of about 48414 daltons and a pi of about 9 54 Clone DNA561 12 has been deposited with the ATCC. and has been assigned deposit number 209883 In the event of a conflict between the sequence of the cDNA insert of the deposit and the sequence described herein, it is understood that the deposit contains the correct sequence
1 1 Isolation of cDNA clones encoding PROl 1 12
The extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins ii om the Swiss-Protpublic protein database were used to search expressed sequence tag (EST) databases, following the ECD homology search described above 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 clustering and assembling of the public and private ESTs into one or several consensus sequences to create a candidate sequence -vas performed using repeated cycles of the computer program phrap Candidate sequences with a sufficient score were further examined with the computer program blast or blast-2 (Altschul, SF, and W Gish (1996), Local alignment statistics ed R Doolittle, Methods in Enzymology266 460-^80) to confirm the novelty of the sequence A candidate, or initial consensus sequence was formed from sequences identified in an Incyte database which fit the signal algorithm
Based on the discoveries and information provided herein, Merck EST AA223646, clone 650953, from library 318, a human neuroepithelium tissue library, was further examined DNA sequencing of the clone gave UNQ555 (DNA57702-1476) (Figure 21 , SEQ ID NO 21). which includes the full-length DNA sequence for a PRO 1 1 12
The entire nucleotide sequence of UNQ555 (DNA57702-1476) is shown in Figure 21 (SEQ ID NO.21 ) Clone UNQ555 (DNA57702-1476) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 20-22 and ending at the stop codon at nucleotide positions 806-808 of SEQ ID NO 21 (Fιgure21) The predicted polypeptide precursor is 262 amino acids long (Figure 22, SEQ ID NO 22) The full-length PROl 1 12 protein shown in Figure 22 has an estimated molecular weight of about 29379 daltons and a pi of about 8 93 Figure 22 also shows the approximate locations of the signal peptide and transmembranedomains Clone UNQ555 (DNA57702-I476) was deposited with the ATCC on 9 June 1998 and assigned ATCC Deposit No 209951
12 Isolation of cDNA clones encoding human PRO509
To isolate a cDNA for HVEM, a bacteπophage library of human retinal cDNA (commercially available from Clontech) was screened by hybridization with a synthetic oligonucleotide probe based on an EST sequence (GenBank locus AA021617), which showed some degree of homology to members of the TNFR family Five positive clones (containing cDNA inserts of 1 8-1 9kb) were identified in the cDNA library, and the positive clones were confirmed to be specific by PCR using the above hybridization probe as a PCR primer Single phage plaques containing each of the five positive clones were isolated by limiting dilution and the DNA was purified using a Wizard Lambda Prep DNA purification kit (commercially available from Promega)
The cDNA inserts from three of the five bacteπophage clones were excised from the vector arms by digestion with EcoRI, gel-puπfied,and subcloned into pRK5 and sequenced on both strands The three clones contained an identical open reading frame (with the exception of an intron found in one of the clones)
The entire nucleotide sequence of HVEM (DNA50148) is shown in Figure 23 (SEQ ID NO 23) The cDNA contained one open reading frame with a translational initiation site assigned to the ATG codon at nucleotide positions 86-88 The surroundingsequence at this site is in reasonable agreement with the proposed consensus sequence for initiation sites [Kozak, J Cell Biol . 1 15 887-903 (1991)] The open reading frame ends at the termination codon TGA at nucleotide positions 925-927
The piedicted amino acid sequence of the full length HVEM (UNQ329) contains 283 amino ai-Hs (See Fig 24, SEQ ID NO 24) A putative transmembrane region of the HVEM comprises amino acids 201- 225 of Fig 24 and a putative cytoplasmic region of the HVEM comprises amino acids 226-283 of Fig 24 The sequence differs from the HVEM sequence reported in Montgomery et al , supra, in at least two amino acids as shown in Fig 26, codon 108 encodes a seπne and codon 140 encodes an alanine An alignment (using the Align™ -.mputer program) of a 58 amino acid long cytoplasmic region of HVEM with other known members of the human TNF receptor family showed some sequence similarity, in particular to CD40 ( 12 identities) and LT-beta receptor ( 1 1 identities)
13 Isolation of cDNA clones encoding PRQ853
Using the ECD homology search described above, a consensus sequence was determined Bazsed on the consensus sequence, ohgonucleotides were synthethized I) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for the PR0853 sequence (UNQ419) RNA for constmction ofthe cDNA libraries was isolated from human fetal kidney tissue (LIB228) The cDNA libraries used to isolate the cDNA clones were contmcted by standard methods using commercially available reagents such as those from Invitrogen. San Diego, CA
DNA sequencing of the clones isolated as described above gave the full-length DNA sequence for a PR0853 [herein designated as DNA48227] (Figure 25, SEQ ID NO 25) and the derived PR0853 protein (UNQ419) (Figure 26, SEQ ID NO 26)
The entire nucleotide sequence of DNA48227 is shown in Figure 25 (SEQ ID NO 25) Clone DNA48227-1350 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 128- 130 and ending at the stop codon at nucleotide positions 1259-1261 (Figure 25) The predicted polypeptide precursor is 377 amino acids long (Figure 26)(SEQ ID NO 26) The full-length PR0853 (UNQ419) protein shown in Figure 26 (SEQ ID NO 26) has an estimated molecular weight of about 40,849 daltons and a pi of about 7 98 Important regions of the amino acid sequence of UNQ419 include the signal peptide, corresponding to amino acids from about 1 to about 16 of SEQ ID NO 26. the glycosaminoglycanattachmentsite. corresponding to amino acids from about 46 to about 49 of SEQ ID NO 26, and two sequences typical of the short-chain alcohol dehydrogenase family, corresponding to amino acids from about 37 to about 49 and about 1 14 to about 124 of SEQ ID NO 26, respectively Clone DNA48227- 1350 has been deposited with the ATCC and is assigned ATCC deposit no 209812
14 Isolation of cDNA clones encoding human PRQ882
PR0882 (UNQ448) is identical with cardiotrophin- 1 The amino acid sequence of this 201 amino acid protein is present in the public Dayhoff database under Accession Nos P R83967, P W29238 and CTF1 HUMAN. among others, and is shown in Figure 28 The nucleotide of the DNA encoding the PR0882 (UNQ448) is shown in Figure 29 (SEQ ID NO 29) Cardiotrophin- 1 has also been disclosed in WO9730146, published on 21 August 1997 and W09529237, published on 2 November 1995
EXAMPLE 2 Gene Amplification This example shows that the PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-. PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encodιng genes are amplified in the genome of certain human lung, colon and/or breast cancers and/or cell lines Amplification is associated with overexpression of the gene product, indicating that the binding specificities for at least two different •nigens In the present case, one of the binding specificities is for the PRO201. PR0292 PR0327, PR01265. PR0344, PR0343. PR0347, PR0357, PR0715. PRO10 I 7, PROl 1 12, PRO509, PR0853 or PR0882 proteins are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast zrά other cancers Therapeutic agent may take the form of antagonists of binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 -, PR0292-, PR0327- , PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-. PRO 1 1 12-, PRO509-, PR0853- or PR0882-encodιng genes, for example, muπne-human chimeric, humanized or human antibodies against a binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347. PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide
The starting material for the screen was genomic DNA isolated from a variety cancers The DNA is quantitated precisely, eg fluorometπcally As a negative control, DNA was isolated from the cells often normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown) The 5' nuclease assay (for example, TaqMan™) and real-time quantitative PCR (for example, ABI Pπzm 7700 Sequence Detection System™ (Perkin Elmer, Applied Biosystems Division, Foster City, CA)), were used to find genes potentially amplified in certain cancers The results were used to determine whetherthe DNA encoding binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327, PR01265 PR0344, PR0343, PR0347. PR0357 PRO715. PRO1017. PRO1 1 12 PRO509, PR0853 or PR0882 is over-represented m any of the primary lung or colon cancers or cancer cell lines or breast cancer cell lines that were screened The primary lung cancerswere obtained from individualswith tumors of the type and stage as indicated in Table 1 An explanation of the abbreviations used for the designation of the primary tumors listed in Table 1 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore
The results of the Taqman™ are reported in delta (Δ) CT units One unit corresponds 1 PCR cycle or approximately a 2-fold amplification relative to normal, two units corresponds to 4-fold. 3 units to 8-fold amplification and so on Quantitation was obtained using pπmers and a Taqman™ fluorescent prove derived from the binding specificities for at least two different antigens In the present case, one of the binding specificitiesis for the PRO201-, PR0292-, PR0327-, PR01265-, PR0344-. PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-, PROl 1 12-, PRO509-, PR0853- or PR0882-encodιng gene Regions of binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO201 , PR0292, PR0327. PRO 1265. PR0344. PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 which are most likely to contain unique nucleic acid seαuences and which are least likely to have spliced out introns are preferred for the primer and probe derivation, e g 3-untranslated region The sequences for the primers and probes (forward, reverse and probe) used for the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 . ' 2, PRO509, PR0853 or PR0882 gene amplification were as follows PRO201 (DNA30676) 30676 tm f
5'-CGCAGACACCCTTCTTCACA-3' (SEQ ID NO 32)
: 676 tm r 5'-CGACTCCTTTGGTCTCTTCTGG-3' (SEQ ID NO 33) 30676 tm p 5'-CCGGGACCCCCAGGTTTTTGC-3' (SEQ ID NO 34)
PRQ292 (DNA35617) 35617 tm f 5'-GATCCTGGG CGACGTCTTC-3' (SEQ ID NO 35)
35617 tm p
5'-TCGGCCGCTACTACACTGTGTTTGACC-3' (SEQ ID NO 36) 35617 tm r 5'-GCCCACCCTGTTGTTGTCA-3' (SEQ ID NO 37)
PRQ327 (DNA38 I 13) 381 13 tm f 5'-CTCAAGAAGCACG CGTACTGC-3' (SEQ ID NO 38) 381 13.tm.p
5'-CCAACCTCAGCTTCCGCCTCTACGA-3' (SEQ ID NO: 39)
381 13.tm.r
5'-CATCCAGGCTCGCCACTG-3' (SEQ ID NO: 40)
PRO1265 (DNA60764): 60764.tm.fl
5'-TGACCTGGCAA AGG AAG A A-3' (SEQ ID NO: 41 )
60764.tm.p l
5'-CAGCCACCCTCCAGTCCAAGG-3' (SEQ ID NO: 42) 60764.tm.rl
5'-GGGTCGTGTTTTGGAGAGA-3' (SEQ ID NO: 43)
PRQ344 (DNA40592): 40592.tm.fl
5'-TGGCAAGGAATGGGAACAGT-3' (SEQ ID NO: 44) 40592.tm.pl
5'-ATGCTGC CAGACCTGAT CGCAGACA-3' (SEQ ID NO: 45)
40592.tm.rl
5'-G GGCAGAAATC CAGCCACT-3' (SEQ ID NO: 46)
PRQ343 (DNA43318): 43318.tm.fl
5'-TCTACATCAGCCTCTCTGCGC-3' (SEQ ID NO: 47)
43318.tm.pl
5'-CGATCTTCTCCACCCAGGAGCGG-3' (SEQ ID NO: 48)
43318.tm.rl 5'-GGAGCTGCACCCCTTGC-3' (SEQ ID NO: 49)
PRQ347 (DNA44176): 44176.tm.fl
5'-CCCTTCGCCTGCTTTTGA-3' (SEQ ID NO: 50)
44176.tm.pl 5'-GCCATCTAATTGAAGCCCATCTTCCCA-3' (SEQ ID NO: 51 ) 44176.tm.rl 5'-CTGGCGGTGT CCTCTCCTT-3' (SEQ ID NO: 52)
PRQ357 (DNA44804): 44804.tm.fl 5'-CCTCGGTCTCCTCATCTGTGA-3' (SEQ ID NO: 53)
44804.tm.p l
5'-TGGCCCAGCTGACGAGCCCT-3' (SEQ ID NO: 54)
44804. tm.rl 5'-CTCATAGGCACTCGGTTCTGG-3' (SEQ ID NO: 55)
PR0715 DNA52722): 52722.tm.fl
5'-TGGCTCCCAGCTTGGAAGA-3' (SEQ ID NO: 56)
52722.tm.pl 5'-CAGCTCTTGGCTGTCTCCAGTATGTACCCA-3' (SEQ ID NO: 57)
52722.tm.rl 5'-GATGCCTCTGTTCCTGCACAT-3' (SEQ ID NO: 58)
PRO 1017 (DNA561 12 : 561 12.tm.fl
5'-CCTCCTCCGAGACTGAAAGCT-3' (SEQ ID NO: 59)
561 12.tm.pl
5'-TCGCGTTGCTTTTTCTCGCGTG-3' (SEQ ID NO: 60)
561 12.tm.rl 5'-GCGTGCGTC AGGTTCCA-3" (SEQ ID NO: 61 )
PRO 1 1 12 (DNA57702): 57702.tm.fl
5'-GTCCCTTCACTGTTTAGAGCATGA-3' (SEQ ID NO: 62)
57702.tm.pl 5'-ACTCTCCCCCTCAACAGCCTCCTGAG-3' (SEQ ID NO: 63) 57702.tm.rl 5'-GTGG TCAGGGCAGA TCCTTT-3' (SEQ ID NO: 64)
PRO509 (DNA50148): 50148.tm.fi 5'-GGAGGAGACAATACCCTCATTCA-3' (SEQ ID NO: 65)
50148.tm.p l
5'-AGCAGCCGTCGCTCCAGGTATCTC-3' (SEQ ID NO: 66)
50148.tm.rl 5'-CCA GGTGGACAGCCTCTTTC-3' (SEQ ID NO: 67)
PRQ853 (DNA48227) 48227 tm fl
5'-GGCACTTCATGGTCCTTGAAA-3' (SEQ ID NO 68)
48227 tm pi
5'-CGGATGTGTGTGAGGCCATGCC-3' (SEQ ID NO 69) 48227 tm rl
5'-GAAAGTA ACCACGGAGG TCAAGAT-3' (SEQ ID NO 70)
PRQ882 (DNA58125) 58125 tm f 1
5'-TTCCCAGCCTCTCTTTGCTTT-3" (SEQ ID NO 71 ) 58125 tm pi
5'-TGCCCCGTTCTCTTAACTCTTGGACCC-3' (SEQ ID NO 72)
58125 tm rl
5'-TCAGACGGAGTTACCATGCAGA-3' (SEQ ID NO 73)
The 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor amplification in real time Two oligonucleotidepπmers are used to generate an amplicon typical of a PCR reaction A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe During the amplification reaction, the TAQ DNA polymerase enzyme cleaves the probe in a template-dependent manner The resultant probe fragments disassociate in solution, and signal from 'he released reporter dye is free from the quenching effect of the second fiuorophore One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism
7700TM Sequence Detection The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer The system amplifies samples in a 96-well format on a thermocycler During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD The system includes software for mnning the instmment and for analyzing the data
5' Nuclease assay data are initial ly expressed as Ct. or the threshold cycle Th is is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence The ΔCt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer DNA results to normal human DNA results Table 1 describes the stage. T stage and N stage ofvaπous primary tumors which were used to screen the PRO201, PR0292. PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO1017, PROl 112, PRO509, PR0853 or PR0882 compounds of the invention Table 1 Primary Lung and Colon Tumor Profiles
Figure imgf000059_0001
Figure imgf000060_0001
DNA Preparation
DNA was prepared from cultured cell lines, primary tumors, normal human blood The isolation was performed using purification kit, buffer set and protease and all from Quiagen, according to the manufacturer's instmctions and the description below Cell culture Ivsis
Cells were washed and trypsinizedat a concentration of 7 5 x 10s per tip and pelleted by centπfiiging at 1000 rpm for 5 mιnutesat 4°C, followed by washing again with 1/2 volume of PBS recentπfugation The pellets were washed a third time, the suspended cells collected and washed 2x with PBS The cells were then suspended into 10 mL PBS Buffer Cl was equilibrated at 4°C Quiagen protease #19155 was diluted into 6 25 ml cold ddH20 to a final concentration of 20 mg/ml and equilibrated at 4°C 10 mL of G2 Buffer was prepared by diluting Quiagen RNAse A stock (100 mg/ml) to a final concentration of 200 μg/ml
Buffer C 1 ( 10 mL, 4°C) and ddH20 (40 mL.4°C ) were then added to the 10 mL of cell suspension, mixed by inverting and incubated on ice for 10 minutes The cell nuclei were pelleted by centπfuging in a Beckman swinging bucket rotor at 2500 rpm at 4°C for 15 minutes The supernatant was discarded and the nuclei were suspended with a vortex into 2 mL Buffer C 1 (at 4°C) and 6 mL ddH-O, followed by a second 4°C centrifugation at 2500 rpm for 15 minutes The nuclei were then resuspended into the residual buffer using 200 μl per tip G2 buffer ( 10 ml) was added to the suspended nuclei while gentle vortexing w as applied Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds Quiagen protease (200 μl, prepared as indicated above) was added and incubated at 50°C for 60 minutes The incubation and centrifugation was repeated until the lysates were clear (e g , incubating additional 30-60 minutes, pelleting
Figure imgf000061_0001
Solid human tumor sample preparation and sis Tumor samples were weighed and placed into 50 ml conical tubes and held on ice Processing was limited to no more than 250 mg tissue per preparation ( 1 tip/preparation) The protease solution was freshly prepared by diluting into 6 25 mi cold ddH-0 to a final concentration of 20 mg/ml and stored at 4°C G2 buffer (20 ml) was prepared by diluting DNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock) The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds using the large tip of the polytron in a laminar-flow TC hood to order to avoid inhalation of aerosols and held at room temperature Between samples, the polytron was cleaned by spinning at 2 x 30 seconds each in 2L ddH,0, followed by G2 buffer (50 ml) If tissue was still present on the generator tip, the apparatus was disassembled and cleaned Quiagen protease (prepared as indicated above, I 0 ml) was added followed by vortexing and incubation at 50°C for 3 hours The incubation and centrifugation was repeated until the lysates were clear ( g , incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min , 4°C) Human blood preparation and lysis Blood was drawn from healthv volunteers using standard infectious agent protocols and citrated into
10 ml samples per tip Quiagen protease was freshly prepared by dilution into 6 25 ml cold ddH .O to a final concentration of 20 mg/ml and stored at 4°C G2 buffer was prepared by diluting RNAse A to a final concentration of 200 μg/ml from 100 mg/ml stock The blood ( 10 ml) was placed into a 50 ml conical tube and 10 ml C 1 buffer and 5ύ ml ddH:0 (both previously equilibrated to 4°C) were added, and the components mixed by inverting and held on ice for 10 minutes The nuclei were pelleted with a Beckman swinging bucket rotor at 2500 rpm, 4°C for 15 minutes and the supernatant discarded With a vortex, the nuclei were suspended into 2 ml C l buffer (4°C) and 6 ml ddH:0 (4°C) Vortexing was repeated until the pellet was white The nuclei were then suspended into the residual buffer using a 200 μl tip G2 buffer ( 10 ml) were added to the suspended nucl* while gently vortexing, followed by vigorous vortexing for 30 seconds Quiagen protease was added (200 μl) and incubated at 50° C for 60 minutes The incubation and centrifugation was repeated until the lysates were clear (e g , incubating additional 30-60 minutes pelleting at 3000 x g for 10 mιn , 4°C)
Purification of cleared lysates ( 1 ) Isolation of genomic DNA Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) with 10 ml QBT buffer QF elution buffer was equilibrated at 50°C The samples were vortexed for 30 seconds, then loaded onto equilibrated tips and drained by gravity The tips were washed with 2 \ 15 ml QC buffer The DNA was eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 ml QF buffer (50°C) Isopropanol ( 10 5 ml) was added to each sample, the tubes covered with parafin and mixed by repeated inversion until the DNA precipitated Samples were pelleted by centrifugation in the SS-34 rotor at 15.000 rpm for 10 minutes at 4°C The pellet location was marked, the supernatant discarded, and 10 ml 70% ethanol (4°C) was added Samples were pelleted again by centrifugation on the SS-34 rotor at 10.000 rpm for 10 minutes at 4°C The pellet location was marked and the supernatant discarded The tubes were then placed on their side in a drying rack and dried 10 minutes at 37°C. taking care not to overdry the samples After drying, the pellets were dissolved into 1 0 ml TE (pH 8 5) and placed at 50°C for 1 -2 hours Samples were held overnιghtat 4°C as dissolution continued The DNA solution was then transferred to 1 5 ml tubes with a 26 gauge needle on a tuberculin syringe The transfer was repeated 5x in order to shear the DNA Samples were then placed at 50°C for 1 -2 hours (2) Quantitation of genomic DNA and preparation for gene amplification assav
The DNA levels in each tube were quantified by standard A260, A280 spectrophotometry on a 1 20 dilution (5 μl DNA + 95 μl ddH20) using the 0 1 ml quartz cuvetts in the Beckman DU640 spectrophotometer A260/A280 ratios were in the range of 1 8-1 9 Each DNA samples was then diluted further to approximately 200 ng/ml in TE (pH 8 5) If the original material was highly concentrated (about 700 ng/μl), the material was placed at 50°C for several hours until resuspended
FluorometπcDNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelinesas modified below This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes The Hoechst dye working solution (#H33258. 10 μl. prepared within 12 hours of use) was diluted into 100 ml 1 x TNE buffer A 2 ml cuvette was filled with the fluorometer solution, placed into the machine, and the machine was zeroed pGEM 3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solution and calibrated at 200 units An additional 2 μl of pGEM 3Zf(+) DNA was then tested and the reading confirmed at 400 +/- 10 units. Each sample was then read at least in triplicate When 3 samples were found to be within 10% of each other, their average was taken and this value was useα as the quantification value The fluorometπcly determined concentration was then used to dilute each sample to 10 ng/μl in ddH20 This was done simultaneously on all template samples for a single TaqMan plate assay, and with enough material to run 500-1000 assays The samples were tested in triplicate with Taqman™ primers and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls The diluted samples were us d σrovided that the CT value of normal human DNA subtracted from test DNA was -7-I' 1 CT The diluted, lot-qualified genomic DNA was stored in 1 0 ml aliquots at -80°C Aliquots which were subsequentlvto be used in the gene amplification assay were stored at 4°C Each 1 ml aliquot is enough for 8-9 plates or 54 tests
Gene amplification assay The PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343. PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509. PR0853 or PR0882 compounds of the invention were screened in the following primary tumors and the resulting ΔCt values are reported in Table 2 Table 2 ΔCt values in lung and colon primary tumor and cell line models
en
I
Figure imgf000063_0001
Figure imgf000063_0002
en
Figure imgf000064_0001
en
CO
I
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
en en
I
15
20
Figure imgf000067_0002
en en
I 10
Figure imgf000068_0001
10
en
I
20
Figure imgf000069_0001
10
en oo
I
Figure imgf000070_0001
PRO201 PRQ327 and PRO l 265
PRO201 (UNQ175) (DNA30676), PRO-327 (UNQ288)(DNA381 13) and PR01265 (UNQ636)(DNA60764) were also reexamined along with selected tumors irom the above initial screen with framework mapping Figure 32 and Table 3 indicate the chromosomal mapping ot the framework markers that were used in the present example The tramework markers are located approximately every 20 megabases and were used to control aneuploidy
PRO201 (UNQ 175XDNA30676), PR0327 (UNQ288)(DNA381 13) and PR01265 (UNQ636)(DNA60764) were also reexamined with epicenter mapping The markers indicated in Tables 4A, 4B and 4Care located in close proximity (in the genome) to DNA30676. DNA381 13 and DNA60764. respectively, and are used to assess the relative amplification in the immediate vicinity of Chromosome 19 wherein the respective molecule is located. The distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers One cR is very roughly equivalent to 20 kilobases. The marker SHGC-35441 is the marker found to be the closest to the location on chromosome 19 where DNA30676 maps, is closest to DNA381 13 and SHGC-33698 is closest to DNA60764.
Figure imgf000071_0001
Table 4A
Figure imgf000071_0002
Table 4B Epicenter Markers Along Chromosome 19 used for DNA381 13
Figure imgf000072_0001
Table 4C Epicenter Markers Along Chromosome 19 used for DNA60764
Figure imgf000072_0002
The Ct values of the above described framework markers along Chromosome 19 relative to PRO201 PR0327 and PRO 1265 are indicated for selected tumors in Table 5 A, 5B and 5C, respectively
Table 5A Amplification of framework markers relative to DNA30676 ( Ct)
Figure imgf000072_0003
Figure imgf000073_0001
Figure imgf000074_0001
Table 5C Amplification of framework markers relative to DNA60764 ( Ct)
Figure imgf000074_0002
Table 6 6 land 6 2 indicate the Ct values for results of epicenier mapping relative to DNA30676 DNA381 13 and DNA60764 respectivelv indicating the relative amplification in the region more immediate to the actual location of DNA30676 DNA381 1 and DNA670764 along chromosome 19
Table 6
Figure imgf000075_0001
Figure imgf000076_0001
Table 6 1 Amplification of epicenter markers relame to DNA381 13 ( Ct)
Figure imgf000076_0002
Figure imgf000077_0001
Table 6 2 indicates the Ct \alues for the results of epicenter mapping relative to DNA60764 indicating relative amplification in the region more immediate to the actual location of DNA60764 along chromosome 19 DNA34353 DNA40620 and DNA54002 are other independently identified molecules which have been observed to map to to the same region of chromosome 19 as DNA60764
Table 6 2 Amplification of epicenter markers relative to DNA60764 ( Ct)
Figure imgf000077_0002
Figure imgf000078_0001
PRQ292
PR0292 (UNQ266) (DNA35617) was also examined with framework mapping Figure 33 and Table 7 indicate the chromosomal mapping of the framework markers that were used in this analysis T e framework markers are located approximately every 20 megabases and were used to control aneuploidy
Table 7 Framework Marker used on Chromosome 1 1 for DNA35617
Figure imgf000078_0002
The ΔCt values of the above described framework markers along Chromosome 1 1 relative for PR0292 are indicated for selected tumors in Table 8
Table 8 Amplification ot Framework Markers relative to DNA35617
Figure imgf000078_0003
Figure imgf000079_0001
PRQ343 and PRQ882
PR0343 (UNQ302) (DNA433 I 8) and PR0882 (UNQ448) (DNA58125) were also examined with both framework and epicenter mapping Figure 34 and Table 9 indicate the chromosomal mapping of the framework markers that were used in this analysis The framework markers are located approximately every 20 megabases and were used to control aneuploidy Tables 9 1 and 9 2 in indicate the epicenter markers used for the mapping of DNA43318 and DNA58125 The markers shown in Tables 9 1 and 92 are located in close proximity (in the genome) to DNAs DNA43318 and DNA58125, respectivelv, and are used to assess the relative amplification in the immediate vicinitv of Chromosome 16 wherein the respective molecules map The distance between individual markers in measured in centiravs (cR) which is a radiation breakage unit roughly equal to a
Figure imgf000080_0001
roughly equiv alent to 20 kilobases The markers AFMa061 \ b5 and SHGC 36123 are located the closest to the location on chromosome 16 where DNA43318and DNA58125. respectively, map
Table 9 Framework Marker used on Chromosome 16 for DNA4 1 and DNA58125
Figure imgf000080_0002
Table 9 1 Epicenter markers Along Chromosome 16 used for DNA43318
Figure imgf000080_0003
Table 9 2 Epicenter markers along Chromosome 16 used for DNA58125
Figure imgf000080_0004
The ΔCi values ol the Iramevvork markers of Table 9 along Chromosome 16 relative tor PRO.343 and PR088. are indicated for selected tumors in Table 10.
Table 10 Amplification of Framework Markers relative to DNA4331 8 and DNA58125
Figure imgf000081_0001
Figure imgf000082_0001
Table 10 1 and 10 2 indicate the ΔCt values from epicenter mapping relative to DNA43318 and DNA58125, respectively indicating the relative amplification in the region more immediate to the actual location of the respective molecules along chromosome 16
Table 10 1 Amphticaiion ol epicenter markers relative to DNA43318 (ΔCt)
Figure imgf000083_0001
Figure imgf000084_0001
Table 10 2 Amplification of epicenter markers relative to DNA58125 (ΔCt)
Figure imgf000084_0002
Figure imgf000085_0001
PRO1017
PRO 1017 (UNQ500) (DNA561 12) was also examined with framework mapping. Figure 35 and Table 1 1 indicate the chromosomal mapping of the framework markers that were used in this analysis The framework markers are located approximately every 20 megabases and were used to control aneuploidy.
PROI017 (U Q500)(DNA561 12) was also examined with epicenter mapping. Table 1 1.1 indicates the epicenter markers which are located in close proximity to DNA561 12 which were employed to assess the relative amplification in the immediate vicinity of chromosome 7 wherein DNA561 12 is located. The distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases. The marker SHGC-22698 is the marker found to be the closest to the location on chromosome 7 where DNA561 12 maps.
Table 1 1 Framework Marker used on Chromosome 7 for DNA 561 12
Figure imgf000085_0002
Table 1 1.1 Epicenter Markers along Chromosome 7 used for DNA561 12
Figure imgf000086_0001
Table 1 1 2 indicated the DCt values for the results of epicenter mapping relative to DNA561 12, indicating the relative amplification in the region more immediate to the actual location of DNA561 12 along chromosome 7
Table 11 2 Amplification of epicenter markers relative to DNA561 12 (ΔCt)
Figure imgf000086_0002
Figure imgf000087_0001
The ΔCt values of the above described framework markers along Chromosome 7 relative DNA56112 is indicated for selected tumors in Table 12
Table 12 Amplification of Framework Markers relative to DNA561 12
Figure imgf000087_0002
Figure imgf000088_0001
PR0715 and PRQ853
PR0715 (UNQ383) (DNA52722) and PR0853 (UNQ419) (DNA48227) were also reexamined with both framework and epicenter mapping Figures 36A and 36B and Table 13 indicate the chromosomal localizations of the framework markers that were used for the procedure The framework markers are located approximately every 20 bases and were used to control aneuploidy Tables 14A and 14B indicate the epicenter mapping markers that were used in the procedure The epicenter markers were located in close proximity to DNA52722 and DNA48226, respectively and are used to determine the relative DNA amplification in the immediate vicinity of DNA52722 and DNA48226 The distance between individual markers is measured in centirays, which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers One cR is very roughly equivalent to about 20 kilobases In both figures 14Aand 14B, "BAC" means bacterial artificial chromosome The ends of a BAC clone which contained the gene of interest were sequenced TaqMan primers and probes were made from this sequence, which are indicated in the respective tables BAC clones are typically 100 to 150 Kb, so these primers and probes can be used as nearby markers to probe DNA from tumors In figure 14 A, the marker SHGC-31370 is the marker found to be the closest to the location on chromosome 17 where DNA52722 maps In figure 14B, the marker SHGC-37126 is the marker found to be the closes to the location on chromosome 17 where DNA48227 maps
Table 13 Framework Markers Used Along Chromosome 17 for DNA52722 and DNA48227
Figure imgf000088_0002
Table 14A Epicenter Markers Used on Chromosome 17 in Vicinity of DNA52722
Figure imgf000088_0003
Figure imgf000089_0001
Table 15A Epicenter Markers Used on Chromosome 17 in Vicinity of DNA48227
Figure imgf000089_0002
Table 16 indicates the ΔCt values of the above described framework markers along chromosome 17 relative to DNA52722 and DNA48227 for selected tumors. While not shown, the similar ΔCt values for the framework markers in the analysis of DNA48227 were reported.
Table 16 Amplification of Framework Markers Relative to DNA52722
Figure imgf000089_0003
Figure imgf000090_0001
Table 18 indicates the ΔCt values for the indicated epicenter markers indicating the relative amplification along chromosome 17 in the immediate vicinity of DNA52722
Table 18 Amplification ol Epicenter Markers Relative to DNA52722
Figure imgf000091_0001
Figure imgf000092_0001
Tables 18A and 18B indicate the ΔCt values for the indicated epicenter markers, indicating the relative amplification of selected lung and colon tumors, respectively, along chromosome 17 in the immediate vicinity of DNA48227
Table 18A Amplification of Epicenter Markers in vicinity of DNA48227on chromosome 17 in selected lung tumors
Figure imgf000092_0002
Figure imgf000093_0001
Table 18B Amplification of Epicenter Markers in viciniiv ol DNA48227on chromosome 17 in selected colon tumors
Figure imgf000093_0002
PRQ357
PR0357 (UNQ324 DNA44804) was reexamined with selected tumors from the above initial screen with framework mapping Figure 40 and Table 19 indicate the chromosomal mapping of the framework markers that were used in the present example The framework markers are located approximately every 20 megabases and were used to control aneuploidv
PR0357 (UNQ324 DNA44804) was also examined with epicenter mapping The markers indicated in Table 20 are located in close proximitv (in the genome) to DNA44804 and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein DNA44804 is located The distance between individual markers is measured in centirays (cR) which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between the two markers One cR is very roughly equivalent to 20 kilobases The marker SHGC-6154 is the marker tound to be the closest to the location on chromosome 16 where DNA44804 maps
Table 19 Framework markers for DNA44804
Figure imgf000094_0001
Table 20 Epicenter markers for DNA44804 along chromosome 16
Figure imgf000094_0002
The ΔCt values of the above described framework markers along chromosome 16 relative to DNA44804 is described in Table 21
Table 21
Figure imgf000095_0001
Table 22 indicates the ΔCt values for the results oi epicenter mapping relative to DNA44804 indicating the relative amplification in the region more immediate to the actual location of DNA44804 along chromosome 16
Table 22 Amplification of epicenter markers relative to DNA44804
Figure imgf000095_0002
Figure imgf000096_0001
DISCUSSION and CONCLUSION PRO201
The ΔCt values for DNA30676 (PRO201 , UNQ 175) in a variety of lung and colon tumors are reported in Table 2 A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of
DNA30676 occurred in primary lung tumors SRC727 (LT3), SRC732 (LT10), SRC733 (LTI 1), SRC735 (LT13), SRC736 (LT15) SRC737 (LT16) SRC738 (LT17), SRC740 (LT19), SRC741 (LT21) primary colon tumors SRC742 (CT2), SRC743 (CT3), SRC744 (CT8), SRC745 (CT10), SRC746 (CT12), SRC747 (CT14), SRC748 (CT15), SRC749 (CT16), SRC750 (CT17), SRC751 (CTI), SRC752 (CT4), SRC753 (CT5), SRC754 (CT6), SRC755 (CT7), SRC756 (CT9), SRC757 (CTI 1 ), SRC758 (CTI 8), lung tumor cell lines SRC769 (Calu-1), SRC770 (Calu 6), SRC771 (HI 57), SRC772 (H441), SRC774 (SKEMS-1), SRC832 (H522), SRC833 (H810), and colon tumor cell lines SRC778 (Colo320), SRC828 (Colo205) The ΔCt values and average ΔCt values for the primary lung tumors hits are 2 05, 1 08, 1 63, 2 13, 2 58, 1 10, 1 63, 3 37, 1 3, primary colon tumors are 3 81, 2 3, 1 97, 3 01 , 2 46, 3 34, 2 58, 2 46, 2 64, 1 57, 3 39, 2 95, 2 81, 2 77, 2 63, 3, 2 23, lung tumor cell lines are 2 37, 2 61 , 2 39, 2 07, 2 91, 2 07, colon tumor cell lines 1 95, 1 26 This represents an increase in gene copy of approximately 4 14 2 11, 3 10,
4 38, 5 98, 2 14, 3 10, 10 34, 2 46 for the pπmarv lung tumors, 14 03, 4 92, 3 92, 8 06, 5 50, 10 13, 5 98,
5 50, 623, 2 97, 10 48, 7 73, 7 01 , 6 82, 6 19, 8 0, 4 69 for the pπmarv colon tumors, 5 17, 6 10, 5 24,
6 19, 7 52, 4 20 tor the lung tumor cell lines, and 3 86, 2 39 for the colon tumor cell lines Amplification has been confirmed by framework mapping for DNA30676 in pπmary lung tumors LT3, LTI 5-LT 18 and in primary colon tumors CT2 CT3, CT8, CTI O CT12, CT14, CT15, CT16. CT 17 The reported ΔCt values for the primary lung tumors are I 04. 1 43. 1 35, 1 51 and 1.22. while the primary colon tumors report 2 81 , 2 03, 1 39. 2 21. 1 93, 2 37, 1 27, 1 76 and I 65. Relative to normal tissue, this represents approximately a 2 06. 2 69. 2 55, 2 85. and 2.32 fold increase for the lung tumors, and a 7.01. 4 08. 2.62, 4.63, 3.81 , 5 17. 2 41 , 3 39. 3 14 fold increase tor the colon tumors Epicenter mapping for DNA30676 resulted in confirmation of significant amplification: in primary lung tumors LT3, LTI . LTI 5. LTI 6, LTI 8. and in primary colon tumors CTI . CT3, CT4. CT5, CT6, CT7, CT8, CT9, CT10. CTI I . CT12, CT14, CT15, CT 16, CT17 and CT18 The reported ΔCt values for the primary lung tumors were 1.87, 1.44, 1.99, 1 72 and 1 09. while the primary colon tumors indicated ΔCt and average ΔCt values of 2.56, 2.19, 3.22, 2.83. 2 93. 2 43, 1 95, 2 72. 3 12, 3.26, 2.77, 2.88, 2.60. 2.61 , 2.24 and 2 48 Relative to normal tissue, this represents a 3.66, 2.71 , 3.97, 3.29, 2.13-fold increase in gene copy for the lung tumors and a 5.90, 4 56, 9.32, 7 1 1 , 7 62, 5.39. 3 86, 6 59, 8.69, 9.58, 6 82. 7.36, 6.06, 6.1 1 , 4.72, 5.58-fold increase increase in gene copy for the colon tumors.
In contrast, the amplification of the closest known framework markers (with one exception, i.e. S50)(Table 5 A) or epicenter markers (Table 6) does not occur to a greater extent than that of DNA30676 This strongly suggests that DNA30676 is the gene responsible for the amplification of the particular region on Chromosome 19 Because amplification of DNA30676 occurs in various lung and colon tumors and cell lines (especially colon), it is highly probable to play a significant role in tumor formation or growth As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA30676 (PRO201 ) would be expected to have utility in cancer therapy.
PRQ292:
The ΔCt values for DNA35617 (PR0292, UNQ266) in a variety of lung and colon tumors are reported in Table 2. A ΔCt value of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA35617 occurred in primary lung tumors: LT12, LT13, LT15, LT19, LT21 , in primary colon tumors CT2, CT8. CT10, CT14; in lung tumor cell lines H441 and H810, in colon tumor cell lines SW620, Colo320, HT29 and LS 174T. The ΔCt and average ΔCt values for these hits are: ( 1 ) primary lung tumors: 1 35, 1 93, 2.15, 2.33, 1.42; (2) primary colon tumors: 3.54, 1.52, 1.63, 1.58; (3) lung tumor cell lines: 1.13, 1.08; and (4) colon tumor cell lines: 1.41 , 1.34, 1.10, 1.50. Relative to normal tissue, this represents approximately a 2.55, 3.81 , 4.44, 5.03, 2.67-fold increase, respectively, in gene copy for the primary lung tumor hits, a 1 1.63, 2.87, 3.09, 2.99-fold increase, respectively, in gene copy for the primary colon tumors, a 2 19 and 2.1 1 -fold increase in gene copy for the lung tumor cell lines, and a 2.66. 3.53, 2.14 and 2.83-foId increase in gene copy for the colon tumor cell lines.
Amplification of DNA35617 was also confirmed by framework mapping. Table 8 indicates that significant amplification of DNA35617 was confirmed in LT12, LT13, LT15, LT16 and CT2. CT8, CT10 and CT14. The reported ΔCt values for the primary tumors were 1.61 , 1.95, 2.86 and 1.68, while for the primary colon tumors the values were 3.75, 1.76, 1.68 and 1.75 These represent a 3.05, 3.86, 7.26, 3.20- fold increase in gene copy for the lung tumors and a 13.45, 3 39, 3.20 and 3.36-fold increase in gene copy for the colon tumors In contrast, the amplification of the closest known framework markers (Table 8) does not occur to a greater extent that that of DNA35617. This strongly suggests that DNA356 I 7 is the gene responsible for the amplification of the particular region on chromosome 17 Because amplification of DNA35617 occurs in various tumor tissues, especially colon tumors, it is highly probably to plav a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g . antibodies) directed against the protein encoded by DNA35617 would be expected to have utility in cancer therapy
PRQ327.
The ΔCt values for DNA381 13 (PR0327)(UNQ288) in a variety of lung and colon tumors are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA381 13 occurred in primary lung tumors: SRC734 (LT12), SRC735 (LT13), SRC736 (LT15), SRC737 (LT16), SRC738 (LT17), SRC740 (LT19); primary colon tumors: SRC742 (CT2), SRC743 (CT3), SRC744 (CT8), SRC745 (CTIO), SRC746 (CT12), SRC747 (CT14), SRC748 (CT15), SRC749 (CT16), SRC750 (CT17), SRC751 (CTI), SRC752 (CT4), SRC753 (CT5), SRC754 (CT6), SRC756 (CT9), SRC757 (CTI 1), SRC758
(CTI 8), lung tumor cell lines SRC771 (HI 57), SRC772 (H441 ), SRC773 (H460), SRC774 (SKMES-1), SRC832 (H522), SRC833 (H810); colon tumor cell lines: SRC777 (SW620), SRC778 (Colo320), SRC830 (HCC2998), SRC831 (KM 12).
The ΔCt values and average ΔCt values for the primary lung tumor hits are: 2.57, 2.09, 3 44, 1.91 , 1.43, 3.58; primary colon tumors are: 3 64, 1.12, 1.64, 2.53, 1.88, 2.51, 1.30, 1.47, 1.71, 1.37, 2.22, 2.50, • 2.28, 1.58, 3.33 and 1.07; lung tumor cell lines 1.65, 2.23, 1.12, 1.18, 1.02 and 1.1 ; and colon tumor cell lines 2.24, 1.01, 1.23 and 1.61. This represents an increase in gene copy of approximately: 5 94, 4.26, 10 85, 3.75, 2.69, 1 1.96 for the primary lung tumors; 12.47, 2.17, 3.12, 5.78, 3.68, 5.67, 2.46, 2.77, 3.27, 2.58, 4 66, 5.66, 4.86, 2.99, 10 06, 2.10 for the primary colon tumors; 3 14, 4.69, 2.17. 2.27, 2 03. 2.14 for the lung tumor cell lines, and 4.73, 2.01. 2.35 and 3.05 for the colon tumor cell lines
Amplification has been confirmed by framework mapping for DNA381 13 in primary lung tumors LT10 and in primary colon tumors CT2, CT3, CT8, CT10, CT12, CT14, CT16. The reported ΔCt values for the pπmary lung tumor is 1 45; the primary colon tumors are 2.94, 1.23, 1.45, 1.72, 1.60, 2.03 and 1.07. Relative to normal tissue, this represents approximately a 2.73-fold increase for the lung tumor and a 7.67, 2.34, 2.73, 3.29, 3.03, 4 08 and 2.10-fold increase for the colon tumors. Epicenter mapping resulted in confirmation of significant amplification in: primary lung tumors LT12, LT13, LT15, LT16, LT17 and in pπmary colon tumors CTI , CT2, CT3, CT4, CT5, CT6, CT8, CT9, CT10, CTI 1 , CT12, CT14, CT16. The reported ΔCt values for the primary lung tumors are. 1.57, 1.22, 2.47, 2.23, 1.02 and for the primary colon tumors are 1.38, 2 94, 1.23, 2.23, 2 51, 2.29, 1 45. 1.59, 1.72, 3.34, 1 6, 2.03, 1 07 and 1 08. Relative to normal tissue, this represents a 2.97, 2.33, 5 54, 4 69. 2.03-fold increase in gene copy for the lung tumors and a 2.60, 7.67, 2.35, 4.69, 5.70, 4.89, 2.73, 3 01 , 3.29, 10 13, 3.03, 4.08. 2.10, 2.1 1-fold increase in gene copy for the colon tumors. With the exception of S41. amplification of the closest markers to DNA381 13 does not occur to a greater extent than that of DNA381 1 itself This supports that notion that DNA381 13 is the gene which is driving the amplification of this particular region of Chromosome 19 However, the amplification of marker S41 (which does not map closely to DNA381 13) could be an independent amplification event or even an error in the ordering of the markers Because amplification of DNA381 13 occurs in various lung and colon tumors and cell lines, it is highly probably to play a significant role in tumor formation and growth. As a result, antagonists (e g., antibodies) directed against the protein encoded by DNA381 13 (PR0327) would be expected to have utility in cancer therapy
PRO 1265: The ΔCt values for DNA60764 (PR01265)(UNQ636) in a variety of lung tumors are reported in
Table 2. A ΔCt value of > l was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA60765 occurred in primary lung tumors LT3, LT12, LT13. LT15, LT16 and LT17 The ΔCt values of these hits are 1.03, 2.17, 2.24, 3.51 , 3.32 and 1.02. This represents an increase in gene copy of approximately 2.04, 4.50, 4.72, 11.39, 9.99 and 2.03
Amplification has also been confirmed framework mapping for DNA60764 in LT16. The reported ΔCt value was 1.37, which represents a 2 58 fold increase in gene copy relative to normal tissue. Epicenter mapping has also confirmed amplification of DNA60764 in LT12, LT13, LT15, LT16, CTI , CT4, CT5, CT7 and CTI 1. These tumors report ΔCt values of 2.35, 2.37, 3.88, 3.32 in the lung tumors and 1.74, 1.86, 3.28, 1.29 and 2.32 in the colon tumors. Relative to normal tissue, this represents an increase in gene copy of approximately 5.10, 5.17, 14.72 and 9.98 in the lung tumors and 3.34, 3.63, 9.71 , 2.45 and 4.99 in the colon tumors.
In contrast, the amplification of the closest known framework markers, epicenter markers and the comparison sequences does not occur to a greater extent than that of DNA60764. This strongly suggests that DNA60764 is ghe gene responsible for the amplification of the particular region in Chromosome 19 Because amplification of DNA60764 occurs in various lung and colon tumors, it is highly probably to play a significant role in tumor formation or growth. As a result, antagonists (e g., antibodies) directed against the protein encoded by DNA60764 would be expected to have utility in cancer therapy.
PRQ344: The ΔCt values for DNA40592 (PR0344, UNQ303) in a variety of lung and colon tumors are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA40592 occurred in primary lung tumors LT13 and LT19, in primary colon tumors CT2. CT14, CT15. CTI, CT4, CT5, CTI 1. The ΔCt values and average ΔCt of these hits are 1.34, 1.52 in the lung tumors, and 1.84, 1.16, 1.17, 1.24, 1.46, 1.51 and 1 35. This represents approximately a 2 53, 2.87 and 3.58, 2.23, 2.25, 2.36,
2.75, 2.85, 2.5-fold increase, respectively, in gene copy relative to normal tissue. Because amplification of DNA40592 occurs in various tumors, it is likely assocaited with tumor formation or growth. As a result, antagonists, (e g . antibodies) directed against the protein encoded by DNA40592 (PR0344 would be expected to be useful in cancer therapy
PRQ343
The ΔCt values for DNA43318 (PRO343)(UNQ302) in a variety of lung and colon tumors are reported in Table 2. A ΔCt of >1 was typicallv used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA43318 occurred in primary lung tumor: LT13, LT15, LT19, CT2, CT8, CTIO. CT12, CT14, CT15, CT16. CT17 and CT5. The ΔCt and average ΔCt values for the primary lung tumor hits are 1 67, 1.47 and 1.66. while the colon tumor hits are 2.72, 1.43, 1.68, 1.33, 2.02, 1.73, 1 16, 1.5, 1.34. This represents an increase in gene copy of approximately 3.18. 2.77 and 3.16 -fold for the lung tumors and 6.59, 2.69, 3.20, 2.51 , 4.06, 3.31 , 2.23, 2.83 and 2.53 -fold for the colon tumors.
Amplification has been confirmed by framework mapping for DNA43318: in primary lung tumors LT12, LT13, LT15, LT16 and LT18: and in primary colon tumors CT2, CT4, CT5, CT8. CTIO, CT14, CT15 and CT16 The reported ΔCt values are 1 16. 1.72, 2.73, 1 46 and 1.06 for the lung tumors and 3.54, 1 17, 2.63, 1.44, 1.36, 1.68, 1.07 and 1.01 for the colon tumors. Relative to normal tissue, this represents about a 2.23, 3.29, 6.63, 2.75 and 2.08-fold increase in gene copy for the lung tumors, and about a 11.63, 2.25, 6.19, 2.71 , 2.57, 3.20, 2.10 and 2.01-fold increase for the colon tumors. Epicenter mapping for DNA43318 confirmed significant amplification in LT12, LT13, LT15, LT16, CT4, CT5, CT6, CTI 1 and CT2. The reported ΔCt values are 1.32, 1.94, 3.07, 1.83, 1.02, 2.40, 3.78, 1.51 and 2.48. Relative to normal tissue, this represents a 2.50, 3.84, 8.40, 3.56-fold increase in the lung tumors and a 2.03, 5.28, 13.74, 2.85, 5.58-fold increase in gene copy for the colon tumors.
In contrast, the amplification of the closest known framework and epicenter markers (with one exception, i.e, PI 07, Table 10.1) does not occur to a greater extent than that of DNA43318. This strongly suggests that DNA43318 is the gene responsible for the amplification of the particular region of Chromosome 16 Because amplification of DNA43318 occurs in various lung and colon tumors, it is highly probably to play a significant role in tumor formation or growth As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA43318 would be expected to have utility in cancer therapy.
PRQ347
The ΔCt values for DNA44176 (PR0347, UNQ306) in a variety of lung and colon tumors are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA44176 occurred in primary lung tumors LT15 and primary colon tumors CT2, CT8, CT14, CT15, CT16, CT5 and CTI 1 The ΔCt values and average ΔCt of these hits are 1.76 in the lung tumors, and 1.85, 1.12, 1.27, 1.07, 1.19, 1.80. 1.42 in the colon tumors. This represents approximately a 3.39. 3.61. 2 17. 2 41. 2.10. 2.28. 3 48. 2.67-fold increase, respectively, in gene copy relative to normal tissue.
Because amplification of DNA44176 occurs in various tumors, it is likely associated with tumor formation or growth. As a result, antagonists, (e.g., antibodies) directed against the protein encoded by DNA44176 (PR0347) would be expected to be useful in cancer therapy
PR0357-
The ΔCt values for DNA44804 (PR0357)(UNQ314) in a variety of lung and colon tumor and cell lines are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA44804 occurred in primary lung tumors: LT9, LT10, LTI 1, LT12. LT13, LT15, LT16, LT17, LT19, LT21 ; in primary colon tumors CT2, CT8, CTIO, CT14, CT15, CT16, CTI , CT4, CT5, CT6, CT7 and CT11. The ΔCt and average ΔCt values of the lung tumor hits are: 1.42, 1.63, 1.47, 1.42, 1.52, 2.18, 1.23, 1.71, 2.47. 1.66: and the colon tumor hits are 2.01 , 1.1 , 1.42, 1.5, 1.25, 1.05, 1.04, 1.27, 1.69, 1.08, 1.73, 1.83. This represents an increase in gene copy of approximately 2.68, 3 09, 2.77, 2.68, 2.87, 4.53, 2.36, 3.27, 5.54 and 3.1 -fold, respectively, for the lung tumors and a 4.03, 2.14, 2.68, 2.83, 2.38, 2.07, 2.06, 2.41, 3.23, 2.11, 3.31, 3.56-fold increase, respectively, for the colon tumors.
Amplification has been confirmed by framework mapping for DNA44804 in primary lung tumors LT3, LT10, LTI 1, LT13, LT15. LT17, LT19 and LT21. The reported ΔCt values for these hits are 1.15, 1.21, 1.71 , 1.96, 2.32, 3.01 , 1.64. 3 03 and 1.33. These represent a 2.22, 2.31, 3.27, 3.89, 4.99, 8.06, 3.12, 8.17and 2.51 -fold increase in gene copy in the respective primary lung tumors Epicenter mapping for DNA44805 resulted in confirmation of significant amplification in primary lung tumors 1.27, 1.42, 1.67, 2.36, 1.17 and 2.29, which represents an increase in gene copy of 2.41 , 2.68, 3.18, 5.13, 2.25, 4.89-fold, respectively.
In contrast, the amplification of the closest known framework markers (from Table 21 ) and epicenter markers (Table 22) does not occur to a greater extent that that of DNA44804. This strongly suggests that DNA44804 is the gene responsible for the amplification of the particular region on chromosome 16. Because amplification of DNA44804 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA44804 would be expected to have utility in cancer therapy.
PRQ715:
The ΔCt values for DNA52722 (PR0715)(UNQ383) in a variety of lung and colon tumor and cell lines are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA52722 occurred in LT12, LT13. LT15, LT17, LT19, CT2, CT3, CT8, CT10, CT14, CT15, CT16, CT17, CTI , CT4, CT5, CT6, CT7, CTI 1, CT18. The ΔCt and average ΔCt values for the pπmary lung tumors are 1.29, 1.51. 1.35, 1.22 and 1.55, while the primary colon tumors report values of 1.64, 1.12, 1 32 1 97 1 42 1 53 1 28 1 31 1 32 1 59 1 92 1 43 1 23 2 29 1 05 Relative to normal tissue these represent 2 45 2 85 2 55 2 33 2 93 fold increase in gene copv for the lung tumors and 3 12, 2 17 2 50 3 92, 2 68 2 89 2 42 2 48, 2 50, 3 01 3 78 2 69 2 34 4 89 2 07 fold increase in gene copy tor the colon tumors Significant amplification has been confirmed bv framework mapping for DNA52722 in primary lung tumors LTI 1 LT13, LT15, LT16, LT17 and LT18 and in pπmarv colon tumors CT2, CT3 CT8 CT10 CT14 CT15 and CT16 The reported ΔCt values for the lung tumors are 1 03, 1 37, 1 75 1 1 1 , 1 14 1 04 and for the colon tumors 1 29 1 06 1 08 1 60, 1 05 1 36 and 1 06 Relative to normal tissue, the represents approximately a 2 04, 2 58 3 36, 2 16 2 20 2 06 -fold increase in gene copy for the lung tumors and a 2 44, 208, 2 11 , 3 03, 207, 2 57 and 208-fold increase in gene copy for the colon tumors Significant amplification was also confirmed by epicenter mapping for DNA52722, wherein hits were observed in LTI 1 , LT13, LT15, LT16, LT17 LT18, CTI, CT3, CT4, CT5, CT6, CT7, CT10, CTI 1 CT14, CT15, CT16 and CTI 8 The reported ΔCt values for the primary lung tumors were 1 07, 1 29, 1 33, 1 1 1, 1 13, 1 21, while the colon tumors indicate 1 22 1 19, 1 26, 1 79, 1 71, 1 19, 1 86, 2 29, 1 05 1 27, 1 03 and 1 06 Relative to normal tissue, this represents a 2 10 2 45, 2 51, 2 16, 2 19, 2 31 -fold increase in the lung tumors and a 2 33, 2 28, 2 39, 3 46 3 27, 2 28, 3 63, 4 89, 2 07, 2 41 , 2 04 and 2 08-fold increase in the colon tumors
In marked contrast, the amplification of the closest known framework and epicenter markers does not occur to a great extant than that of DNA52722 This strongly suggests that DNA52722 is the gene responsible for the amplification of the particular region on Chromosome 17 Because amplification of DNA52722 occurs in various lung and colon tumors and cell lines, it is highly probably to play a significant role in tumor formation or growth As a result, antagonists (e g , antibodies) directed against the protein encoded by DNA52722 (PR0715) would be expected to have utility in cancer therapy
PRO1017 The ΔCt values for DNA561 12 (PRO 1017, UNQ500) in a variety of lung and colon tumors are reported in Table 2 A ΔCt value of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA56112 occurred in primary lung tumors LTla, LT3, LT6, LT7, LT9, LT10, LTI 1, LT12, LT13, LT15, LT16, LT17. LT18, LT19, LT21 , primary colon tumors CT2, CT3, CT8 CT10, CT12, CT14, CT15, CT16, CT17, CT4, CT5, CT6, CT9, CTI 1 The ΔCt and average ΔCt values for (1 ) the primary lung tumor hits are 1 78, 2 37, 2 79, 1 11 , 2 73, 3 51 , 1 96, 2 20, 1 90 2 24, 1 78, 1 76, 1 24 and 2 24, and (2) pπmary colon tumor hits are 3 13, 2 74, 1 78, 2 67, 2 29, 2 87, 2 30, 1 75, 1 82, 1 10 2 20, 1 1 1 , 1 28 and 1 47 This represents an increase in gene copy of approximately 3 43, 5 17, 6 92, 2 16 6 63, 1 1 39, 3 89, 4 59 3 73, 4 72 3 43, 3 39, 2 36 and 4 72 -fold, respectively for the lung colon tumors and 8 75, 6 68 3 43 6 35, 4 89, 7 31 , 4 92, 3 36, 3 53, 2 14, 4 59, 2 16, 2 43and 2 77-fold, respectively for the primary colon tumors Amplification has also been confirmed by tramework mapping tor DNA561 12 in primary lung lu ors LT3. LT4, LT7, LT9. LT10. LTI 1. LT12. LT13. LT15. LT16, LT18 and LT22 The reported ΔCt values are 1.21 , 1.59. 1 40, 1 07, 1 98. 1.15. 2.31 , 1 83, 2.79. 2 22, 1.06 and 1 18. These values represent a 2 31 , 3 01 , 2 64, 2 10, 3 95, 2 22. 4 96. 3 56. 6 92. 4 66. 2 08 and 2 27-fold increase, respectively, in gene copy for the indicated lung tumors Epicenter mapping for DNA561 12 indicated in confirmation of amplification in- primary lung tumors LT12. LT13, LT15. LT16 and LT18 and in primary colon tumors' CT5, CT8, CT10, CT12, CT14, CT16 and CT17 The ΔCt values for the primary lung tumors are. 1.47, 1.51 , 2 27, 1.62 and 1.03; and the primary colon tumors are 1.29, 1.46, 2.28, 1.84. 2.56. 1 22 and 1.52 These values represent a 2.77, 2.85, 4.82, 3 07, 2 04-fold increase, respectively, in the lung tumors and a 2 45. 2 75, 4.86, 3.58, 5.90, 2.33 and 2 87-fold increase, respectively, in the colon tumors
In contrast, the amplification of the closest known framework markers (from Table 12) and epicenter markers (Table 1 1.2) does not occur to a greater extent that that of DNA561 12. This strongly suggests that DNA561 12 is the gene responsible for the amplification of the particular region on chromosome 17. Because amplification of DNA561 12 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g.. antibodies) directed against the protein encoded by DNA56112 would be expected to have utility in cancer therapy.
PROl 1 12:
The ΔCt values for DNA57702 (PROl 1 12, UNQ555) in a variety of lung and colon tumors are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA57702 occurred in lung tumors LT10, LTI 1, LT12, LT13, LT15, LT17, LT18 and in colon tumors CT2, CT8,
CT10, CT12, CT14, CT15, CT16, CTI , CT4, CT5, CT6 and CTI 1.
The ΔCt values and average ΔCt of these hits are 1 13, 1.52, 1.19, 1.63, 1.77. 1.45 and 1.25 for the lung tumor and 2.26, 1.06, 1.57, 1.31 , 1 89, 1.46, 1.25, 1 24. 1.53. 1.97, 1.57 and 2.28 for the colon tumors. This represents approximately a 2.19, 2.87, 2.28, 3 09, 3.41, 2.73, 2.38-fold amplification, respectively, for the lung tumor, and
4.79, 2.08, 1.57, 2.97, 3.70, 2.75, 2.38, 2.36, 2.89, 3.92, 2.96, 4.5- fold amplification, respectively, for the colon tumors, in gene copy relative to normal tissue. Because amplification of DNA57702 occurs in various tumors, it is likely associated with tumor formation or growth. As a result, antagonists, (e.g., antibodies) directed against the protein encoded by
DNA57702 (PROl 1 12) would be expected to be useful in cancer therapy.
PRO509-
The ΔCt values for DNA50148 (PRO509, UNQ329) in a variety of lung and colon tumors are reported in Table 2. A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA50148 occurred in primary colon tumors CT15. CT17, CT6, CTI 1 and CT18. The ΔCt values and average ΔCt ot these hits are I 16 1 02 1 01 1 34 1 35 which represents a 2 23 2 03 2 01. 2 53, 2 55-fold increase respectively in gene copv relative to normal tissue
Because amplification of DNA50148 occurs in various tumors it is likely associated with tumor formation or growth As a result, antagonists, (e g , antibodies) directed against the protein encoded by DNA50148 (PRO509 would be expected to be useful in cancer therapv
PRQ853
The ΔCt values for DNA48227 (PR0853)(UNQ419) in a variety of lung and colon tumors and cell lines are reported in Table 2 A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA48227 occurred in pπmary lung tumors LT12 and lung cell line H522, as well as in primary colon tumors CT2, CT3, CT8 CT10, CT12, CT14, CT15, CT16, CT17, CTI , CT4, CT5, CT7 and CT1 1 The ΔCt and average ΔCt values for these hits are 1 03, 1 02, 2 4, 1 52, 1 55, 1 97, 1 36, 1 75, 1 75, 1 1 1 , 1 11 ,
1 09, 1 16, 1 95, 1 14, 1 88 This represents an increase in gene copy of approximately 2 04 for the primary lung tumor LT12, 2 03 for the lung tumor cell line H522 and 5 28, 2 87, 2 93, 3 92, 2 57, 3 36, 3 36, 2 16, 2 16, 2 13, 2 23, 3 86, 2 20, 3 68 for the primary colon tumors
Confirmation of amplification was not confirmed in epicenter mapping for DNA48227 for pπmary lung tumors, but was seen in primary colon tumors CTI , CT2, CT3, CT4, CT5, CT6, CT7, CT8, CT9, CT10, CTI 1, CTI 2, CTI 4, CTI 5, CTI 7 and CTI 8 The reported ΔCt values in Table 18B are 1 09,
2 10, 1 09, 1 12, 2 33, 1 35, 1 34, 1 22, 1 66, 1 96, 1 03, 1 69, 1 77 and 1 25 This represents a 2 13, 4 29, 2 13, 2 17, 5 03, 2 55, 2 53, 2 33, 3 16, 3 89, 2 04, 3 23, 3 41 and 2 38 -fold amplification, relative to normal tissue, for these tumors
In contrast, the amplification of the closest known framework markers (from Table 16) and epicenter markers (Table 18B) does not occur to a greater extent that that of DNA48227 This strongly suggests that DNA48227 is the gene responsible for the amplification of the particular region on chromosome 17 Because amplification of DNA47227 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth As a result, antagonist (e g , antibodies) directed against the protein encoded by DNA48227 would be expected to have utility in cancer therapy
PRQ882 The ΔCt value for DNA58125 (DNA882, UNQ448) in a variety of lung and colon tumors and cell lines is reported in Table 2 A ΔCt value of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA58125 occurred in primary lung tumors LTla LT3, LT6, LT9, LT10, LTI 1 , LT12, LT13, LT15. LT17, LT19, primary colon tumors CT2, CT3 CT8 CT10, CT12, CT14, CT15, CT16, CT1, CT4, CT5, CT1 1 , lung tumor cell lines H441 , and colon tumor cell lines SW620, Colo320, HT29, SKCOl , SW403, LSI47T, Colo205, HCT15, HCC2998 and KM 12 The ΔCt and average ΔCt values for the primary lung tumor hits are 1 18, 1 26, 1 10, 1 81 , I 95, 1 29, 1 07, 1 36, 1 58, 1 45, 1 87, the colon tumor hits report 2 18, 1 64, 1 10. 1 64. 1 05. 1 62. 1 25. 1 29. 1 17. 1 1. 2.03, 2 13. lung tumor cell lines
1 08, and colon tumor cell lines 1 61. 1 25, 1 54. 1 70. 2 00, 1 19. 1.66. 1.41. 1.19, 1 32 This represents an amplification in gene copy of the tollowing ( 1 ) Primary lung tumors. 2.27, 2 39, 2.14, 3 51. 3 86. 2 44, 2.10, 2.57, 2.99, 2 73 and 3 65 -fold increase, respectively, (2) Primary colon tumors: 4 53, 3 12. 2.14, 3 12. 2 07. 3.07, 2.38, 2.45. 2.25, 2.14, 4 08. 2 50 -fold increase, respectively; (3) lung tumor cell line-
2 1 1-fold increase; and (4) colon tumor cell line. 3 05. 2 38, 2.91 , 3.25, 4.0, 2.28, 3 16, 2.66, 2 28. 2.50- fold increase, respectively
Amplification has been confirmed by framework mapping for DNA58125 in primary lung tumors LT3, LT13, LT15, LT17, LT18, CTI . CT2, CT3, CT4, CT5, CT6, CT8, CT10, CT12, CT14, CT15, CTI 8 The reported ΔCt values for the primary lung tumors are 1.02, 1.33, 1.83, 1.03 and 1 08. while the pπmary colon tumors values are 1.08, 2.27, 1 34, 1.13, 2.17, 1.41 , 1.23, 1.74, 1.13, 1.74. 1.30 and 1.04. Relative to normal tissue, this represents approximately a 2.03, 2.51, 3.56, 2.04. 2.1 1 -fold increase, respectively, for the lung tumors, and a 2.1 1. 4.82, 2.53, 2.19, 4.50, 2.66, 2.34, 3.34, 2.19, 3 34, 2.46 and 2.06-fold increase, respectively, for the colon tumors. Epicenter mapping further confirmed significant amplification in primary colon tumors LTI 2,
LT13, LT15, LT16, LT17 and in primary colon tumors CTI, CT4, CT6, CT7, CT9, CTI 1, CT2, CT8, CT10 and CT16. The reported ΔCt values in the primary lung tumors are 1.02, 1.52, 2.04, 1.09 and 1.32, while the colon tumor values are 2.29, 1.49, 1.83, 1.20, 1.67, 1.02, 1.07, 2.27, 1.50 and 2.24 Relative to normal tissue, this represents approximately a 2.03, 2.87, 4.1 1 , 2.13, 2.50-fold increase, respectively, in gene copy for the lung tumors and a 4.89, 2 81 , 3.56, 2.30, 3.18, 2.03, 2.10, 4.82, 2.83, 4 72-fold increase, respectively, for the colon tumors.
In contrast, the amplification of the closest known framework markers (from Table 10) and epicenter markers (Table 10.2) does not occur to a greater extent that that of DNA58125. This strongly suggests that DNA58125 is the gene responsible for the amplification of the particular region on chromosome 16. Because amplification of DNA58125 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA58125 would be expected to have utility in cancer therapy.
EXAMPLE 3 In situ Hybridization
In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping In situ hybridization was 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, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37αC, and further processed for in situ hybridization as described by Lu and Gillett, supra. A ["-P] 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-Riboprohe synthesis
6 0 μl ( 125 mCi) of ' P-UTP ( Amersham BF 1002. SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried "P-UTP, the following ingredients were added- 2.0 μl 5x transcription buffer l .O μl DTT ( lOO mM)
2.0 μl NTP mix (2.5 mM : 10 μl, each of 10 mM GTP. CTP & ATP + 10 μl H:0) 1.0 μl UTP (50 μM) l .O μl Rnasin
1.0 μl DNA template ( lμg) 1.0 μl H2O
1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually) 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 minutes. 90 μl TE ( 10 mM Tπs pH 7.6/l mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon- 50 ultrafiltration unit, and spun using program 10 (6 minutes). 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 run 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 CC 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 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in - 70°C freezer one hour to overnight P-Hvhπdization
Pretreatment of frozen sections The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes. The trays were placed in 55 °C incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H20). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37 CC (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNase-lree 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 Pretreatment of paraffin-embedded sections The slides were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes 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; 37°C, 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.
Prehvbridtzation The slides were laid out in plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 μi of hybridization buffer (3.75g Dextran Sulfate + 6 ml SQ H-O), vortexed and heated in the microwave for 2 minutes with the cap loosened Alter cooling on ice, 18 75 ml formamide 3 75 ml 20 x SSC and 9 ml SQ H O were added the tissue was vortexed well, and incubated at 42°C for 1 -4 hours
Hybridization 1 0 x 10' cpm probe and 1 0 μl tRNA (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 vortexing, 50 μ I ''P mix were added to 50 μl prehybπdization on slide The slides were incubated overnight at 55 °C
Washes Washing was done 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0 25M EDTA V,=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 2x 10 minutes with 2 x SSC, EDTA at room temperature The stringency wash conditions were as follows 2 hours at 55 °C, 0 1 x SSC,
EDTA (20 ml 20 x SSC + 16 ml EDTA, VF4L)
DNA381 13 (Prolactin Rceptor Homlog)
DNA38113-p1 GGA TTC TAA TAC GAC TCA CTA TAG GGC CCC CCT GAG CTC TCC CGT
GTA DNA38113-p2 CTA TGA AAT TAA CCC TCA CTA AAG GGA AGG CTC GCC ACT GGT CGT
AGA
High expression was observed in developing mouse and human fetal lung (Fig 37), while normal adult lung, including bronchial epithelium was negative Expression was also seen in human fetal trachea, including with high probability, smooth muscle cells (Fig 38) Expression was also observed in non- trophoblastic cells in the human placenta (Fig 39) These data are consistent with a potential role in bronchial development
DNA44804 (ALS Homolog)
608 p1 GGA TTC TAA TAC GAC TCA CTA TAG GGC TGC CCG CAA CCC CTT CAA CTG
608 p2 CTA TGA AAT TAA CCC TCA CTA AAG GGA CCG CAG CTG GGT GAC CGT GTA
Low to moderate expression at sites of bone formation in fetal tissues and in the malignant cells of an osteosarcoma Expression was also observed at low level in the placenta and umbilical cord
DNA52722 (TNF Homolog) dna52722p1 GGA TTC TAA TAC GAC TCA CTA TAG GGC CGC CCC GCC ACC TCC T dna52722p2 CTA TGA AAT TAA CCC TCA CTA AAG GGA CTC GAG ACA CCA CCT GAC CCA
52722 p3 GGA TTC TAA TAC GAC TCA CTA TAG GGC CCA AGG AAG GCA GGA GAC
TCT 52722 p4 CTA TGA AAT TAA CCC TCA CTA AAG GGA CTA GGG GGT GGG AAT GAA
AAG High expression levels were observed in many tissues, including placenta, osteoblasts. injured renal tubules, injured liver colorectal liver matastasis and gall bladder. Tested sample had acetominophen induced liver injury and hepatic cirrhosis
DNA48227 (Reductase Homolog) 607.p 1 : GGA TTC TAA TAC GAC TCA CTA TAG GGC CCA ACA GCG GCA TCG GAA AGA 607.p2. CTA TGA AAT TAA CCC TCA CTA AAG GGA GGA GCA CCA GCC AAG CCA ATG
Elevated expression was observed in the mucosa of the chimp stomach.
EXAMPLE 4 Use of PRO201. PRQ292. PRQ327. PRQ1265. PRQ344. PRQ34-3, PRQ347. PRQ357. PRQ715, PRO1017. PROl 1 12. PRO509. PRQ853 or PRQ882 as a hybridization probe
The following method describes use of a nucleotide sequence encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO201 (Fig. I, SEQ ID NO: 1 ), PR0292 (Fig. 3, SEQ ID NO: 3), PR0327 (Fig. 5, SEQ ID NO: 5), PR01265 (Fig. 7, SEQ ID NO: 7), PR0344 (Fig. 9, SEQ ID NO: 9), PR0343 (Fig. 1 1 , SEQ ID NO: 1 1), PR0347 (Fig. 13, SEQ ID NO: 13), PR0357 (Fig. 15, SEQ ID NO. 15), PR0715 (Fig. 17, SEQ ID NO: 17), PRO 1017 (Fig. 19, SEQ ID NO: 19), PRO l 1 12 (Fig. 21 , SEQ ID NO: 21 ), PRO509 (Fig. 23, SEQ ID NO: 23), PR0853 (Fig. 25, SEQ ID NO: 25) or PR0882 (Fig. 27, SEQ ID NO: 27) is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in human tissue cDNA libraries or human tissue genomic libraries
Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882-deπved 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 0.1 x SSC and 0 l % SDS at 42"C. DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343. PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can then be identified using standard techniques known in the art.
EXAMPLE 5 Expression of PRQ201. PRQ292. PRQ327. PRQ1265. PRQ344, PRQ343. PRQ347. PRQ357. PRQ715. PRO I 017. PRO l 1 12. PRO509. PRQ853 or PRQ882 in E. coli This example illustrates preparation of an unglycosvlated form of PRO201 PR0292 PR0327. PRO 1265 PR0344 PR0343, PR0347. PR0357. PR0715, PRO1017. PROl 1 12, PRO509. PR0853 or PR0882 by recombinant expression in E coli
The DNA sequence encoding PRO201 PR0292. PR0327, PR01265. PR0344, PR0343. PR0347 PR0357. PR0715. PRO1017. PROl 1 12 PRO509, PR0853 or PR0882 (SEQ ID
NOs 1. 3, 5, 7, 9, 1 1 , 13. 15. 17, 19. 21. 23, 25, 27, respectively) is initially amplified using selected PCR primers The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector A variety of expression vectors may be employed An example of a suitable vector is pBR322 (derived from E colt, see Bolivar et al , Gene. 2 95 ( 1977)) which contains genes for ampicilhn and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated The PCR amplified sequences are then hgated into the vector The vector will preferably include sequences which encode for an antibiotic resistance gene, a tip promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO201, PR0292, PR0327. PRO 1265, PR0344, PR0343, PR0347, PR0357. PR0715. PRO1017. PROl 1 12, PRO509, PR0853 or PR0882 coding region, lambda transcriptional terminator, and an argU gene
The hgation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, duπng which the expression promoter is turned on
After culturing the cells for several more hours, the cells can be harvested by centrifugation The cell pellet obtained by the centrifugation can be solubihzed using various agents known in the art. and the solubihzed PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein
EXAMPLE 6
Expression of PRO201. PRQ292. PRQ327, PRO 1265, PRQ344 PRQ343 PRQ347, PRQ357, PRQ715. PRO1017. PROl 1 12. PRO509. PRQ853 or PRQ882 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PRO201 , PR0292, PR0327, PR01265, PR0344, PRO-343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA is hgated into pRK5 with selected restriction enzymes to allow insertion of the PRO20I , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA using hgation methods such as described in Sambrook et al . supra The resulting vector is called pRK5- PRO20 I pRK5-PR0292. pRK5-PR0327. pRK5-PR01265. pRK5-PR0344. pRK5-PR0343. pRK5- PR0347. pRK5-PR0357, pRK5-PR0715. pRK- 1017. pRK5-PR01 1 12., pRK5-PRO509. pRK5-PR0853. pRK5-PR0882 In one embodiment, the selected host cells may be 293 cells Human 293 cells (ATCC
CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 μg pRK5-PRO201. pRK5-PR0292. pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347. pRK5-PR0357, pRK5-PR0715, pRK- 1017, pRK5-PR01 1 12., pRK5-PRO509, pRK5-PR0853. pRK5- PR0882 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 Tπs-HCl, 0.1 mM EDTA, 0.227 M CaCl- 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 minutes at 25"C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37"C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μO/ml 3,S-cysteιne and 200 μCi/ml "S-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 dried and exposed to film for a selected period of time to reveal the presence of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 , or EBAF-2 polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays In an alternative technique. PRO201 , PR0292, PR0327, PR01265. PR0344, PR0343,
PR0347, PR0357. PR0715, PRO 1017, PRO l 1 12, PRO509, PR0853 or PR0882 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Nati. Acad. Sci., 12:7575 ( 1981 ). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO201 , pRK5-PR0292, pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5-PR0357, pRK5-PR0715, pRK- 1017, pRK5-PR01 1 12., pRK5-PRO509, pRK5- PR0853, pRK5-PR0882 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- mtroduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transfemn. After about four days, the conditioned media is centπfuged and filtered to remove cells and debris. The sample containing expressed PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343, PR0347, PR0357, PR0715, PROl 017, PRO l 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
In another embodiment. PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343, PR0347, PR0357, PR0715, PRO 1017. PRO 1 1 12, PRO509. PR0853 or PR0882 can be expressed in CHO cells The pRK5-PRO201 , pRK5-PR0292. pRK5-PR0327. pRK5-PR01265. pRK5-PR0344 pRK5-PR0343. PRK5-PR0347 pRK5-PR0357, pRK5-PR0715. pRK- 1017. pRK5-PR01 1 12 , pRK5- PRO509, pRK5-PR0853. pRK5-PR0882 vector can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as "S-methionine After determining the presence of PRO201. PR0292. PR0327. PRO 1265. PR0344, PR0343. PR0347, PR0357, PR0715, PRO1017. PRO l 1 12. PRO509, PR0853 or PR0882 polypeptide. the culture medium may be replaced with serum tree medium Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343. PR0347, PR0357. PR0715. PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 can then be concentrated and purified by any selected method.
Epitope-tagged PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017. PROl 1 12, PRO509, PR0853 or PR0882 may also be expressed in host CHO cells. The PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PRO.347. PR0357, PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector. The poly-His tagged PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged PRO201. PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017. PROl 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as by Nι2+-chelate affinity chromatography. PR0292 (UNQ266) and PRO509 (UNQ329) were expressed in CHO cells by a transient expression procedure. In addition, PRO1017 (UNQ500) and PRO509 (UNQ329) were stably expressed in CHO cells.
Stable expression in CHO cells was performed using the following procedure. The proteins were expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins were fused to an IgGI constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs were subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons ( 1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's The vector used expression in CHO cells is as described in Lucas et al , Nucl. Acids Res. 24- 9 (1774-1779 ( 1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen) DospeJ or Fugene J (Boehπnger Mannheim) The ceils were grown and described in Lucas et al , supra Approximately 3 x 107 cells are frozen in an ampule for further growth and production as described below
The ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing The contents were pipetted into a centrifuge tube containing 10 mLs of media and centπfuged at 1000 rpm for 5 minutes The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0 2 μm filtered PS20 with 5% 0 2 μm diafiltered fetal bovine serum) The cells were then aliquoted into a 100 L spinner containing 90 mL of selective media Alter 1-2 days, the cells were transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37 °C After another 2-3 days, a 250 mL, 500 mL and 2000 L spinners were seeded with 3 x 10s cells/mL The cell media was exchanged with fresh media by centrifugation and resuspension in production medium Although any suitable CHO media may be employed, a production medium described in US Patent No 5,122,469. issued June 16, 1992 was actually used 3L production spinner is seeded at 1 2 x 10' cells/mL On day 0, the cell number pH were determined On day 1 , the spinner was sampled and sparging with filtered air was commenced On day 2, the spinner was 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% polydimethyisiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) Throughout the production, pH was adjusted as necessary to keep at around 7 2 After 10 days, or until viability dropped below 70%, the cell culture was harvested by centπfugtion and filtering through a 0 22 μm filter The filtrate was either stored at 4°C or immediately loaded onto columns for purification
For the poly-His tagged constructs, the proteins were purified using a Ni NTA column (Qiagen) Before purification, lmidazole was added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4CC After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) constructs of were purified from the conditioned media as follows The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elulion with 100 mM citric acid, pH 3 5 The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tπs buffer, pH 9 The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity was assessed by SDS polyacrylamide gels and by N terminal amino acid sequencing bv Edman degradation PR0292 (UNQ266 PR0327 (UNQ288). PR0344 ( UNQ303). PR0347 (UNQ306). PR0357 (UNQ I 4), PR0853 ( UNQ419) were also produced by transient expression in COS cells
EXAMPLE 7 Expression of PRO201 PRQ292. PRQ327 PROl 265. PRQ344 PRQ343 PRQ347 PRQ357. PRQ715 PRO1017. PRO l 1 12. PRO509. PRQ853 or PRQ882 in Yeast The following method describes recombinant expression of PRO201. PR0292, PR0327, PR01265, PRO-344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in yeast First, yeast expression vectors are constructed for intracellular production or secretion of
PRO201 , PR0292, PR0327, PROI 265, PR0344, PRO-343, PR0347. PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 from the ADH2/GAPDH promoter DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO201 , PR0292, PR0327, PRO 1265, PRO-344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 For secretion, DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO.347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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 PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715. PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 Yeast cells, such as yeast strain AB 1 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% tπchloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
Recombinant PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO l 1 12, PRO509, PR0853 or PR0882 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 may further be purified using selected column chromatography resins.
- I l l - EXAMPLE 8
Expression of PRQ201. PRQ292. PRQ327. PROl 265. PRQ344 PRQ343 PRQ347. PRQ357. PRQ71 .
PRO1017 PROl 1 12. PRO509. PRQ853 or PRQ882 in Baculovirus-Infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected insect cells.
The sequence coding for PRO201. PR0292. PR0327, PR01265, PR0344. PR0343. PR0347, PR0357. PR0715, PRO10I 7. PROl 1 12, PRO509. PR0853 or PR0882 is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and lmmunogiobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 or the desired portion of the coding sequence of PRO201 , PR0292. PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 [such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular] is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 171 1 ) using hpofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28'C, the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors- A Laboratory Manual. Oxford: Oxford University Press (1994). Expressed poly-His tagged PRO201. PR0292, PR0327, PR01265, PR0344. PR0343,
PR0347, PR0357, PR0715. PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 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., Nature. 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9, 12.5 mM MgCl2; 0.1 mM EDTA, 10% glycerol, 0.1 % NP-40; 0.4 M KCI), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation. and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A 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 loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate: 300 mM NaCl. 10% glycerol, pH 6 0), which elutes nonspecifically bound protein After reaching A2S I baseline again, the column is developed with a 0 to 500 M Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with
Figure imgf000114_0001
to alkaline phosphatase (Qiagen) Fractions containing the eluted Hιslπ-tagged PRO201. PRO292 PR0327 PR01265 PR0344. PR0343. PR0347 PR0357. PR0715 PRO1017, PRO! 1 12. PRO509. PR0853 or PR0882. respectively are pooled and dialyzed against loading buffer
Alternatively, purification ot the IgG tagged (or Fc tagged) PRO201. PR0292. PR0327. PR01265, PR0344. PR0343. PR0347. PR0357. PR0715, PRO1017. PROl 1 12. PRO509. PR0853 or PR0882 can be performed using known chromatography techniques, including for instance. Protein A or protein G column chromatography
PR0327 (UNQ288). PR0344 (UNQ303). PRO509 (UNQ329) were expressed in baculovirus infected Sf9 insect cells While the expression was actually performed in a 0 5-2 L scale, it can be readily scaled up for larger (e g 8 L) preparations The proteins were expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region was fused to an IgGI constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms
Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb PH IgG for IgG fusions and pb PH.His.c for poly-His tagged proteins), and the vector and Baculogold® baculovirus DNA (Pharmingen) were co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 171 1 ), using Lipofectin (Gibco BRL) pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences The cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated for 5 days at 28°C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28°C. The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN) for histidme tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining
The first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown m ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were incubated for 3 days at 28°C. The supernatant was harvested and filtered Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed. The conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters For the poly-His tagged constructs, the protein construct were purified using a Ni-NTA column (Qiagen) Before purification, imidazole was added to the conditioned media to a concentration of 5 mM The conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 03 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4°C After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol. pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid. pH 3 5 The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 mL of 1 M Tπs butler, pH 9 The highly purified protein was subsequentlv desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N terminal amino acid sequencing by Edman degradation Additionally, PROl 265 (UNQ636), PR0344 (UNQ303), PR0882 (UNQ448) were expressed by a modified baculovirus procedure in hιgh-5 cells
The DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5 '-of) of an epitope tag contained with a baculovirus expression vector Such epitope tags include poly-his tags and lmmunogiobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl-1 (Novagen) The pIEl-1 and pIEl-2 vectors are designed for constitutive expression of recombinant proteins from the baculovirus lei promoter in stably-transformed insect cells (1) The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lel-mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIEl-1 and pIEl-2 include the te translation initiation site and can be used to produce fusion proteins Briefly, the desired sequence or the desired portion of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5' and 3' regions The 5' primer may incorporate flanking (selected) restriction enzyme sites The product was then digested with those selected restriction enzymes and subcloned into the expression vector For example, derivatives of pIEl-1 can include the Fc region of human IgG (pb PH IgG) or an 8 histidine (pb PH His) tag downstream (3'-of) the desired sequence Preferably, the vector construct is sequenced for confirmation
Hι5 cells are grown to a confluency of 50% under the conditions of, 27°C, no CO:, NO pen/strep For each 150 mm plate, 30 ug of pIE based vector containing the sequence was mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Bιoscιences # 14401 -78P (note this media is light sensitive)), and in a separate tube, 100 ul of CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium The two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2ml of DNA/CellFECTIN mix and this is layered on Hι5 cells that have been washed once with Ex-Cell media The plate is then incubated in darkness for 1 hour at room temperature The DNA CellFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remove excess CellFECTIN 30 ml of fresh Ex-Cell media was added and the cells are incubated for 3 days at 28C The supernatant was harvested and the expression of the sequence in the baculovirus expression vector was determined by batch binding of 1 ml of supernatent to 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining The conditioned media from the transfected cells (0 5 to 3 L) was harvested bv centrifugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein comprising the sequence is purified using a Ni-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes. pH 7 4 buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 48C After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was then subsequently desalted into a storage buffer containing 10 M Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column wais washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 mL of 1 M Tπs buffer, pH 9 The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity of the sequence was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary
EXAMPLE 9
Preparation of Antibodies that Bind PRO201 , PRQ292. PRQ327, PRO 1265. PRQ344. PRQ343 PRQ347. PRQ357. PRQ715. PRO1017. PROl 1 12. PRO509. PRQ853 or PRQ882 This example illustrates preparation of monoclonal antibodies which can specifically bind PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347. PR0357. PR0715. PRO1017, PRO 1 1 12. PRO509, PR0853 or PR0882
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra Immunogens that may be employed include purified PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, fusion proteins containing PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, and cells expressing recombinant PRO201 , PR0292, PR0327, PR01265, PR0344, PRO-343, PR0347, PR0357, PR0715, PROI017, PROl 112, PRO509, PR0853 or PR0882 on the cell surface Selection of the immunogen can be made by the skilled artisan without undue experimentation
Mice, such as Balb/c, are immunized with the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or lntrapeπtoneally 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 hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected ad|uvant Thereafter, tor several weeks, the mice mav also be boosted with additional immunization injections Serum samples mav be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect ami- PRO201. PR0292. PR0327. PRO 1265. PR0344. PR0343, PR0347. PR0357, PR0715, PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 antibodies After a suitable antibody titer has been detected, the animals ' positive" for antibodies can be injected with a final intravenous inaction of PRO201 , PR0292. PR0327 PRO 1265, PR0344, PR0343, PR0347, PR0357. PR0715. PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 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 munne myeloma cell line 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 Determination of "positive" hybndoma cells secreting the desired monoclonal antibodies against PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 is within the skill in the art
The positive hybndoma cells can be injected lntrapeπtoneally into syngeneic Balb/c mice to produce ascites containing the anti- PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 monoclonal antibodies. Alternatively, the hybπdoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed * * * *
Deposit of Material
The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd . Manassas, VA 20110-2209, USA (ATCC)
Material ATCC Pep No Deposit Date DNA30676-1223 209567 12/23/97 DNA381 13-1230 209530 12/10/97 DNA60764-1533 203452 1 1/10/98 DNA40592-1242 209492 1 1/21/97 DNA43318-1217 209482 1 1/21/97 DNA44176- 1244 209532 12/10/97 DNA44804-1248 209527 12/10/97 DNA52722-1229 209570 1/7/98 DNA561 12-1379 209883 5/20/98 DNA57702-1476 209951 6/9/98 DNA48227-1350 209812 4/28/98
16 These deposits were made under the prov isions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treat) ) 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 Inc 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 according to 35 USC § 122 and the Commissioners rules pursuant thereto (including 37 CFR §1 14 with particular reference to 886 OG 638) The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims

Claims

Claims
1 An isolated antibody binding a PRO201 PR0292 PRO-327 PROl 265. PR0344. PR0343, PR0347. PR0357 PR0715 PRO1017 PRO l 1 12, PRO509, PR0853 or PR0882 polypeptide
2 The antibody of claim 1 which induces death of a cell overexpressing a PRO201 , PR0292, PR0327, PRO 1265, PR0344 PR0343, PR0347, PR0357 PR0715, PRO1017. PROl 1 12,
PRO509, PR0853 or PR0882 polypeptide
3 The antibody of claim 2 wherein said cell is a cancer cell
4 The antibody of claim 1 which is a monoclonal antibody
5 The antibody of claim 4. which has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues
6 The antibody of claim 5 which is labeled
7 The antibody of claim 6 which is immobilized on a solid support
8 The antibody of claim 1 which is an antibody fragment, a single-chain antibody, or an anti- ldiotypic antibody 9 A composition comprising an antibody of claim 1 in admixture with a pharmaceutically acceptable carrier
10 The composition of claim 9 comprising growth inhibitory amount of said antibody
11 The composition of claim 10 further comprising a second antibody or a cytotoxic or chemotherapeutic agent 12 A method for determining the presence of a PRO201 , PR0292, PR0327, PRO 1265, PRO.344,
PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide comprising exposing a cell suspected of containing the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 112, PRO509, PR0853 or PR0882 polypeptide to an ant╬╣-PRO201 , ant╬╣-PR0292, ant╬╣-PR0327, anti-PRO 1265, ant╬╣-PR0344, ant╬╣-PR0343, ant╬╣-PR0347, ant╬╣-PR0357, ant╬╣-PR0715, anti-PRO 1017, anti-
PRO 1 1 12, ant╬╣-PRO509 ant╬╣-PR0853 or ant╬╣-PR0882 antibody and determining binding of said antibody to said cell
13 A method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide (a) in 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 expression level in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained
14 A method of diagnosing tumor in a mammal, comprising (a) contacting an PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715,
PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 antibody with a test sample of tissue cells obtained from the mammal . and (b) detecting the formation of a complex between the anti- PRO201 , ant╬╣-PR0292, ant╬╣-PR0327. anti-PROl 265, ant╬╣-PR0344, ant╬╣-PR0343, ant╬╣-PR0347, ant╬╣-PR0357, ant╬╣-PR0715, ant╬╣-PRO1017, anti-PROl 1 12, ant╬╣-PRO509, ant╬╣-PR0853 or anti- PR0882 antibody and the PRO201 PR0292. PR0327, PRO 1265 PR0344 PR0343, PR0347, PR0357 PR071 PRO l 017. PRO l 1 12. PRO509. PR08 or PR0882 polvpeptide in the test sample 15 The method of claim 14 wherein said lest sample is obtained from an individual suspected to have neoplastic cell growth or proliferation 16 A cancer diagnostic kit, comprising an ant╬╣-PRO201. ant╬╣-PR0292, ant╬╣-PR0327. anti-PRO 1265. ant╬╣-PR0344. ant╬╣-PR0343, ant╬╣-PR0347. ant╬╣-PR0357. ant╬╣-PR0715. ant╬╣-PRO I 017. anti- PROl 1 12, ant╬╣-PRO509, ant╬╣-PR0853 or ant╬╣-PR0882 antibody and a earner in suitable packaging
17 The kit of claim 16 further comprising instructions for using said antibody to detect the PRO 187, PR0533, PR0214, PRO240, PR021 1 , PRO230, PR0261 , PR0246. or EB AF-2 polypeptide
18 A method for inhibiting the growth of tumor cells comprising exposing a cell which overexpresses a PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide to an effective amount of an agent inhibiting the expression and/or activity ot the PRO201 , PR0292, PR0327,
PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide.
19. The method of claim 18 wherein said agent is an ant╬╣-PRO201 , ant╬╣-PR0292, ant╬╣-PRO.327, anti-
PR01265, ant╬╣-PR0344, ant╬╣-PR0343, ant╬╣-PR0347, ant╬╣-PRO-357, ant╬╣-PR0715, anti- PRO 1017, anti-PRO 1 1 12, ant╬╣-PRO509, ant╬╣-PR0853 or ant╬╣-PR0882 antibody
20 The method of claim 19 wherein said tumor cells are further exposed to radiation treatment or a cytotoxic or chemotherapeutic agent.
21. An article of manufacture, comprising: a container, a label on the container; and a composition comprising an active agent contained within the container; wherein the composition is effective for inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, and the active agent in the composition is an agent inhibiting the expression and/or activity of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PRO.347, PRO-357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide
22. The article of manufacture of claim 21 wherein said active agent is an ant╬╣-PRO201 , anti- PR0292, ant╬╣-PR0327, anti-PRO 1265, ant╬╣-PRO.344, ant╬╣-PR0343, ant╬╣-PR0347, ant╬╣-PR0357, ant╬╣-PR0715, ant╬╣-PRO1017, anti-PROl 1 12. ant╬╣-PRO509, ant╬╣-PR0853 or ant╬╣-PR0882 antibody 23 A method for identifying a compound capable of inhibiting the expression or activity of a
PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347. PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide, comprising contacting a candidate compound with a PRO201. PR0292. PR0327. PRO 1265, PR0344. PR0343. PR0347. PR0357. PR0715 PRO1017. PROl 1 12. PRO509, PR0853 or PR0882 polypeptide under conditions and for a time sufficient to allow these two components to interact The method of claim 23 wherein said candidate compound or said PRO201. PR0292, PR0327. PR01265, PR0344, PR0343, PR0347, PR0357. PR0715, PRO1017, PROl 1 12. PRO509, PR0853 or PR0882 polypeptide is immobilized on a solid support. The method of claim 24 wherein the non-immobilized component carries a detectable label.
PCT/US1999/000106 1996-11-06 1999-01-05 Compositions and methods for the treatment of tumor WO1999035170A2 (en)

Priority Applications (343)

Application Number Priority Date Filing Date Title
AU22122/99A AU2212299A (en) 1998-01-05 1999-01-05 Compositions and methods for the treatment of tumor
US09/918,585 US20030060406A1 (en) 1997-10-17 2001-07-30 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US09/978,188 US20030139328A1 (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
US09/978,189 US6972325B2 (en) 1997-10-17 2001-10-15 PRO273 polypeptides
US09/978,298 US20030134785A1 (en) 1997-10-17 2001-10-15 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
US09/978,194 US20030195333A1 (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
US09/978,191 US20030050239A1 (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
US09/981,915 US20030054986A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,423 US20030069178A1 (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 US20050124789A9 (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
US09/978,681 US20030195148A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/978,802 US20030199674A1 (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
US09/978,375 US7196165B2 (en) 1997-10-17 2001-10-16 PRO363 polypeptides
US09/978,564 US7195760B2 (en) 1997-10-17 2001-10-16 Anti-pro363 antibodies
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
US09/978,585 US20030049633A1 (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
US09/978,697 US20020169284A1 (en) 1997-10-17 2001-10-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/981,915 US7285623B2 (en) 1997-10-17 2001-10-16 PRO337 polypeptides
US09/978,824 US20030055216A1 (en) 1997-10-17 2001-10-17 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
US10/210,028 US20030203446A1 (en) 1998-10-07 2001-10-18 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/165,036 US20050227342A1 (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
US10/165,247 US7112657B2 (en) 1998-10-07 2001-10-19 PRO697 polypeptides
US10/164,749 US20040029218A1 (en) 1998-10-07 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/166,709 US20030104536A1 (en) 1998-10-07 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
US10/162,521 US7067628B2 (en) 1998-03-17 2001-10-19 PRO788 polypeptides
US10/145,017 US20030186365A1 (en) 1998-03-26 2001-10-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/160,502 US7220835B2 (en) 1998-07-30 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
US10/143,029 US7105640B2 (en) 1997-10-17 2001-10-19 Anti-pro792 antibodies
US10/165,067 US7279553B2 (en) 1998-05-13 2001-10-19 PRO1083 polypeptides
US10/145,124 US20030190701A1 (en) 1998-04-30 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
US10/013,922 US20030195345A1 (en) 1997-10-17 2001-10-21 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
US10/017,081 US20030049684A1 (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
US10/020,445 US20030198994A1 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,834 US20030064407A1 (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
US10/002,967 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
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
US10/017,085 US6974696B2 (en) 1997-10-17 2001-10-24 PRO853 nucleic acids
US09/999,833 US6916648B2 (en) 1997-10-17 2001-10-24 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/999,832 US7132283B2 (en) 1997-10-17 2001-10-24 PRO273 polypeptides
US10/013,925 US7037710B2 (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
US10/013,923 US7169912B2 (en) 1997-10-17 2001-10-25 PRO1017 nucleic acids
US10/016,177 US20030073131A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,929 US7019124B2 (en) 1997-10-17 2001-10-25 PRO788 nucleic acids
US10/013,921 US20030068648A1 (en) 1997-10-17 2001-10-25 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/013,928 US20030215905A1 (en) 1998-10-07 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
US10/013,917 US7029874B2 (en) 1998-03-17 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
US10/013,927 US7189529B2 (en) 1997-10-17 2001-10-25 PRO792 nucleic acids
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
US09/991,163 US20020132253A1 (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
US09/991,854 US20030059780A1 (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
US09/990,442 US20020132252A1 (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
US09/990,443 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
US09/993,667 US20030022187A1 (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
US09/993,469 US20030068623A1 (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
US09/990,726 US20030054359A1 (en) 1997-06-16 2001-11-14 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/990,562 US20030027985A1 (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
US09/991,073 US20020127576A1 (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
US09/990,456 US20020137890A1 (en) 1997-03-31 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
US09/997,529 US7309761B2 (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
US09/997,601 US7189814B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,529 US20030134284A1 (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
US09/997,666 US20030027163A1 (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
US09/997,628 US20030059782A1 (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
US09/998,156 US20030044806A1 (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
US09/997,641 US20030224358A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,428 US20030027162A1 (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
US09/997,349 US7034106B2 (en) 1997-06-16 2001-11-15 Pro1159 polypeptides
US09/997,641 US7112656B2 (en) 1997-06-16 2001-11-15 PRO1312 polypeptides
US09/997,384 US7119177B2 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,601 US20030054404A1 (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
US09/997,585 US7166282B2 (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
US09/998,041 US7309775B2 (en) 1997-06-16 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
US09/997,384 US20030087305A1 (en) 1997-06-16 2001-11-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/997,542 US20030068647A1 (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
US09/997,614 US20030124531A1 (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
US09/991,157 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
US09/991,181 US6913919B2 (en) 1997-06-16 2001-11-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/991,150 US20030194760A1 (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
US09/990,441 US7041804B2 (en) 1997-06-16 2001-11-16 Antibodies to PRO1387 polypeptides
US09/989,729 US20030059831A1 (en) 1997-06-16 2001-11-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US09/989,722 US20020072067A1 (en) 1997-06-16 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
US09/989,734 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
US09/989,735 US6972185B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO844 polypeptides
US09/989,732 US7037679B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO1184 polypeptides
US09/989,726 US7018811B2 (en) 1997-06-16 2001-11-19 Nucleic acids encoding PRO189 polypeptides
US09/989,723 US20020072092A1 (en) 1997-06-16 2001-11-19 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
US09/989,727 US20020072497A1 (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
US09/989,293 US7034136B2 (en) 1997-06-16 2001-11-20 Nucleic acids encoding PRO1159 polypeptides
US09/989,725 US20030139329A1 (en) 1997-06-16 2001-11-20 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US09/989,730 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
US10/007,194 US7041805B2 (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
US10/006,130 US7098312B2 (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
US10/006,116 US20030082626A1 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,856 US7538086B2 (en) 1998-09-01 2001-12-06 PRO1303 polypeptides
US10/007,236 US7034123B2 (en) 1998-09-01 2001-12-06 Anti-PRO1347 antibodies
US10/006,041 US6951921B2 (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
US10/006,117 US7071304B2 (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
US10/006,485 US7026448B2 (en) 1998-09-01 2001-12-06 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/006,172 US7081514B2 (en) 1998-09-01 2001-12-06 PRO1347 polypeptides
US10/012,754 US7375184B2 (en) 1998-09-01 2001-12-07 PRO1382 polypeptides
US10/012,753 US7488796B2 (en) 1998-09-01 2001-12-07 PRO1269 polypeptides
US10/012,064 US6953841B2 (en) 1999-01-05 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
US10/012,752 US7026455B2 (en) 1998-09-01 2001-12-07 Anti-pro 1343 antibodies
US10/012,137 US20030187189A1 (en) 1998-09-01 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
US10/012,754 US20030187191A1 (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
US10/012,101 US20030187239A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/011,671 US20030096954A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,149 US7038019B2 (en) 1998-09-01 2001-12-07 Antibodies to PRO1410
US10/012,121 US7022817B2 (en) 1998-09-01 2001-12-07 PRO1325 polypeptides
US10/011,833 US6951920B2 (en) 1998-09-01 2001-12-07 PRO1340 polypeptides
US10/011,692 US20030109672A1 (en) 1998-09-01 2001-12-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/012,755 US20030096955A1 (en) 1998-09-01 2001-12-07 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
US10/013,911 US20030187193A1 (en) 1998-09-01 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
US10/013,910 US7057018B2 (en) 1999-01-05 2001-12-10 Pro 1474 polypeptides
US10/013,915 US20030204053A1 (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
US10/013,906 US20030191282A1 (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
US10/013,909 US20030186318A1 (en) 1999-01-05 2001-12-10 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
US10/015,389 US6936436B2 (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
US10/015,671 US6946263B2 (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
US10/015,480 US7074912B2 (en) 1998-09-01 2001-12-11 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
US10/015,387 US20030135034A1 (en) 1998-09-01 2001-12-12 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/015,386 US7022498B2 (en) 1998-09-01 2001-12-12 Pro 1325 nucleic acids
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
US10/015,390 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
US10/017,867 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
US10/017,407 US20030125535A1 (en) 1998-09-01 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
US10/020,063 US20030119097A1 (en) 1999-01-05 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
US10/028,072 US20030004311A1 (en) 1997-06-18 2001-12-19 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
US10/062,923 US20030191283A1 (en) 1998-04-23 2002-01-31 Nsp molecules
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
US10/121,047 US20030077778A1 (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
US10/121,059 US20030190721A1 (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
US10/121,044 US20030190717A1 (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
US10/121,045 US20030073210A1 (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
US10/121,040 US20030082759A1 (en) 1997-03-31 2002-04-11 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
US10/121,056 US20030082760A1 (en) 1997-03-31 2002-04-12 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
US10/121,043 US7220831B2 (en) 1997-03-31 2002-04-12 PRO235 polypeptides
US10/121,058 US20030190720A1 (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
US10/121,054 US20030199054A1 (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
US10/121,049 US20030022239A1 (en) 1997-06-18 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
US10/121,050 US20030054516A1 (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
US10/121,061 US20030082761A1 (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
US10/121,060 US20030190722A1 (en) 1997-03-31 2002-04-12 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
US10/123,213 US20030199057A1 (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
US10/123,771 US20030199060A1 (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
US10/123,292 US20030073211A1 (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
US10/123,261 US20030068796A1 (en) 1997-03-31 2002-04-15 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
US10/123,109 US20030190723A1 (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
US10/123,215 US7291329B2 (en) 1997-03-31 2002-04-15 Antibodies against PRO4406
US10/123,108 US7635478B2 (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
US10/123,213 US7193048B2 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,212 US7276577B2 (en) 1997-03-31 2002-04-15 PRO1866 polypeptides
US10/123,262 US20030049816A1 (en) 1997-03-31 2002-04-15 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,155 US20030068794A1 (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
US10/123,908 US7335728B2 (en) 1997-03-31 2002-04-16 PRO1310 polypeptides
US10/123,909 US7193049B2 (en) 1997-03-31 2002-04-16 PRO862 polypeptides
US10/123,907 US7084258B2 (en) 1997-03-31 2002-04-16 Antibodies against the PRO862 polypeptides
US10/123,904 US20030022328A1 (en) 1997-03-31 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US20030087344A1 (en) 1997-06-18 2002-04-16 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/123,905 US7285625B2 (en) 1997-06-18 2002-04-16 PRO536 polypeptides
US10/123,913 US20030203462A1 (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
US10/123,906 US20030190726A1 (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
US10/123,912 US20030100087A1 (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
US10/123,911 US7408032B2 (en) 1997-03-31 2002-04-16 PRO1188 polypeptides
US10/124,823 US20030199062A1 (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
US10/124,820 US20030190729A1 (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
US10/124,816 US20030190728A1 (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
US10/124,814 US7105335B2 (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
US10/125,805 US20030194794A1 (en) 1997-03-31 2002-04-17 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
US10/124,821 US20030199023A1 (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
US10/124,818 US20030082763A1 (en) 1997-03-31 2002-04-17 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/124,819 US7285626B2 (en) 1997-03-31 2002-04-17 PRO1076 polypeptides
US10/125,927 US20030190731A1 (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
US10/125,931 US20030199063A1 (en) 1997-03-31 2002-04-19 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/125,932 US7317079B2 (en) 1997-03-31 2002-04-19 PRO812 polypeptides
US10/125,922 US7309762B2 (en) 1997-03-31 2002-04-19 PRO1360 polypeptides
US10/127,831 US20030082689A1 (en) 1997-03-31 2002-04-22 Secreted and transmembrane polypeptides and nucleic acids encoding the same
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
US10/131,825 US7282566B2 (en) 1997-03-31 2002-04-24 PRO1779 polypeptide
US10/131,823 US7304132B2 (en) 1997-03-31 2002-04-24 PRO1693 polypeptides
US10/137,867 US20030207349A1 (en) 1997-03-31 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
US10/137,868 US20030082764A1 (en) 1997-03-31 2002-05-03 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,020 US20030207415A1 (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
US10/140,023 US20030207416A1 (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
US10/140,024 US20040058424A1 (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
US10/139,980 US7247710B2 (en) 1997-03-31 2002-05-06 PRO4395 antibodies
US10/140,809 US20030207418A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/140,865 US20030207420A1 (en) 1997-03-31 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
US10/140,921 US7317080B2 (en) 1997-03-31 2002-05-07 PRO4303 polypeptides
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
US10/140,860 US7307151B2 (en) 1997-03-31 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
US10/140,925 US20030073215A1 (en) 1997-03-31 2002-05-07 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/141,754 US7361732B2 (en) 1997-03-31 2002-05-08 PRO4400 polypeptides
US10/141,755 US7297764B2 (en) 1997-03-31 2002-05-08 PRO4318 polypeptides
US10/141,760 US7342104B2 (en) 1997-03-31 2002-05-08 Antibodies against the PRO4320 polypeptide
US10/141,701 US20030207421A1 (en) 1997-03-31 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
US10/143,113 US7329730B2 (en) 1997-03-31 2002-05-09 PRO4348 polypeptides
US10/143,114 US20030036180A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,425 US20030207424A1 (en) 1997-03-31 2002-05-09 Secreted and transmembrane polypeptides and nucleic acids encoding the same
US10/142,430 US7309766B2 (en) 1997-03-31 2002-05-09 PRO5774 polypeptides
US10/142,417 US7304133B2 (en) 1997-03-31 2002-05-09 PRO4389 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
US10/143,032 US7408033B2 (en) 1997-03-31 2002-05-10 PRO5995 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
US10/147,492 US20030082765A1 (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
US10/147,519 US20030077791A1 (en) 1997-03-31 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
US10/158,782 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
US10/160,498 US20030073216A1 (en) 1997-03-31 2002-05-30 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
US10/213,199 US7381809B2 (en) 1998-09-09 2002-08-05 Compositions and methods for the treatment of immune related diseases
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
US11/341,175 US7468427B2 (en) 1997-03-31 2006-01-27 Antibodies to PRO1275 polypeptide
US11/796,725 US20090197301A1 (en) 1998-09-01 2007-04-27 Secreted and transmembrane polypeptides and nucleic acids encoding the same

Applications Claiming Priority (12)

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US7044098P 1998-01-05 1998-01-05
US60/070,440 1998-01-05
US8350098P 1998-04-29 1998-04-29
US60/083,500 1998-04-29
US8641498P 1998-05-22 1998-05-22
US60/086,414 1998-05-22
US8874298P 1998-06-10 1998-06-10
US60/088,742 1998-06-10
US10778398P 1998-11-10 1998-11-10
US60/107,783 1998-11-10
US10930498P 1998-11-20 1998-11-20
US60/109,304 1998-11-20

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US28429199A A-371-Of-International 1996-11-06 1999-04-12
PCT/US1999/020111 Continuation-In-Part WO2000012708A2 (en) 1997-03-31 1999-09-01 Further pro polypeptides and sequences thereof
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

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