EP1185642A2 - Compositions and methods for the treatment of tumor - Google Patents

Compositions and methods for the treatment of tumor

Info

Publication number
EP1185642A2
EP1185642A2 EP00930756A EP00930756A EP1185642A2 EP 1185642 A2 EP1185642 A2 EP 1185642A2 EP 00930756 A EP00930756 A EP 00930756A EP 00930756 A EP00930756 A EP 00930756A EP 1185642 A2 EP1185642 A2 EP 1185642A2
Authority
EP
European Patent Office
Prior art keywords
seq
antι
polypeptide
antibody
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00930756A
Other languages
German (de)
French (fr)
Inventor
David Botstein
Audrey Goddard
Austin L. Gurney
Victoria Smith
Colin K. Watanabe
William I. Wood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to EP06001196A priority Critical patent/EP1683811B1/en
Priority to ES06001196T priority patent/ES2331657T3/en
Priority to EP05024729A priority patent/EP1657256B1/en
Publication of EP1185642A2 publication Critical patent/EP1185642A2/en
Withdrawn legal-status Critical Current

Links

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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • Malignant tumors 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 an 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 variety of forms, characterized by different degrees of invasiveness and aggressiveness Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which 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 would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth
  • tumor suppression genes which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes,
  • a well known mechanism ot 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, ; e is proportionate to the number of copies made
  • Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of va ⁇ ous human malignancies, including breast cancer
  • erbB2 also known as her2. or c-erbB-2
  • c-erbB-2 which encodes a 185-kd transmembrane glycoprotein receptor (p 185 HER2 , HER2) related to the epidermal growth factor receptor EGFR)
  • 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 contribute to tumo ⁇ genesis 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 mav act as predictors of the prognosis of tumor treatment
  • the present invention concerns an isolated antibody which binds to a polypeptide designated herein as a PRO polypeptide
  • the isolated antibody specifically binds to a PRO polypeptide
  • the antibody induces the death of a cell which expresses a PRO polypeptide
  • the cell that expresses the PRO polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type
  • the antibody is a monoclonal antibody, which preferably has non-human complementa ⁇ tv determining region (CDR) residues and human framework region (FR) residues
  • CDR non-human complementa ⁇ tv determining region
  • 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 a humanized antibody which binds, preferably specifically, to a PRO polypeptide
  • the invention concerns a composition ot matter which comprises an antibody which binds, preferably specifically, to
  • the invention concerns isolated nucleic acid molecules which encode anti-PRO antibodies and vectors and recombinant host cells comprising such nucleic acid molecules
  • the invention concerns a method tor producing an anti-PRO antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes the antibody under conditions sufficient to allow expression of the antibody, and recovering the antibody from the cell culture
  • the invention further concerns antagonists of a PRO polypeptide that inhibit one or more of the biological and/or immunological functions or activities of a PRO polypeptide
  • the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PRO polypeptide or the complement thereof
  • the isolated nucleic acid molecule is preferably DNA, and hybridization preferably occurs under stringent hybridization and wash 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 transcription and/or translation of the respective amplified genes, or as antisense primers in amplification reactions
  • sequences can be used as part of a ribozyme and/or a triple helix sequence which, in turn, may be used in regulation of the amplified genes
  • the invention provides a method for determining the presence of a PRO polypeptide in a sample suspected of containing a PRO polypeptide, wherein the method comprises exposing the sample to an anti-PRO antibody and determining binding of the antibody to a PRO polypeptide in the sample.
  • the invention provides a method for determining the presence of a PRO polypeptide in a cell, wherein the method comprises exposing the cell to an anti-PRO antibody and determining binding of the antibody to the cell
  • the present invention concerns a method of diagnosing tumor in a mammal, comprising detecting the level of expression of a gene encoding a PRO 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 as compared to the control sample, is indicative of 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 anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-PRO antibody and a PRO polypeptide in the test sample, wherein the formation of a complex is indicative of the presence ot a tumor in said mammal
  • the detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type A larger quantity of complexes formed in the test sample indicates the presence 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, tor 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-PRO antibody and a carrier (e g , a buffer) in suitable packaging
  • a carrier e g , a buffer
  • the kit preferably contains instructions for using the antibody to detect the presence ot a PRO polypeptide in a sample suspected of containing the same
  • the invention concerns a method for inhibiting the growth of tumor cells comprising exposing tumor eel Is which express a PRO polypeptide to an effective amount of an agent which inhibits a biological and or immunological activity and/or the expression of a PRO polypeptide, wherein growth of the tumor cells is thereby inhibited
  • the agent preferably is an anti-PRO antibody, a small organic and inorganic molecule, peptide, phosphopeptide. antisense or ribozyme molecule, or a triple helix molecule
  • the agent, e g , the anti-PRO antibody induces cell death
  • the tumor cells are further exposed to radiation treatment and or a cytotoxic or chemotherapeutic agent
  • the invention concerns an article of manufacture, comprising a container, a label on the container, and a composition composing an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO polypeptide as compared to a normal cell of the same tissue type
  • the active agent in the composition is an agent which inhibits an activity and/or the expression of a PRO polypeptide
  • the active agent is an anti-PRO antibody or an antisense oligonucleotide
  • the invention also provides a method for identifying a compound that inhibits an activity of a PRO polypeptide, compnsmg contacting a candidate compound with a PRO polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether a biological and/or immunological activity of the PRO polypeptide is inhibited
  • either the candidate compound or the PRO polypeptide is immobilized on
  • the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that express the polypeptide, wherein the method comprises contacting the cells with a candidate compound and determining whether the expression of the PRO polypeptide is inhibited
  • this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable for allowing expression ot the PRO polypeptide and (b) determining the inhibition of expression ot said polypeptide
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide
  • the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucle
  • the isolated nucleic acid molecule comp ⁇ ses a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 9
  • the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92%
  • Another aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane doma ⁇ n(s) ot such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein desc ⁇ bed PRO polypeptides are contemplated
  • nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 1 10 nucleotides in length, altemativelv at least about 120 nucleotides in
  • the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% am o acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, altemativelv at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity
  • the invention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives,retemativelv at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO polypeptide hav ing a full-length amino acid sequence as disclosed here
  • the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an ammo acid sequence as hereinbefore described Processes for producing the same are also herein described, wherein those processes comp ⁇ se culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression ot the PRO polypeptide and recovering the PRO polypeptide from the cell culture
  • Another aspect of the present invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
  • the invention concerns antagonists of a native PRO polypeptide as defined herein
  • the antagonist is an anti-PRO antibody or a small molecule
  • the invention concerns a method of identifying antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide
  • the PRO polypeptide is a native PRO polypeptide
  • the invention concerns a composition of matter comprising a PRO polypeptide. or an antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier
  • the carrier is a pharmaceutically acceptable carrier
  • Another embodiment of the present invention is directed to the use of a PRO polypeptide. or an antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide. an antagonist thereof or an anti-PRO antibody
  • the invention provides ⁇ ectors comprising DNA encoding any ot the herein described polypeptides
  • Host cells comprising any such vector are also provided
  • the host cells may be CHO cells.
  • E coli, yeast, or Baculovirus-intected insect cells A process for producing anv ot the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture
  • the invention provides chimeric molecules comprising any of the herein described polypeptides tused to a heterologous polypeptide or amino acid sequence Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin
  • the invention provides an antibody which specifically binds to any of the above or below described polypeptides
  • the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
  • Figure 1 shows the nucleotide sequence (SEQ ID NO 1 ) ot a cDNA containing a nucleotide sequence encoding native sequence PRO5800, wherein the nucleotide sequence (SEQ ID NO 1 ) is a clone designated herein as DNA 108912-2680 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 2 shows the amino acid sequence (SEQ ID NO 2) of a native sequence PRO5800 polypeptide as derived from the coding sequence of SEQ ID NO 1 shown in Figure 1
  • Figure 3 shows the nucleotide sequence (SEQ ID NO 3) of a cDNA containing a nucleotide sequence encoding native sequence PRO6000, wherein the nucleotide sequence (SEQ ID NO 3) is a clone designated herein as DNA102880-2689 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 4 shows the amino acid sequence (SEQ ID NO 4) of a native sequence PRO6000 polypeptide as derived from the coding sequence of SEQ ID NO 3 shown in Figure 3
  • Figure 5 shows the nucleotide sequence (SEQ ID NO 5) of a cDNA containing a nucleotide sequence encoding native sequence PRO6016 wherein the nucleotide sequence (SEQ ID NO 5) is a clone designated herein as DNA96881 -2699
  • Also presented in bold font and underlined are the positions of the respecm e sta ⁇ and stop codons
  • Figure 6 shows the amino acid sequence (SEQ ID NO 6) of a native sequence PRO6016 polypeptide as derived from the coding sequence of SEQ ID NO 5 shown in Figure 5
  • Figure 7 shows the nucleotide sequence (SEQ ID NO 7) of a cDNA containing a nucleotide sequence encoding native sequence PRO6018, wherein the nucleotide sequence (SEQ ID NO 7) is a clone designated herein as DNA98565-2701 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 8 shows the ammo acid sequence (SEQ ID NO 8) of a native sequence PRO6018 polypeptide as derived from the coding sequence of SEQ ID NO 7 shown in Figure 7
  • Figure 9 shows the nucleotide sequence (SEQ ID NO 9) of a cDNA containing a nucleotide sequence encoding native sequence PR06496 wherein the nucleotide sequence (SEQ ID NO 9) is a clone designated herein as DNA 1 19302-2737 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 10 shows the amino acid sequence (SEQ ID NO 10) of a native sequence PR06496 polypeptide as derived from the coding sequence of SEQ ID NO 9 shown in Figure 9
  • Figure 11 shows the nucleotide sequence (SEQ ID NO 1 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PR07154, wherein the nucleotide sequence (SEQ ID NO 1 1 ) is a clone designated herein as DNA 108760-2740 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 12 shows the amino acid sequence (SEQ ID NO 12) of a native sequence PR07154 polypeptide as derived from the coding sequence of SEQ ID NO 1 1 shown in Figure 1 1
  • Figure 13 shows the nucleotide sequence (SEQ ID NO 13) of a cD A containing a nucleotide sequence encoding native sequence PR07170 wherein the nucleotide sequence (SEQ ID NO 13) is a clone designated herein as DNA 108722-2743 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 14 shows the amino acid sequence (SEQ ID NO 14) of a native sequence PRO7170 polypeptide as derived from the coding sequence of SEQ ID NO 13 shown in Figure 13
  • Figure 15 shows the nucleotide sequence (SEQ ID NO 15) of a cDNA containing a nucleotide sequence encoding native sequence PR07422, wherein the nucleotide sequence (SEQ ID NO 15) is a clone designated herein as DNA1 19536-2752 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 16 shows the amino acid sequence (SEQ ID NO 16) of a native sequence PR07422 polypeptide as derived from the coding sequence of SEQ ID NO 15 shown in Figure 15
  • Figure 17 shows the nucleotide sequence (SEQ ID NO 17) of a cDNA containing a nucleotide sequence encoding native sequence PR07431 , wherein the nucleotide sequence (SEQ ID NO 17) is a clone designated herein as DNA1 19542-2754 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 18 shows the amino acid sequence (SEQ ID NO 18) of a native sequence PR07431 polypeptide as derived from the coding sequence of SEQ ID NO 17 shown in Figure 17
  • Figure 19 shows the nucleotide sequence (SEQ ID NO 19) of a cDNA containing a nucleotide sequence encoding native sequence PR07476 wherein the nucleotide seq uence (SEQ ID NO 19) is a clone designated herein as DNA115253-2757 Also presented in bold font and underlined are the positions of the respective start and stop codons
  • Figure 20 shows the amino acid sequence (SEQ ID NO 20) of a native sequence PR07476 polypeptide as derived from the coding sequence of SEQ ID NO 19 shown in Figure 19
  • 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 "
  • amplicon Usually, the amount of the messenger RNA (mRNA) produced.
  • mRNA messenger RNA
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by 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, gastrointestinal cancer, pancreatic cancer, glioblastoma. cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endomet ⁇ al 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
  • treatment refers to both therapeutic treatment and prophylactic or preventati ve 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, cattle, pigs sheep, etc Preferably, the mammal is human
  • Car ⁇ ers as used herein include pharmaceutically acceptable carriers, excipients or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution
  • physiologically acceptable car ⁇ ers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins.
  • hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccha ⁇ des.
  • Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction ot cells
  • the term is intended to include radioactive isotopes (e g , I l I 12 ' Y 9U 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
  • chemotherapeutic agents include ad ⁇ amycin, doxorubicin, epirubicin, 5-fluorourac ⁇ l, cytosine arabinoside ("Ara- C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pac taxel (Taxol. B ⁇ stol-Mvers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer, Antony, Rnace), toxotere methotrexate.
  • teniposide daunomycin, carminomycin, aminopte ⁇ n, dactinomycin mitomycins, esperamicins (see, U S Pat No 4,675,187), 5-FU, 6-th ⁇ oguan ⁇ ne, 6-mercaptopu ⁇ ne, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards
  • hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onap ⁇ stone
  • 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 in vitro or in vivo
  • the 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 v cas (vinc ⁇ stine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin
  • Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and
  • Doxorubicin is an anthracychne antibiotic
  • the full chemical name of doxorubicin is (8S-c ⁇ s)-10-[(3- am ⁇ no-2.3.6-t ⁇ deoxy-c--L-lyxo-hexapyranosyl)oxy]-7 8,9, 10-tetrahydro-6 8 1 l-t ⁇ hvdroxv-8-(hydroxyacetyl)-l methoxy 5, 12-naphthacened ⁇ one
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators
  • lymphokines monokines include lymphokines monokines, and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone. N-methionvl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsu n relaxin, prorelaxin, glycoprotem hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteimzing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin.
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • LH luteimzing hormone
  • TPO thrombopoietin
  • TGFs nerve growth factors
  • TGFs transforming growth factors
  • EPO erythropoietin
  • CSFs colony stimulating factors
  • 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 less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form
  • the prodrugs of this invention include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containingprodrugs, D-ar ⁇ uno acid-modified prodrugs, glysocylated prodrugs, ⁇ -lactam-containing
  • a “therapeutically effective amount”, in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects ( 1 ) inhibition, to some extent, of tumor growth including, slowing down and complete growth arrest, (2) reduction in the number of tumor cells, (3) reduction in tumor size, (4) inhibition (/ e . reduction, slowing down or complete stopping) of tumor cell infiltration into pe ⁇ pheral organs, (5) inhibition (.
  • a "therapeutically effective amount" of a PRO polypeptide antagonist for purposes ot treatment of tumor may be determined empirically and in a routine manner
  • a 'growth inhibitory amount of a PRO antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e g , cancer cell either ... vitro or in vivo
  • a 'growth inhibitory amount ' of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • a "cvtotoxic amount of a PRO antagonist is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitro or in A cytotoxic amount ' of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
  • PRO polypeptide and PRO as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (. e , PRO/number) refers to specific polypeptide sequences as described herein
  • the PRO polypeptides desc ⁇ bed herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
  • a ' native sequence PRO polypeptide comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means
  • the term ' native sequence PRO polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring alle c variants of the polypeptide
  • the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures
  • the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that
  • the PRO polypeptide "extracellular domain' or "ECD” refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and or cytoplasmic domains and preferably, will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them
  • PRO polypeptide variant means an active PRO polypeptide as defined above or below having at least about 80% ammo acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without the signal peptide. as disclosed herein or anv other fragment of a full-length PRO polypeptide sequence as disclosed herein
  • Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted at the N- or C-terminus of the full-length nativ e amino acid sequence Ordina ⁇ lv .
  • a PRO polypeptide variant will hav e at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% ammo acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity.
  • PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 am o acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids
  • Percent (%) amino acid sequence identity' with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, In
  • ALIGN-2 program is publicly available through Genentech, lnc South San Francisco California or may be compiled from the source code provided in Table 1
  • the ALIGN-2 program should be compiled for use on a UNIX operating system preferably digital UNIX V4 OD All sequence comparison parameters are set b ⁇ the ALIGN-2 program and do not vary
  • % amino acid sequence identitv of a given amino acid sequence A to with or against a given amino acid sequence B is calculated as follows
  • % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res . 25 3389-3402 (1997))
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows
  • a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of 5 interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence ot interest (- e . the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B". the amino acid sequence
  • amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest
  • PRO variant polynucleotide or "PRO variant nucleic acid sequence ' means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as
  • a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide. as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein
  • a PRO va ⁇ ant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about
  • nucleic acid sequence identity alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least
  • nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence
  • PRO polypeptide sequence as disclosed herein a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein an extracellular domain ot a PRO polypeptide with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence
  • PRO variant polynucleotides are at least about 30 nucleotides in length, altemativelv at least
  • nucleotides in length alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more
  • Percent (%) nucleic acid sequence identity with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN.
  • ALIGN-2 or Mega gn (DNASTAR) software
  • ALIGN-2 or Mega gn (DNASTAR) software
  • % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Ine , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087
  • the ALIGN-2 program is publicly available through Genentech, Ine , South San Francisco. California or may be compiled from the source code provided in Table 1
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
  • % nucleic acid sequence identity of a given nucleic acid sequence C to with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comp ⁇ ses a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows
  • % nucleic acid sequence identity used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997))
  • NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov
  • % nucleic acid sequence identity of a given nucleic acid sequence C to, with or against a given nucleic acid sequence D is calculated as follows
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynu
  • PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in the accompanying figures
  • PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
  • Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest
  • % value of positives ot a given amino acid sequence A to, with, or against a given am o acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to with or against a given amino acid sequence B) is calculated as follows 100 times the fraction X/Y
  • isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components ot its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes In preferred embodiments, the polypeptide will be purified (1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator.
  • Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step
  • an "isolated" nucleic acid molecule encoding a PRO polypeptide or an “isolated” nucleic acid encoding an anti-PRO antibody 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 PRO-encoding nucleic acid or the anti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free ot association with all components with which it is naturally associated
  • An isolated PRO-encoding nucleic acid molecule or an anti- PRO-encoding 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 PRO-encoding nucleic acid molecule or the anti-PRO encoding nucleic acid molecule as it exists in natural cells
  • an isolated nucleic acid molecule encoding a PRO polypeptide or an isolated nucleic acid molecule encoding an anti-PRO antibody includes PRO-nucle
  • Nucleic acid is "operably linked' when it is placed into a functional relationship with another nucleic acid sequence
  • DNA tor a presequence or secretory leader is operably linked to DNA tor a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ot the sequence, or a ⁇ bosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • operably linked means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase
  • enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites If such sites do not exist the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
  • antibody is used in the broadest sense and specifically covers, for example, single anti-PRO monoclonal antibodies (including antagonist and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments ot anti-PRO antibodies (see below)
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, / e , the individual antibodies comprising the population are identical except tor possible naturally-occurring mutations that may be present in minor amounts
  • Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures
  • Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hvbridizable sequence the higher
  • St ⁇ ngent conditions or ' high st ⁇ ngency conditions
  • 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 ⁇ nylpyrrohdone/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).
  • Modely stringent conditions may be identified as described by Sambrook et al , Molecular Cloning A Laboratory Manual, New York Cold Sp ⁇ ng Harbor Press, 1989, and include the use ot washing solution and hybridization conditions (e g , temperature ionic strength and % SDS) less stringent 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 mM t ⁇ sodium citrate). 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution. 10% dextran sulfate.
  • epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag polypeptide '
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
  • the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes
  • Suitable tag polypeptides generally hav e at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
  • Active or activity for the purposes herein refers to form(s) of PRO polypeptides which retain a biological and/or an immunological activity/property of a native or naturally-occurring PRO polypeptide, wherein "biological ' activity refers to a function (either inhibitory or stimulatory) caused by a native or naturally occurring PRO polypeptide other than the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally-occurring PRO polypeptide and an immunological" activity refers to the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally-occurring PRO polypeptide
  • Bioactivity in the context of an antibody or another antagonist 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 or otherwise interfere with the transcription or translation of a PRO polypeptide
  • 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
  • biological activity in the context of a PRO polypeptide means the ability of a PRO polypeptide to induce neoplastic cell growth or uncontrolled cell growth
  • immunological activity means immunological cross-reactivity with at least one epitope of a PRO polypeptide
  • immunological cross-reactivity means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO polypeptide having this activity with polyclonal antisera raised against the known active PRO 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 (e g , antibody) identified, to the corresponding PRO polypeptide is significantly higher (preferably at 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 10-t ⁇ mes 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 partially or fully blocks, inhibit
  • Antibodies are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity Polvpeptides 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 ( ⁇ ? g , 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 daltons, composed ot two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one co alent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced intrachain 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, 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 amino 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
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al . NIH Publ No 91 -3242. Vol I. pages 647-669 (1991 ))
  • the constant domains are not involved directly in binding an antibody to an antigen but exhibit various effector functions, such as participation ot the antibody in antibody-dependent cellular toxicity
  • hyperva ⁇ able region ' 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 ⁇ ty determining region ' or "CDR" (i e , residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain.
  • 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 etal Protein Eng , 8(10) 1057- 1062 [ 1995]), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual 'Fc" fragment, whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab')-, fragment that has two antigen-combining sites and is still capable of cross-linking antigen "Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site
  • This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (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 1 domain including one or more cysteines 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') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
  • immunoglobulins can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , IgG2, IgG3, IgG4, IgA, and IgA2
  • the heavy-chain constant domains that correspond 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
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies. i e the individual antibodies comprising the population are identical except for possible naturall v occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigemc site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hyb ⁇ doma culture, uncontaminated by other immunoglobulins The modifier monoclonal indicates the character of the antibody as being obtained from a substantiallv 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 mav be made by the hyb ⁇ doma method
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al , Nature.
  • the monoclonal antibodies herein specificallv 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 ot the cha ⁇ n(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as tragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4,816,567 Morrison et al , Proc Natl Acad Sci USA. 81 6851 -6855 [1984])
  • Humanized forms ot non-human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived trom non-human immunoglobulin
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR ot 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 modifications are made to further refine and maximize antibody performance
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that ot a human immunoglobulin
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-binding region of the antibody is de ⁇ ved 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
  • an "isolated' antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components ot its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes
  • the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal am o 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
  • 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, 1-131, 1-123, 1-125, Y-90, Re- 188, Re- 186, At-211 , Cu-67, B ⁇ -212, and Pd- 109
  • the label may also be a non-detectable entity such as a toxin
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere
  • solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccharides (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate, in others it is a purification column (e g., an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those desc ⁇ bed in U S Patent No. 4,275,149
  • a “liposome” is a small vesicle composed of various types of lipids, phospho pids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto and, optionally, a chemotherapeutic agent) to a mammal.
  • a drug such as a PRO polypeptide or antibody thereto and, optionally, a chemotherapeutic agent
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid 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 immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i e , is ' heterologous '), and an immunoglobulin constant domain sequence
  • the adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand
  • the immunoglobulin constant domain sequence in the lmmunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM
  • Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
  • Tables 2A-2D show hypothetical exemplifications tor using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO " represents the amino acid sequence of a hypothetical PRO polypeptide of interest.
  • “Comparison Protein” represents the amino acid sequence of a polypeptide against which the "PRO” polypeptide ot interest is being compared
  • PRO-DNA represents a hypothetical PRO-encoding nucleic acid sequence of interest
  • “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA” nucleic acid molecule of interest is being compared
  • "X", “Y”, and “Z” each represent different hypothetical amino acid residues
  • "N", “L” and “V” each represent different hypothetical nucleotides.
  • filel and file2 are two dna or two protein sequences.
  • Max file length is 65535 (limited by unsigned short x in the jmp struct)
  • a sequence with 1/3 or more ot its elements ACGTU is assumed to be DNA
  • the program may create a imp file in /tmp to hold info about traceback.
  • static nm matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char oouutt[[22]][[IP LINE] ; / * output line */ static char starfP 1 ]; /* set by stars() *//
  • *ps[ ⁇ ] toupper(*ps[ ⁇ ]), po[ ⁇ ] + + , ps[ ⁇ ] + + ,
  • *py+ + *px; else if ( ⁇ slower(*px))
  • *py+ + toupper(*px); if ( ⁇ ndex("ATGCU",*(py-l))) natgc + + ; ⁇ ⁇
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides In particular.
  • cDNA encoding PRO polypeptides has been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed However for sake ot simplicity, in the present specification the proteins encoded by the herein disclosed nucleic acid sequences as well as all further native homologues and variants included in the foregoing definition of PRO polypeptides will be referred to as "PRO" regardless of their origin or mode of preparation
  • cDNA clones have been deposited with the ATCC
  • the actual nucleotide sequence ot the clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art
  • the predicted amino acid sequences can be determined from the nucleotide sequences using routine skill For the PRO polypeptides and encoding nucleic acid described herein, Applicants have identified what are believed to be the reading frames best identifiable with the sequence information available
  • PRO polypeptide variants can be prepared.
  • PRO polypeptide variants can be prepared by introducing approp ⁇ ate nucleotide changes into the PRO DNA and/or by synthesis of the desired PRO polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO polypeptide. such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
  • Variations in the native full-length sequence PRO polypeptide or in various domains of the PRO polypeptide described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5.364.934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO polypeptide that results in a change in the amino acid sequence of the PRO polypeptide as compared with the native sequence PRO polypeptide
  • the ⁇ a ⁇ at ⁇ on is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO polypeptide
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the number ot amino acid sequence changes made in regions of high homology Amino acid substitutions can be the result ot replacing one amino acid with another amino acid having similar structural
  • Insertions or deletions may optionally be in the range ot about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence PRO polypeptide fragments are provided herein Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, tor example, when compared with a full-length native protein Certain fragments lack amino acid residues that are not essential tor a desired biological activity of the PRO polypeptide
  • PRO polypeptide fragments may be prepared by any ot a number of conventional techniques Desired peptide fragments may be chemically synthesized
  • An alternative approach involves generating PRO polypeptide fragments by enzymatic digestion e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or bv digesting the DNA with suitable restriction enzymes and isolating the desired fragment
  • Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • Ohgonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR
  • PRO polypeptide fragments share at least one biological and or immunological activity with the native PRO polypeptide
  • conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened
  • Phe (F) leu. val lie. ala tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr, phe tyr Tyr (Y) tip, phe thr. ser phe Val (V) lie, leu met, phe. ala, norleucine leu
  • Substantial modifications in function or immunological identity of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, tor example, as a sheet or helical conformation (b) the charge or hydrophobicity ot the molecule at the target site or (c) the bulk of the side chain Naturally occurring residues are divided into groups based on common side chain properties (1) hydrophobic norleucine, met, ala. val. leu, lie (2) neutral hydrophilic cys, ser, thr,
  • Such substituted residues also may be introduced into the conservative substitution sites or. more preferably, into the remaining (non-conserved) sites
  • the variations can be made using methods known in the art such as ohgonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al , Nucl Acids Res . 13 4331 (1986), Zoller et al , Nucl Acids Res K ) 6487 ( 1987)], cassette mutagenesis [Wells et al . Gene. 34 315 (1985)], restriction selection mutagenesis [Wells etal , Philos Trans R Soc. London SerA.317 415 (1986)] or other known techniques can be pei formed on the cloned DNA to produce the PRO variant DNA
  • Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence
  • preferred scanning amino acids are relatively small, neutral amino acids
  • amino acids include alanine, glycine, serine, and cysteine
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science 244 1081-1085 ( 1989)]
  • Alanine is also typically preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed positions [Creighton. The Proteins, (W H Freeman & Co . N Y ), Chothia, J Mol Biol 150 1 ( 1976)] If alanine substitution does not yield adequate amounts of ⁇ a ⁇ ant. an lsote ⁇ c amino acid can be used
  • Covalent modifications ot the PRO polypeptide are included within the scope of this invention
  • One type of covalent modification includes reacting targeted amino acid residues ot a PRO polypeptide with an organic de ⁇ vatizing agent that is capable ot reacting with selected side chains or the N- or C- terminal residues ot the PRO polypeptide
  • Derivatization with bitunctional agents is useful for instance, tor crosslinking the PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies and vice versa
  • Commonly used crosslinking agents include e g , ] ] -b ⁇ s(d ⁇ azoacetyl)-2-phenylethane. glutaraldehyde.
  • N- hydroxysuccinimide esters for example, esters with 4-az ⁇ dosahcyl ⁇ c acid, homobif unctional lmidoesters including disuccimmidyl esters such as 3 3'-d ⁇ th ⁇ ob ⁇ s(succ ⁇ n ⁇ m ⁇ dylprop ⁇ onate) bitunctional maleimides such as bis-N- male ⁇ m ⁇ do-l ,8-octane and agents such as methyl-3-[( ⁇ -az ⁇ dophenyl)d ⁇ th ⁇ o]prop ⁇ o ⁇ m ⁇ date
  • Another type of covalent modification of the PRO polypeptide included within the scope ot this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended tor purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO polypeptides (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide
  • the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions ot the various carbohydrate moieties present
  • Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence
  • the alteration may be made, for example, by the addition of, or substitution by, one or more se ⁇ ne or threonine residues to the native sequence PRO polypeptide (for O-linked glycosylation sites)
  • the PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids
  • Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e.g , in WO 87/05330 published 11 September 1987, and in Aplin andW ⁇ ston. CRC C ⁇ t Rev Biochem . pp 259-306 (1981) Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and desc ⁇ bed, for instance, by Hakimuddin.
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al Meth Enzvmol 138 350 (1987)
  • PRO polypeptides comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes in the manner set forth in U S Patent Nos 4 640,835, 4.496,689, 4,301 , 144, 4,670,417, 4,791 ,192 or 4.179,337
  • the PRO polypeptides of the present invention may also be modified in a way to form a chimeric molecule comprising the PRO polypeptide fused to another, heterologous polypeptide or amino acid sequence
  • such a chimeric molecule comprises a fusion of the PRO polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the amino- or carboxyl-terminus ot the PRO polypeptide.
  • the presence of such epitope-tagged forms of the PRO polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the PRO polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type ot affinity matrix that binds to the epitope tag
  • Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidme (poly-His) or poly-histidine-glycine (poly- His-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol 8 2159-2165 ( 1988)], the c-myc tag and the
  • Tag polypeptides include the Flag-peptide [Hopp e a/ , B ⁇ oTechnology. 6 1204- 1210 (1988)1, the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an -tubulin epitope peptide [Skinner et al . J Biol Chem . 266 15163-15166 ( 1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA 87 6393-6397 (1990)]
  • the chimeric molecule may comprise a fusion of the PRO polypeptide with an immunoglobulin or a particular region of an immunoglobulin
  • an immunoglobulin also referred to as an "lmmunoadhesin”
  • a fusion could be to the Fc region of an IgG molecule
  • the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule
  • the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgGl molecule
  • PRO 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, CA (1969), Mer ⁇ field, J Am Chem Soc .
  • In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer s instructions Various portions of the PRO polypeptide may be chemically synthesized separately and combined using chemical or enzvmatic methods to produce the full-length PRO polypeptide
  • DNA encoding the PRO polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue such as described in the Examples
  • the PRO-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis
  • Probes such as antibodies to the PRO polypeptide, or ohgonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures such as described in Sambrook et al . Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989)
  • An alternative means to isolate the gene encoding the PRO polypeptide 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 ot 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 ot labeling are well known in the art. and include the use of radiolabels like ⁇ P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supia
  • 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 amino acid or nucleotide level within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein
  • Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA
  • Host cells are transfected or transformed with expression or cloning vectors described herein for PRO polypeptide 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
  • principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach. M Butler, ed (IRL Press, 1991) and Sambrook et al , supra
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include prokarvote veast or higher eukarvote cells
  • Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram-negative or Gram-positn e organisms for example Enterobacte ⁇ aceae such as E coli
  • Various £ coli strains are pubhclv available such as £ coh Y ⁇ 2 strain MM294 (ATCC 31 446), £ coli XI 776 (ATCC 31 ,537) £ co/.
  • strain W31 10 ATCC 27,325) and £ coli strain K5 772 (ATCC 53,635)
  • Other suitable prokarvotic host cells include Enterobacte ⁇ aceae such as £-.c/7£/-c/-- ⁇ 2 e g , E coli Enterobacter Etwinia Klebsiella Pioteus Salmonella, e g Salmonella nphtmutium, Serratia, e g , Sen alia marcescans, and Shigella as well as Bacilli such as B subtilis and -5 licheniformis (e g , B hcheniformis 41 P disclosed in DD 266 710 published 12 April 1989), Pseuaomonas such as P aeruginosa, and Sti eptom ces
  • B subtilis and -5 licheniformis e g , B hcheniformis 41 P disclosed in DD 266 710 published 12 April 1989
  • Pseuaomonas such as P
  • Suitable host cells tor the expression of glycosylated PRO polypeptides are derived from multicellular organisms
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9 as well as plant cells
  • ot useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV 1 line transformed by S V40 (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 Natl Acad Sci USA.
  • mice sertoh cells TM4, Mather Biol Reprod 23 243-251 (1980)
  • human lung cells W 138, ATCC CCL 75
  • human liver cells Hep G2. HB 8065
  • mouse mammary tumor MMT 060562 ATCC CCL51
  • the selection of the appropriate host cell is deemed to be within the skill in the art
  • nucleic acid e g , cDNA or genomic DNA
  • the nucleic acid (e g , cDNA or genomic DNA) encoding the PRO polypeptide may be inserted into a replicable vector for cloning (amplification ot the DNA) or for expression
  • a replicable vector for cloning (amplification ot the DNA) or for expression
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety ot procedures
  • DNA is inserted into an appropriate restriction endonuclease s ⁇ te(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 transc ⁇ ption termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation
  • the PRO 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 PRO-encoding DNA that is inserted into the vector
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders
  • yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharonnces and Kluw erom ⁇ ces ⁇ -factor leaders the latter desc ⁇ bed in U S Patent No 5,010,182), or acid phosphatase leader, the C albicans glucoamylase leader
  • 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 critical nutrients not available from complex media, e g , the gene encoding D-alanine racemase tor Bacilli
  • suitable selectable markers tor mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid such as DHFR or thymidine kinase
  • An approp ⁇ ate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Proc Natl Acad Sci USA. 77 4216 ( 1980)
  • a suitable selection gene for use in yeast is the tip] gene present in the yeast plasmid YRp7 [Stinchcomb et al Nature. 282 39 (1979), Kings an et al , Gene.
  • the tip] gene provides a selection marker for a mutant strain ot yeast lacking the ability to grow in tryptophan, for example ATCC No 44076 or PEP4- 1 [Jones, Genetics 85 12 (1977)]
  • Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis
  • Promoters recognized by a variety ot potential host cells are well known Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al , Nature, 275 615 (1978), Goeddel et al , Nature, 281 544 (1979)], alkaline phosphatase, a tryptophan (tip) promoter system [Goeddel.
  • Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding the PRO polypeptide
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 (1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg .7 149 (1968), Holland, Biochemistry, H 4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokmase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, t ⁇ osephosphate isomerase, phosphoglucose isomerase, and glucok ase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2.
  • lsocytochrome C acid phosphatase. degradative enzymes associated with nitrogen metabolism metallothionein.
  • PRO polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (S V40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
  • viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus
  • Enhancers are cis-acting elements of DNA. usually about from 10 to 300 bp, that act on a promoter to increase its transcription
  • Many enhancer sequences are now known from mammalian genes (globin.
  • an enhancer from a eukaryotic cell virus examples include the S V40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus earh promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PRO coding sequence, but is preferably located at a site 5' from the promoter
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3'. untranslated regions of eukaryotic or viral DN As or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion ot the mRNA encoding the PRO polypeptide
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription ot mRNA [Thomas. Proc Natl Acad Sci USA. 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for immunohisto
  • PRO polypeptides may be recovered from culture medium or from host cell lvsates If membrane-bound it can be released from the membrane using a suitable detergent solution (e g , T ⁇ ton-X 100) or by enzymatic cleavage Cells employed in expression of the PRO polypeptide can be disrupted by va ⁇ ous physical or chemical means, such as freeze thaw cycling, sonication. mechanical disruption, or cell lysing agents
  • PRO polypeptide may be desired to purify the PRO polypeptide from recombinant cell proteins or polypeptides
  • the following procedures are exemplary ot suitable purification procedures bv 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 PRO polypeptide
  • Various methods of protein purification may be employed and such methods are known in the art and described tor example in Deutscher, Methods in Enzvmology. 182 (1990). Scopes. Protein Purification Principles and Practice Sp ⁇ nger-Verlag New York ( 1982) The purification step(s) selected will depend, tor example, on the nature ot the production process used and the particular PRO polypeptide produced
  • 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 original 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 quantitative modifications of the genetic mate ⁇ al Qualitative modifications include DNA mutations, in which coding sequences are altered resulting in a structurally and/or functionally different protein Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, i 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
  • MTX cytotoxic drug methotrexate
  • Gene amplification is most commonly encountered in the development of resistance to cytotoxic drugs (antibiotics for bacteria and chemotherapeutic agents 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
  • cytotoxic drugs antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells
  • 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 ot the most common genetic changes observed in human malignancies are mutations ot the p53 protein p53 controls the transition of cells from the stationary (Gl ) to the rephcative (S) phase and prevents this transition in the presence of DNA damage
  • Gl stationary
  • S rephcative
  • one of the main consequences ot disabling p53 mutations is the accumulation
  • the amplification of DNA sequences may indicate a specific functional requirement as illustrated in the DHFR experimental system Therefore, the amplification of certain oncogenes in malignancies points toward a causative role ot these genes in the process ot malignant transformation and maintenance of the transformed phenotype
  • 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 ot neoplastic cells
  • Members of the gene family of growth factor receptors have been found to be amplified in various types ot 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 therapy and the amplification of the grow th factor receptor homologue ERB2 in breast cancer Lastly, genes involved in intracellular signaling and control of cell cycle progression
  • PCR polymerase chain reaction
  • cytogenetic analysis and CGH represent screening methods to survev 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
  • 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, thy us, testis, ovary, uterus, etc , tumor, or tumor cell lines with pooled DNA from healthy donors Quantitative PCR was performed using a TaqManTM instrument (ABI) Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs
  • Human lung carcinoma cell lines include A549 (SRCC768). Calu- 1 (SRCC769), Calu-6 (SRCC770), HI 57 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832),and H810 (SRCC833), all available from ATCC
  • Primary human lung 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, SRCC724 (adenocarcinoma.
  • SRCC725 squamous cell carcinoma, abbreviated as "SqCCa)(LTla)
  • SRCC726 (adenocarc ⁇ noma)(LT2)
  • SRCC727 (adenocarc ⁇ noma)(LT3)
  • SRCC728 (adenocarc ⁇ noma)(LT4)
  • SRCC729 (squamous cell carc ⁇ noma)(LT6)
  • SRCC730 (adeno/squamous cell carc ⁇ noma)(LT7)
  • SRCC731 adenocarc ⁇ noma)(LT9)
  • SRCC732 (squamous cell carc ⁇ noma)(LT10)
  • SRCC733 (squamous cell carc ⁇ noma)(LTl l)
  • SRCC734 (adenocarc ⁇ noma)(LT12), SRCC735 (adeno/squamous cell carc ⁇ noma)(LT13), SRCC736 (
  • SW620 lymph node metastasis of colon adenocarcinoma. SRCC777
  • Colo320 carcinoma, SRCC778
  • HT29 adenocarcinoma. SRCC779
  • HM7 a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr Robert Warren, UCSF
  • CaWiDr adenocarcinoma, SRCC781
  • HCT1 16 carcinoma, SRCC782
  • SKCOl adenocarcinoma, SRCC783
  • SW403 adenocarcinoma, SRCC784
  • LS174T carcinoma, SRCC785
  • Colo205 carcinoma, SRCC828
  • HCT15 carcinoma, SRCC829
  • HCC2998 a, SRCC830
  • KM12 carcinoma, SRCC831
  • CT3 SRCC743
  • CT8 SRCC744
  • CT10 SRCC745)
  • CT12 SRCC746)
  • CT14 SRCC747
  • CT15 SRCC748
  • CT16 SRCC749
  • CT17 SRCC750
  • CT1 SRCC751
  • CT4 SRCC752
  • CT5 SRCC753
  • CT6 SRCC754
  • CT7 SRCC755
  • CT9 SRCC756
  • CT1 1 SRCC757
  • CTI 8 SRCC758
  • CT19 adenocarcinoma, SRCC906)
  • CT20 adenocarcinoma, SRCC907)
  • CT21 adenocarcinoma. SRCC908).
  • CT22 (adenocarcinoma, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (adenocarcinoma. SRCC91 1 ), CT25 (adenocarcinoma. SRCC912), CT26 (adenocarcinoma, SRCC913), CT27 (adenocarcinoma, SRCC914).
  • CT28 (adenocarcinoma, SRCC915), CT29 (adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31 (adenocarcinoma, SRCC918), CT32 (adenocarcinoma.
  • CT35 adenocarcinoma. SRCC921
  • CT36 adenocarcinoma. SRCC922
  • human colon tumor centers designated SRCC1051 [HF-000499].
  • SRCC1052 [HF-000539], SRCC1053 [HF-000575], SRCC1054 [HF-000698], SRCC1059 [HF-000755], SRCC1060 [HF-000756], SRCC1 142 [HF-000762], SRCC1 144 [HF-000789], SRCC1 146 [HF-000795] and SRCC1 148[HF-00081 1 ]
  • 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), and SKBR3 (SRCC767), and human breast tumor center designated SRCC1057 [HF-000545] Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1 100, SRCC1 101 , and human breast-met-lung-NS tumor designated SRCC893 [LT 32] Human rectum tumors include SRCC981 [HF-000550] and SRCC982 [HF-000551] Human kidney tumor centers include SRCC989 [HF-00061 1 ] and SRCC1014 [HF-000613] Human testis tumor center
  • Human lymph node tumors include SRCC1004 [HF-000854], SRCC1005 [HF-000855], and SRCC1006 [HF-000856]
  • gene amplification and/or gene expression in various tissues may be measured by conventional Southern blotting.
  • Northern blotting to quantitate the transcription of mRNA (Thomas Proc Natl Acad Sci USA.77 5201 -5205 [ 1980] ), dot blotting (DNA analysis), or in situ hybridization, using an approp ⁇ ately 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 v a ⁇ ous tissues may be measured by immunological methods such as immunohistochemical staining ot 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 ot sample fluids may be either monoclonal or polyclonal. and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PRO DNA and encoding a specific antibody epitope
  • General techniques tor generating antibodies, and special protocols for Northern blotting 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 the neighboring genomic region
  • Selective or preferential amplification at the genomic region to which the 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
  • 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, Ine .
  • 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, thus forming an insoluble three-part complex
  • the second antibodv mav 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 ELISA assay, in which case the detectable moiety is an enzyme
  • the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, tor example
  • Cell-based assays and animal models tor tumors can be used to verify the findings ot the gene amplification assay, and further understand the relationship between the genes identified herein and the development and pathogenesis ot 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, tor example, the breast, colon and lung cancer cells and cell lines listed above
  • cells of a cell type known to be involved in a particular tumor are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth is analyzed
  • Suitable cells include, for example, stable tumor cell lines such as, the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transtected NIH-3T3 cells, which can be transfected with the desired gene
  • Animal models of tumors and cancers include both non- recombinant and recombinant (transgenic) animals
  • Non-recombinant animal models include, for example, rodent, e g , murine models
  • Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e g subcutaneous injection, tail vein injection, spleen implantation, lntrape ⁇ toneal 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 97/3
  • nude mice Probablv the most often used animal species in oncological studies are lmmunodeficient mice and, in particular, nude mice
  • the autosomal recessive nu gene has been introduced into a v ery large number of distinct congenic strains of nude mouse, including, for example, AS W, A/He, AKR, BALB/c B 10 LP, C17. C3H, C57BL, C57, CBA, DBA. DDD, I/st, NC, NFR, NFS, NFS/N. NZB, NZC, NZW, P.
  • the cells introduced into such animals 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) .
  • B 104- 1 -1 cell line stable NIH-3T3 cell line transfected with the neu protooncogene
  • Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmah et al .
  • 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 transplanted s c as solid blocks as needle biopsies by use ot a trochar, or as cell suspensions
  • tumor tissue fragments ot suitable size are introduced into the s c space
  • Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously 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 neuroblastoma cells (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 et al , Cancer Research, 54 4726-4728 ( 1994) and
  • 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 drug 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
  • mice (DeLeo et al , J Exp Med , 146 720 [1977]), which provide a highly controllable model system for studying the anti-tumor activities of va ⁇ ous agents (Palladino et al , J Immunol , 138 4023-4032
  • tumor cells are propagated in vitro in cell culture Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about lOx lO 6 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
  • 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 survive For further information about this tumor model see Zacharski Haemostasis 16 300-320 [1986])
  • One wav ot evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor 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 drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important v a ⁇ able in the description ot tumor growth is the tumor volume doubling time Computer programs for the calculation and description ot 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
  • Recombinant (transgenic) animal 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
  • Animals that 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 microinjection (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 Natl Acad Sci USA.
  • transgenic animals include those that carry the transgene only in part of their cells (“mosaic animals”).
  • the transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems
  • Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA. 89 6232- 6236 (1992)
  • transgene expression in transgenic animals can be monitored by standard techniques For example, Southern 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 immunocvtochemistrv The animals are further examined for signs of tumor or cancer development
  • 'knock out animals can be constructed which have a defectiv e or altered gene encoding a PRO 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
  • cDNA encoding a PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques
  • a portion of the genomic DNA encoding a particular PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
  • Typicallv several kilobases of unaltered flanking DNA are included in the vector [see e g , Thomas and Capecchi, Cell 51 503 (1987) for a description ot homologous recombination vectors]
  • the vector is introduced into an embryonic stem cell line (e g by electropor
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out ' animal
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells ot 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 pathological conditions due to absence of the PRO polypeptide
  • fibrosarcoma adenocarcinoma. lymphoma, chrondroma, leiomyosarcoma of dogs, cats, and baboons
  • mammary adenocarcinoma in dogs and cats is a preferred model as its appearance and behavior are very similar to those in humans
  • the use of this model is limited by the rare occurrence of this type ot tumor in animals
  • Screening assays for drug 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 drug candidates
  • Small molecules contemplated include synthetic organic or inorganic compounds, including peptides preferably soluble peptides, (poly)pept ⁇ de- ⁇ mmunoglobuhn fusions, and.
  • antibodies including, without limitation poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments
  • 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 tor a time sufficient to allow these two components to interact
  • binding assays the interaction is binding and the complex formed can be isolated or detected in the reaction mixture
  • the polypeptide encoded bv the gene identified herein or the drug candidate is immobilized on a solid phase, e g , on a microtiter plate by covalent or non-covalent attachments
  • Non- covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying
  • an immobilized antibody, e 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-im
  • the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions
  • assays include traditional approaches, such as, cross-linking, co- lmmunoprecipitation, and co-purification through gradients or chromatographic columns
  • protein- protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature. 340 245-246 (1989), Chien et al , Proc Natl Acad Sci USA 88 9578-9582 ( 1991)] as disclosed by Chevray and Nathans. Proc Natl Acad Sci USA.
  • yeast GAL4 Many transc ⁇ ptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain
  • the yeast expression system described in the foregoing publications (generally referred to as the ' two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4.
  • GAL1 -lacZ reporter gene under control of a GAL4-act ⁇ vated promoter depends on reconstitution ot G AL4 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 crucial for these interactions
  • a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and tor a time allowing tor the interaction and binding ot the two products
  • 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 ot a complex in the control react ⁇ on(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner
  • the PRO polypeptide mav be added to a cell along w ith the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide
  • antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions tor a competitive inhibition assay
  • the PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art.
  • expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transtect COS cells or other cells that are not responsive to the PRO polypeptide Transfected cells that are grown on glass slides are exposed to labeled PRO polypeptide
  • the PRO polypeptide can be labeled by a variety of means including lodination or inclusion of a recognition site for a site-specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
  • labeled PRO polypeptide can be photoaffinity-hnked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved by PAGE and exposed to X-ray film
  • the labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing
  • the amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor
  • mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound The ability of the compound to enhance or block this interaction could then be measured
  • potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with the PRO polypeptide, and. in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments
  • a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide
  • Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e g , an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation
  • Antisense technology can be used to control gene expression through t ⁇ ple-hehx formation or antisense DNA or RNA.
  • RNA oligonucleotide of from about 10 to 40 base pairs in length
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee etal , Nucl Acids Res . 6 3073 (1979), Cooney etal . Science. 241 456 (1988), Dervan et al Science.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem .
  • oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press Boca Raton, FL, 1988)
  • the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide
  • antisense DNA oligodeoxy ⁇ bonucleotides derived from the translation-initiation site, e g , between about -10 and +10 positions of the target gene nucleotide sequence, are preferred
  • Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more
  • Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-hke molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA followed bv endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology. 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
  • Nucleic acid molecules in t ⁇ ple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides
  • the base composition ot these oligonucleotides is designed such that it promotes t ⁇ ple-hehx formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex
  • Hoogsteen base-pairing rules which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex
  • compositions useful in the treatment ot tumors associated with the amplification of the genes identified herein include, without limitation antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc , that inhibit the expression and/or activ uy ot the target gene product
  • antisense RNA and RNA molecules act to directly block the translation ot mRNA by hybridizing to targeted mRNA and preventing protein translation
  • oligodeoxy ⁇ bonucleotides 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
  • Nucleic acid molecules in triple helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides
  • the base composition of these oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex
  • Hoogsteen base pairing rules which generally require sizeable stretches of pu ⁇ nes or py ⁇ midines on one strand of a duplex
  • Some of the most promising drug 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
  • 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 lntrape ⁇ toneal injections
  • the immunizing agent may include the PRO polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include but are not limited to keyhole limpet hemocyanin.
  • ot adjuvants which may be employed include Freund s complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A. synthetic trehalose dicorynomycolate)
  • the immunization protocol may be selected by one skilled in the art without undue experimentation
  • the anti-PRO antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hyb ⁇ doma methods, such as those described by Kohler and Milstein Nature. 256 495 (1975)
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PRO polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • More preferred immortalized cell line* are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection (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 etal., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO polypeptides.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. 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 heav v and light chains of murine antibodies)
  • the hyb ⁇ doma cells ot the invention serv e as a preferred soui ce ot such DNA
  • the DNA may be placed into expression vectors which are then transfected into host cells such as simian COS cells Chinese hamster ov ai v (CHO) cells 01 myeloma cells that do not otherwise pioduce immunoglobulin protein to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the
  • the antibodies may be monovalent antibodies
  • Methods tor preparing monovalent antibodies are well known in the art For example, one method inv olves recombinant expression of immunoglobulin light chain and modified heavy chain
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking
  • Digestion ot antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art
  • the anti-PRO antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins. immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) ot 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 immunoglobulin are replaced by corresponding non-human residues
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al Nature. 321 522-525 ( 1986) Riechmann et al . Nature, 332 323-329 ( 1988), and Presta. Curr Op Stiuct Biol , 2 593-596 (1992)]
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human These non- human amino acid residues are often referred to as ' import residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers [Jones et al Natui e 321 522 525 ( 1986) Riechmann et al Nature 332 323 327 ( 1988) Verhoeyen et al Science 239 1534- 1536 (1988)] by substituting iodent CDRs or CDR sequences tor the corresponding sequences ot a human antibody Accordingly such humanized antibodies are chimenc antibodies (U S Patent No 4 816,567) wheiein substantially less than an intact human v ariable domain has been substituted by the corresponding sequence from a non human species
  • humanized antibodies are typically human antibodies in
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991 ), Marks et al J Mol Biol , 222 581 (1991 )] The techniques of Cole et al , and Boerner et al , are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss p 77 (1985) and Boerner et al J Immunol , 147(1 ) 86-95 (1991 )] Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed which closely resembles that seen in humans in all respects, including gene rearrangement assembly, and antibody repertoire This approach is described, for example, in U S Patent Nos
  • ADPT Antibody Dependent Enzyme Mediated Prodrug Therapy
  • the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug activating enzv, me which converts a prodrug (e g a peptidyl chemotherapeutic agent see WO 81/01 145) to an active anti-cancer drug See, for example WO 88/07378 and U S Patent No 4 975,278
  • a prodrug activating enzv me which converts a prodrug
  • a prodrug e g a peptidyl chemotherapeutic agent see WO 81/01 14
  • the enzyme component of the immunoconj ugate useful for ADEPT includes any enzyme capable ot 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 lysozyme, human glucuronidase, alkaline phosphatase useful tor converting phosphate-containing prodrugs into free drugs arylsulfatase useful for converting sulfate containing prodrugs into free drugs cytosine deaminase useful for converting non toxic 5-fluorocytos ⁇ ne into the anti cancerdrug5 fluorouracil, proteases, such as serratia protease, thermolysin, subtihsin, carboxypeptidases (e g carboxypeptidase G2 and carbox peptidase A) and cathepsins (such as cathepsms B and L), that are useful tor conv erting peptide containing prodrugs into free drugs, D-alanylcarboxvpeptidases useful for converting prodrugs that contain D amino acid substitu
  • the enzymes of this invention can be covendedly bound to the anti-PRO antibodies by techniques well known in the art such as the use of the heterobifunctional cross-linking agents discussed above.
  • 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 et al., 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 PRO polypeptide the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 [1983]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829. published 13 May 1993. and in Traunecker etal.. EMBO J..
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge, CH2. and CH3 regions. It is prefe ⁇ ed to have the first heavy-chain constant region (CH I ) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • CH I first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.. tyrosine or tryptophan).
  • Compensatory " cavities " of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products uch 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 bispecific antibodies can be prepared using chemical linkage Brennan etal Science 229 81 ( 1985) describe a procedure wherein intact antibodies are proteolvtically 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) de ⁇ vativ es One ot the Fab -TNB derivatives is then reconverted to the Fab - thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ot the other Fab'-TNB derivative to form the bispecific antibody
  • TNB thionitrobenzoate
  • Fab' fragments may be directly recovered from £ coli and chemically coupled to form bispecific antibodies Shalaby et al . J Exp Med , J_75 217 225 (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 ot human cytotoxic lymphocytes against human breast tumor targets Various techniques tor making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol , 148(5) 1547- 1553 ( 1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The
  • Antibodies with more than two valencies are contemplated
  • t ⁇ specif ie antibodies can be prepared Tutt et al , J Immunol , 147 60 ( 1991 )
  • Exemplary bispecific antibodies may bind to two different epitopes on a given polypeptide herein
  • an anti-polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e g , CD2 CD3. CD28 or B7) or Fc receptors tor 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 polypeptide
  • Bispecific antibodies may also be used to localize cvtotoxic agents to cells which express a particular polypeptide
  • These antibodies possess a polypeptide-binding arm and an arm w hich binds a cytotoxic agent or a radionuchde chelator such as EOTUBE DPTA DOTA or TETA
  • Another bispecific antibody ot interest binds the polypeptide and further binds tissue factor (TF) 6 Heterocontugate Antibodies
  • Heteroconjugate antibodies are composed ot two covalently joined 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 ot HIV infection [WO 91/00360, WO 92/200373, EP 03089] It is contemplated that the antibodies may be prepared ...
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond
  • suitable reagents tor this purpose include lminothiolate and methyl-4- mercaptobuty ⁇ midate and those disclosed, tor example in U S Patent No 4.676,980
  • effector function engineering It may be desirable to modify the antibody ot the invention with respect to effector function, so as to enhance the effectiveness ot 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 homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al . J Exp Med . 176 1 191 -1 195 ( 1992) and Shopes.
  • ADCC complement-mediated cell killing and antibody-dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research.53 2560- 2565 (1993)
  • an antibody can be engineered 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)
  • 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, nonbinding 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.
  • Plntolaca amencana proteins PAPI, PAPII. and PAP-S
  • momordica charantia inhibitor curcin crotin.
  • Small molecule toxins include, tor example calicheamicins. maytansinoids, palytoxin and CC 1065
  • radionuclides are av ailable for the production of radioconjugated antibodies Examples include 21 B ⁇
  • Conjugates of the antibody and cvtotoxic agent are made using a variety of bitunctional protein coupling agents such as N-succ ⁇ n ⁇ m ⁇ dyl-3-(2-py ⁇ dvld ⁇ th ⁇ ol ) propionate (SPDP) lminothiolane (IT), bitunctional derivatives of imidoesters (such as dimethvl adipimidate HCL) active esters (such as disucci midvl suberate). aldehydes (such as glutaraldehyde).
  • bitunctional protein coupling agents such as N-succ ⁇ n ⁇ m ⁇ dyl-3-(2-py ⁇ dvld ⁇ th ⁇ ol ) propionate (SPDP) lminothiolane (IT), bitunctional derivatives of imidoesters (such as dimethvl adipimidate HCL) active esters (such as disucci midvl suberate).
  • aldehydes such as glutaralde
  • bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine
  • bis-diazonium derivatives such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzovl)-ethvlened ⁇ am ⁇ ne
  • dnsocvanates such as tolyene 2.6-d ⁇ socyanate
  • a ⁇ cin immunotoxin can be prepared as described in Vitetta et al Science 238 1098 ( 1987) Carbon- 14-Iabeled l - ⁇ soth ⁇ ocyanatobenzyl-3- methyidiethylene t ⁇ aminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation ot radionucleotide to the antibody See, WO94/1 1026
  • the antibody may be conjugated to a receptor ' (such as streptav idin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient tot ⁇ owed by removal ot unbound conjugate from the circulation using a clearing agent and then administration ot A gand (e g , avidin) which is conjugated to a cytotoxic agent (e g , a radionucleotide)
  • a receptor ' such as streptav idin
  • the antibodies disclosed herein may also be formulated as immunohposomes
  • Liposomes containing the antibody are prepared bv methods known in the art, such as described in Epstein et al , Proc Natl Acad Set USA. 82 3688 (1985), Hwang et al Proc Natl Acad Sci USA 77 4030 ( 1980). and U S Patent 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 phosphatidylcholine.
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al J Biol Chem . 257 286-288 (1982) via a disulfide interchange reaction
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome See. Gabizon etal . J National Cancer Inst 81 19).1484 ( 1989)
  • Antibodies specifically binding the product ot an amplified gene identified herein as well as other molecules identified bv the screening assays disclosed hereinbefore can be administered tor the treatment ot tumors, including cancers, in the form of pharmaceutical compositions
  • the protein encoded bv the amplified gene is intracellular and whole antibodies are used as inhibitors internalizing antibodies are preferred
  • hpofections 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
  • an antibody peptide molecules can be designed which retain the ability to bind the target protein sequence
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technologv (see, e g . Marasco et al Proc Natl Acad Sci USA 90 7889-7893 [ 1993])
  • Therapeutic formulations ot the antibody are prepared tor storage bv mixing the antibody having the desired degree ot pu ⁇ tv with optional pharmaceutically acceptable carriers excipients or stabilizers (Remington s Pharmaceutical Sciences. 16th edition Osol. A ed [ 1980]) in the form ot lyophihzed formulations or aqueous solutions Acceptable carriers excipients.
  • buffers such as phosphate, citrate, and other organic acids: antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethomum chloride, benzalkonium chloride, benzetho um 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.
  • hydrophilic polymers such as polyvinylpyrrolidone. amino acids such as glycine, glutamine. asparagine. histidine arginine. or lysine. monosaccha ⁇ des. disaccha ⁇ des and other carbohydrates including glucose, mannose. or dext ⁇ ns.
  • chelating agents such as EDTA, sugars such as sucrose, mannitol. trehalose or sorbitol. salt-forming counter-ions such as sodium, metal complexes (e g . Zn-protein complexes), and or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG)
  • Non-antibody compounds identified by the screening assays ot 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 tor the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent cytokine or growth inhibitory agent Such molecules are suitably present in combination 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 mtertacial polyme ⁇ zation. tor example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules. respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions. nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences. 16th edition, Osol. A ed (1980)
  • the formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
  • sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which mat ⁇ ces are in the form ot shaped articles, e g , films or microcapsules
  • ot sustained-release matrices include polyesters. hydrogels (for example, poly(2-hydroxyethyl-methacrylate). or poly(v ⁇ nylalcohol)), polylactides ( U S Pat No 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate.
  • non-degradable ethylene-v in l acetate degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydro ⁇ ybut ⁇ c acid
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate
  • poly-D-(-)-3-hydro ⁇ ybut ⁇ c acid While polymers such as ethylene-v my 1 acetate and lactic acid-glycohc acid enable release of molecules tor over 100 days, certain hydrogels release proteins tor shorter time periods When encapsulated antibodies remain in the body tor a long time, they may denature or aggregate as a result ot exposure to moisture at 37°C.
  • Rational strategies can be devised tor stabilization depending on the mechanism involved For example, it the aggregation mechanism is discovered to be lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions O Methods ot Treatment
  • the antibodies and other anti-tumor compounds ot the present invention mav be used to treat v a ⁇ ous conditions, including those characterized by ov erexpression 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 malignant tumors (e ? , renal, liv er kidney bladder breast gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, ghoblastomas.
  • leukemias and lymphoid malignancies other disorders such as neuronal, glial, astrocytal. hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and lmmunologic disorders
  • the anti-tumor agents ot 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, lntrape ⁇ toneal, intracerobrospinal. subcutaneous lntra-articular, intrasynovial.
  • Intravenous administration ot the antibody is preferred
  • Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e g , antibodies of the instant invention
  • the patient to be treated with such anti-cancer agents may also receive radiation therapy
  • a chemotherapeutic agent may be administered to the patient
  • Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed , M C. Perry, Williams & Wilkins. Baltimore. MD
  • 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 as onap ⁇ 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 ot the present invention
  • simultaneous administration or administration of the antibody of the present invention first is also contemplated Suitable dosages tor 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 will depend on the type of disease to be treated as defined above the severity and course ot the disease whether the agent is administered tor preventive or therapeutic purposes previous therapy the patient s clinical history and response to the agent, and the discretion ot the attending phvsician
  • the agent is suitably administered to the patient at one time or over a series ot treatments
  • ot antibody is an initial candidate dosage for administration to the patient, whether, tor example bv one or more separate administrations, or by continuous infusion
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more depending on the factors mentioned above
  • the treatment is sustained until a desired suppression of disease svmptoms occurs
  • other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays
  • an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises a container and a label Suitable containers include, tor 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 in j ection needle)
  • the active agent in the composition is usually an anti-tumor agent capable of interfering with the activity ot 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 mav further comp ⁇ se a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer
  • tumor diagnostics or prognostics While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for drug 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 ot tumors.
  • antibodies directed against the protein products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics
  • antibodies can be used to qualitatively or quantitativ eh 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 microscopv flow cytometrv. fluo ⁇ metry, or other techniques known in 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
  • 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 ot the marker gene product in the tissue examined It will be apparent for those skilled in the art that a wide variety ot histological methods are readily available for in situ detection
  • ECD extracellular domain sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
  • BLAST or BLAST2 [Altschul et al . Methods in Enzvmologv 266 460-480 ( 1996)] as a comparison of the ECD protein sequences to a 6 frame translation ot the EST sequences Those compa ⁇ sons 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 ot Washington. Seattle. Washington)
  • DNA 102836 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA 102836 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles ot BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above Based on the DNA 102836 consensus sequence, oligonucleotides vv ere synthesized 1 ) to identify by PCR a cDNA library that contained the sequence ot interest, and 2) for use as probes to isolate a clone of the full-length coding sequence tor PRO5800 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product ot about 100-1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized w hen the consensus sequence is greater than about 1-1 5kbp In
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive hbrarv was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs
  • PCR primers (forward and reverse) were synthesized forward PCR primer 1
  • oligonucleotide hybridization probe was constructed from the consensus DNA 102836 sequence which had the following nucleotide sequence hybridization probe
  • RNA tor construction ot the cDNA libraries was isolated from human fetal liver tissue
  • the cDNA libraries used to isolate the cDNA clones were constructed bv standard methods using commercially available reagents such as those from Invitrogen, San Diego.
  • C A The cDNA w s primed with ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl. sized appropriately by gel electrophoresis. and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD.
  • pRK5B is a precursor of pRK5D that does not contain the Sfil site. see. Holmes et al Science. 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites
  • Clone DNA 102880-2689 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 28-30 and ending at the stop codon at nucleotide positions 580-582 ( Figure 3. SEQ ID NO 3)
  • the predicted polypeptide precursor is 184 am o acids long ( Figure 4.
  • the full-length PRO6000 protein shown in Figure 4 has an estimated molecular weight of about 21.052 daltons and a pi of about 5 01
  • Analysis of the full-length PRO6000 sequence shown in Figure 4 evidences the presence ot a variety of important polypeptide domains as shown in Figure 4, wherein the locations given tor those important polvpeptide domains are approximate as described above
  • Clone DNA 102880-2689 has been deposited w ith ATCC on July 20. 1999 and is assigned ATCC Deposit No PTA-383
  • CA upon ESTs as well as clustered and assembled EST fragments from public (e g , Genbank) and/or private (LIFESEQ*, Incyte Pharmaceuticals, Ine , 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 tor 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 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
  • Clone DNA96881 -2699 contains a single open reading frame ith an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 1005-1007 ( Figure 5 SEQ ID NO 5)
  • the predicted polypeptide precursor is 315 amino acids long ( Figure 6 SEQ ID NO 6)
  • the full-length PRO6016 protein shown in Figure 6 has an estimated molecular weight of about 35,963 daltons and a pi of about 5 38
  • Analysis ot the full-length PRO6016 sequence shown in Figure 6 (SEQ ID NO 6) evidences the presence ot a variety of important polypeptide domains as shown in Figure 6. wherein the locations given tor those important polypeptide domains are approximate as described above
  • Clone DNA96881 -2699 has been deposited with ATCC on August 17 1999 and is assigned ATCC Deposit No PTA-553
  • DNA98565-2701 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech. Ine (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e g , Genbank) and/or private (LIFESEQ* 1 , Incyte Pharmaceuticals, Ine , Palo Alto.
  • the signal sequence algorithm computes a secretion signal score based on the character ot 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 am o acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation parameters) known to be associated with secretion signals
  • LIFESEQ* (Incyte Pharmaceuticals, Palo Alto, CA) database, designated herein as 745575H1
  • EST expressed sequence tag
  • public EST databases e.g., Genbank
  • LIFESEQ ® proprietary EST DNA database
  • Clone DNA98565-2701 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 352-357 and ending at the stop codon at nucleotide positions 3085-3087 ( Figure 7 SEQ ID NO 7).
  • the predicted polypeptide precursor is 91 1 amino acids long ( Figure 8. SEQ ID NO 8)
  • the full-length PRO6018 protein shown in Figure 8 has an estimated molecular weight ot about 99,1 17 daltons and a pl of about 4.62.
  • the extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases
  • the EST databases included a proprietary EST database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 [Altschul et al , Methods in Enzymology.
  • DNA43048 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA43048 In some cases, the DNA43048 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 63-65 and a stop signal at nucleotide positions 2328-2330 (Figure 9. SEQ ID NO 9)
  • the predicted polypeptide precursor is 755 amino acids long, has a calculated molecular weight ot approximately 82.785 daltons and an estimated pi ot approximately 8 71
  • Analysis of the full-length PR06496 sequence shown in Figure 10 (SEQ ID NO 10) evidences the presence of a variety ot important polypeptide domains as shown in Figure 10. wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA 1 19302-2737 has been deposited with ATCC on August 10. 1999 and is assigned ATCC Deposit No PTA-520
  • DNA38237 A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA38237 In some cases, the DNA38237 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as tar as possible using the sources ot EST sequences discussed above.
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 102- 104 and a stop signal at nucleotide positions 1083- 1085 ( Figure 1 1.
  • SEQ ID NO 1 1 The predicted polypeptide precursor is 327 amino acids long, has a calculated molecular weight of approximately 34.348 daltons and an estimated pi ot approximately 7 88 Analysis ot the full-length PR07154 sequence shown in Figure 12 (SEQ ID NO 12) evidences the presence ot a variety ot important polypeptide domains as shown in Figure 12. wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA 108760-2740 has been deposited w ith ATCC on August 17.
  • the signal sequence algorithm computes a secretion signal score based on the character ot the DNA nucleotides surrounding the tirst and optionally the second methionine codon(s) (ATG) at the 5'-end ot 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 ammo 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 ot seven sensors (evaluation parameters) known to be associated with secretion signals
  • DNA 108722-2743 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 1506- 1508 ( Figure 13. SEQ ID NO 13)
  • the predicted polypeptide precursor is 482 amino acids long ( Figure 14.
  • SEQ ID NO 14 The full-length PR07170 protein shown in Figure 14 has an estimated molecular weight of about 49,060 daltons and a pi ot about 4 74 Analysis of the full-length PR07170 sequence shown in Figure 14 (SEQ ID NO 14) evidences the presence of a variety of important polypeptide domains as shown in Figure 14 wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA108722-2743 has been deposited with ATCC on August 17, 1999 and is assigned ATCC Deposit No PTA-552
  • the signal sequence algorithm computes a secretion signal score based on the character ot the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end ot 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 sequence 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
  • EST expressed sequence tag
  • the homology search was performed using the computer program BLAST or BLAST2 (Altshul et al . Methods in Enz y mology.
  • Clone DNA 1 19536-2752 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 47-49 and ending at the stop codon at nucleotide positions 31 1 -313 ( Figure 15. SEQ ID NO 15)
  • the predicted polypeptide precursor is 88 amino acids long ( Figure 16)
  • the full-length PR07422 protein shown in Figure 16 has an estimated molecular weight of about 9.645 daltons and a pi of about 5 45
  • Analysis ot the full-length PR07422 sequence shown in Figure 16 evidences the presence ot a variety of important polypeptide domains as shown in Figure 16. wherein the locations given tor those important polypeptide domains are approximate as described above
  • Clone DNA1 19536-2752 has been deposited with ATCC on August 17. 1999 and is assigned ATCC deposit no PTA-551
  • Isolation ot cDNA Clones Encoding Human PRQ74 ⁇ DNA 1 19542-2754 was identified by apply mg a proprietary signal sequence finding algorithm developed by Genentech. Ine . ( South San Francisco C A) upon ESTs as well as clustered and assembled EST fragments from public (e g . GenBank) and/or priv ate (LIFESEQ 0 , Incyte Pharmaceuticals. Ine . Palo Alto.
  • the signal sequence algorithm computes a secretion signal score based on the character ot the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end ot the sequence or sequence fragment under consideiation
  • 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 It neither meets the requirement, the candidate sequence is not scored
  • the DNA and co ⁇ esponding amino acid sequences surrounding the ATG codon are scored using a set ot seven sensors (evaluation parameters) known to be associated with secretion signals
  • DNA 104392 In light of an observed sequence homology between the DNA 104392 sequence and an EST sequence encompassed within clone no 2201 182 from the Incyte database, clone no 2201 182 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence of this cDNA insert is herein designated as DNA1 19542-2754
  • Clone DNA 1 19542-2754 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 247-249 and ending at the stop codon at nucleotide positions 838-840 (Figure 17 SEQ ID NO 17)
  • the predicted polypeptide precursor is 197 amino acids long (Figure 18)
  • the full-length PR07431 protein shown in Figure 18 has an estimated molecular weight ot about 21.992 daltons and a pi ot about 12 18
  • Analysis ot the full-length PR07431 sequence shown in Figure 18 evidences the presence of a variety ot important polypeptide domains as shown in Figure 18. wherein the locations given for those important polypeptide domains are approximate as described above
  • Clone DNA 1 19542-2754 has been deposited with ATCC on August 31 , 1999 and is assigned ATCC deposit no PTA-619
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular
  • oligonucleotide hybridization probe was constructed from the consensus DNA 102863 sequence which had the following nucleotide sequence hybridization probe.
  • the full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 62-64 and a stop signal at nucleotide positions 701 -703 ( Figure 19. SEQ ID NO 19)
  • the predicted polypeptide precursor is 213 amino acids long, has a calculated molecular weight ot approximately 24.031 daltons and an estimated pi ot approximately 9 59
  • Analysis ot the full-length PR07476 sequence shown in Figure 20 evidences the presence of a variety of important polypeptide domains as shown in Figure 20 wherein the locations given for those important polypeptide domains are approximate as desc ⁇ bed above Clone DNA 1 15253-2757 has been deposited with ATCC on August 31 1999 and is assigned ATCC Deposit No PTA-612
  • PR07431 or PR07476 polypeptides for example, mu ⁇ ne- human chimeric. humanized or human antibodies against a PRO5800. PRO6000. PRO6016, PRO6018. PR06496, PR07154. PRO7170, PR07422. PR07431 or PR07476 polypeptide
  • the starting mate ⁇ al tor the screen was genomic DNA isolated from a variety of cancers
  • the DNA is quantitated precisely, e g , fluoromet ⁇ cally
  • DNA was isolated from the cells of ten 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 whether the DNA encoding PRO5800, PRO6000.
  • PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 is over-represented in 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 individuals with tumors of the type and stage as indicated in Table 4
  • An explanation ot the abbreviations used for the designation of the primary tumors listed in Table 4 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore
  • the results of the TaqMan r are reported in delta ( ⁇ ) Ct units One unit corresponds to 1 PCR cycle or approximately a 2-told amplification relative to normal, two units corresponds to 4-fold 3 units to 8-fold amplification and so on Quantitation was obtained using primers and a TaqManTM fluorescent probe derived from the PRO5800-. PRO6000-, PRO6016-, PRO6018-. PR06496-. PR07154-, PR07170-, PR07422-, PR07431 - or PR07476-encod ⁇ ng gene Regions of PRO5800. PRO6000. PRO6016. PRO6018. PR06496, PR07154.
  • PR07170, PR07422, PR07431 or PR07476 which are most likely to contain unique nucleic acid sequences and which are least likely to have spliced out introns are preferred for the primer and probe de ⁇ v ation. e g .3'-untranslated regions
  • PR07431 or PR07476 gene amplification analysis were as follows
  • PRO5800 (DNA 108912-2680) 108912.tm t l
  • PRO6000 (DNA 102880-2689) 102880 tmfl
  • PRO6016 (DNA96881 -2699) 96881 tmfl 5'-TGGCCTACATGTGTCTTCATC-3 (SEQ ID NO 43) 96881 tmrl
  • PRO7170 (DNA 108722-2743): 108722.tm.fl:
  • PR07431 (DNA 119542-2754): 119542.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 oligonucleotide primers are used to generate an amplicon typical ot 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 the released reporter dye is free from the quenching effect of the second fluorophore
  • the 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700TM Sequence Detection
  • a real-time quantitative PCR device such as the ABI Prism 7700TM Sequence Detection
  • the system consists ot a thermocycler, laser, charge-coupled device (CCD) camera and computer
  • CCD charge-coupled device
  • 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 running the instrument and for analyzing the data
  • Ct 5' Nuclease assay data are initially expressed as Ct. or the threshold cycle This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence
  • ⁇ Ct values are used as quantitative measurement ot 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 4 describes the stage T stage and N stage of various primary tumors which were used to screen the PRO5800, PRO6000, PRO6016, PRO6018 PR06496.
  • Table 4 Primary Lung and Colon Tumor Profiles
  • DNA was prepared from cultured cell lines, primary tumors, and normal human blood The isolation was performed using purification kit, buffer set and protease and all from Qiagen. according to the manufacturer s instructions and the description below
  • Buffer Cl 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 tor 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 bv a second 4"C centrifugation at 2500 rpm for 1 ⁇ 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 was applied Upon completion ot buffer addition vigorous vortexing was applied tor 30 seconds
  • Qiagen protease 200 ⁇ l, prepared as indicated above
  • 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 ml cold ddH ⁇ O 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 ot the polvtron in a laminar-flow TC hood in order to avoid inhalation ot 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 Qiagen protease (prepared as indicated above 1 0 ml) was added, followed by vortexing and incubation at 50°C for 3 hours The incubation and centrifugation were repeated until the lysates were clear (e g , incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min , 4°C)
  • Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) w ith 10 ml QBT butter QF elution butter was equilibrated at 50 C The samples were vortexed for 30 seconds then loaded onto equilibrated tips and drained by gravity The tips w ere washed with 2 x 15 ml QC buffer The DNA was eluted into 30 ml silanized, autoclaved 30 ml Corex tubes w ith 15 ml QF buffer (50°C) Isopropanol ( 10 5 ml) was added to each sample, the tubes covered with paratin and mixed by repeated inversion until the DNA precipitated Samples were pelleted by centrifugation in the SS-34 rotor at 15.000 rpm tor 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 rot
  • the fluoromet ⁇ cly determined concentration was then used to dilute each sample to 10 ng/ ⁇ l in ddH ⁇ O This was done simultaneously on all template samples for a single TaqManTM plate assay, and with enough material to run 500-1000 assays
  • the samples were tested in triplicate with TaqmanTM primers and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls
  • the diluted samples were used prov ided that the CT value ot normal human DNA subtracted from test DNA was +/- 1 Ct
  • the diluted, lot qualified genomic DNA was stored in 1 0 ml ahquots at -80 C Ahquots which were subsequently to be used in the gene amplification assay were stored at 4"C Each 1 ml aliquot is enough for 8-9 plates or 64 tests Gene amplification
  • PRO5800 PRO6000 PRO6016 PRO6018 PR06496 PR07154 PRO7170 PR07422 PR07431 or PR07476 compounds of the invention were screened in the following primary tumors and the resulting ⁇ Ct values are reported in Table 5 Table 5 ⁇ Ct Values in Lung, Colon and Other Primary Tumoi and Cell Line Models
  • ⁇ Ct values tor DNA 108912-2680 in a va ⁇ etv ot tumors are reported in Table 5
  • a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling ot gene copv
  • Table 5 indicates that significant amplification of nucleic acid DNA 108912-2680 encoding PRO5800 occurred in p ⁇ mary lung tumors HF-001644 and HF-001647
  • DNA 108912-2680 Because amplification ot DNA 108912-2680 occurs in various lung tumors, 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 DNA 108912-2680 (PRO5800) would be expected to have utility in cancer therapy
  • PRO6016 (DNA96881 -2699)
  • DNA96881 -2699 occurs in various tumors 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 DNA96881-2699 (PRO6016) would be expected to have utility in cancer therapy
  • PRO6018 (DNA98565-2701 )
  • DNA1 19302-2737 Because amplification of DNA1 19302-2737 occurs in various lung tumors, 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 DNA1 19302-2737 (PR06496) would be expected to have utility in cancer therapy
  • PRQ7154 (DNA 108760-2740)
  • the ⁇ Ct values for DNA 108760-2740 in a variety ot tumors are reported in Table 5 A ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table
  • DNA 108760-2740 Because amplification of DNA 108760-2740 occurs in various lung tumors, 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 DNA 108760-2740 (PR07154) would be expected to have utility in cancer therapy
  • DNA 108722-2743 Because amplification of DNA 108722-2743 occurs in lung tumor, it is highly probable to play a significant role in tumor formation or growth As a result, antagonists (e s> , antibodies) directed against the protein encoded by DNA 108722-2743 (PRO7170) would be expected to have utility in cancer therapy
  • PRQ7476 as a hybridization probe
  • the following method describes use of a nucleotide sequence encoding a PRO5800. PRO6000. PRO6016,
  • PRO6018 -'PR06496.
  • PR07154 PRO7170
  • Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions
  • Hybridization ot radiolabeled PRO5800-, PRO6000- PRO6016-, PRO6018- PR06496- PR07154-, PR07170-, PR07422- PR07431 - or PR07476-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
  • PRQ7431 or PRQ7476 Polypeptides in £ coli This example illustrates preparation ot an unglycosylated form ot PRO5800 PRO6000, PRO6016, PRO6018 PR06496, PR07154 PRO7170 PR07422 PR07431 or PR07476 bv recombinant expression in E
  • the DNA sequence encoding the PRO polypeptide of interest 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 ot expression vectors may be employed
  • An example of a suitable vector is pBR322 (derived from E coli see Bolivar et al Gene 2 95 (1977)) which contains genes tor ampicillin 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 trp promoter a poly-His leader (including the first six STII codons, polv-His sequence, and enterokinase cleavage site) the PRO5800 PRO6000, PRO6016, PRO6018, PRO6496, PRO7154 PRO7170, PR07422, PR07431 or PR07476 coding region lamb
  • the ligation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
  • the cells After culturing the cells for several more hours, the cells can be harvested by centrifugation
  • the cell pellet obtained by the centrifugation can be solubilized using various agents known in the art and the solubilized PRO5800 PRO6000 PRO6016 PRO6018 PR06496 PR07154 PRO7170 PR07422 PR07431 or PR07476 protein can then be purified using a metal chelating column under conditions that allow tight binding ot the protein
  • PRO5800 PRO6000 PRO6016 PRO6018, PR06496 PR07154 PRO7170 PR07422 PR07431 or
  • PR07476 may be expressed in E co/. in a poly His tagged form using the following procedure The DNA encoding
  • PRO5800, PRO6000, PRO6016 PRO6018 PR06496 PR07154, PRO7170 PR07422 PR07431 or PR07476 is initially amplified using selected PCR primers
  • the primers contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector and other useful sequences providing tor efficient and reliable translation initiation rapid purification on a metal chelation column and proteolvtic removal with enterokinase
  • the PCR-amphfied, polv-His tagged sequences are then hgated into an expression vector which is used to transform an E coli host based on strain 52 (W31 10 tuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq)
  • Transformants are first grown in LB containing 50 mg ml carbenicilhn at 30°C with shaking until an O D ot 3-5 at 600 nm is reached Cultures are then diluted 50-100 fold into
  • E coli paste from 0 5 to 1 L fermentations (6 10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM T ⁇ s, pH 8 buffer Solid sodium sultite and sodium tetrathionate are added to make final concentrations ot 0 I M and 0 02 M.
  • the protein is refolded by diluting sample slowly into freshly prepared refolding buffer consisting of 20 M T ⁇ s, pH 8 6, 0 3 M NaCl, 2 5 M urea. 5 mM cysteine, 20 mM glycine and 1 mM EDTA Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml
  • the refolding solution is stirred gently at 4°C for 12-36 hours
  • the refolding reaction is quenched by the addition of TFA to a final concentration of 04% (pH of approximately 3)
  • the solution is filtered through a 0 22 micron filter and acetonitrile is added to 2-10% final concentration
  • the refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0 1 % TFA with elution with a gradient of acetonitrile from 10 to 80% Aliquots of fractions with A, so absorbance
  • PR07431 or PR07476 protein are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution Proteins are formulated into 20 mM Hepes, pH 6 8 with 0 14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered
  • PRQ74 1 or PRQ7476 in mammalian cells This example illustrates preparation ot a potentially glycosylated form ot PRO5800.
  • PRO6018. PR06496 PR07154. PRO7170, PR07422. PR07431 or PR07476 by recombinant expression in mammalian cells The vector pRK5 (see EP 307,247, published March 15. 1989) is employed as the expression vector
  • PRO6016, PRO6018 PR06496, PR07154, PR07170, PR07422 PR07431 or PR07476 DNA is hgated into pRK5 with selected restriction enzymes to allow insertion ot the PRO5800, PRO6000, PRO6016, PRO6018, PR06496.
  • PR07154 PR07170, PR07422, PR07431 or PR07476 DNA using hgation methods such as described in Sambrook et al , supra
  • the resulting vector is called pRK5-[PRO5800, PRO6000 PRO6016. PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476]
  • 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-[PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476] DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al , CeM, 31 543 (1982)] and dissolved in 500 ⁇ l of 1 mM Tris- HCl, 0 1 mM EDTA, 0 227 M CaCl 2 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
  • the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 35 S-cyste ⁇ ne and 200 ⁇ Ci/ml 35 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 the PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422.
  • PR07431 or PR07476 polypeptide The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays
  • PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154 PR07170, PR07422, PR07431 or PR07476 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al . Proc Natl Acad Sci J_2 7575 (1981 ) 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-[PRO5800, PRO6000, PRO6016 PRO6018. PR06496. PR07154, PRO7170, PR07422.
  • PR07431 or PR07476 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 tor four hours The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium and re- lntroduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bov ine insulin and 0 1 ⁇ g/ml bovine transfer ⁇ n After about four days, the conditioned media is cent ⁇ fuged and filtered to remove cells and debris The sample containing expressed PR05800 PRO6000 PRO6016, PRO6018 PR06496, PR07154 PRO7170 PR07422 PR07431 or PR07476 can then be concentrated and purified by any selected method such as dialysis and/or column chromatography
  • PR07431 or PR07476 can be expressed in CHO cells
  • the pRK5-[PRO5800 PRO6000 PRO6016, PRO6018, PR06496 PR07154, PR07170 PR07422, PR07431 or PR07476] v ector can be transfected into CHO cells using known reagents such as CaP0 4 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 35 S- methiomne After determining the presence ot the PRO5800 PRO6000. PRO6016.
  • PR07431 or PR07476 polypeptide the culture medium may be replaced with serum free medium Preferably, the cultures are incubated for about 6 days and then the conditioned medium is harvested The medium containing the expressed PRO5800. PRO6000. PRO6016. PRO6018, PR06496. PR07154, PRO7170, PR07422, PR07431 or PR07476 can then be concentrated and purified by any selected method
  • Epitope-tagged PRO5800. PRO6000. PRO6016. PRO6018. PR06496, PR07154. PR07170. PR07422, PR07431 or PR07476 may also be expressed in host CHO cells
  • the PRO5800. PRO6000, PRO6016. PRO6018, PR06496. PR07154, PR07170. PR07422, PR07431 or PR07476 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 poiy-His tag into a Baculovirus expression vector
  • PR07431 or PR07476 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
  • PR07154, PRO7170, PR07422, PR07431 or PR07476 can then be concentrated and purified by any selected method, such as by N ⁇ 2+ -chelate affinity chromatography
  • Expression in CHO and/or COS cells may also be accomplished by a transient expression procedure PRO5800.
  • PR07476 may be expressed in CHO cells by a transient procedure Stable expression in CHO cells can be performed using the following procedure
  • the proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or in a poly-His tagged form Following PCR amplification the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al . Current Protocols ot Molecular Biology, Unit 3 16.
  • CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA ot interest to allow the convenient shuttling of cDNA' s
  • the vector used tor expression in CHO cells is as described in Lucas et al , Nucl Acids Res . 24 9 (1774-1779 ( 1996), and uses the S V40 early promoter/enhancer to drive expression ot the cDNA of interest and dihydrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance ot the plasmid following transfection
  • the ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing
  • the contents are pipetted into a centrifuge tube containing 10 mis ot media and cent ⁇ tuged at 1000 rpm tor 5 minutes
  • the supernatant is aspirated and the cells are resuspended in 10 ml ot selective media (0 2 ⁇ m filtered PS20 with 57c 0.2 ⁇ m diafiltered fetal bovine serum).
  • the cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media. After I -2 days, the cells are transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37"C.
  • spinners After another 2-3 days. 250 ml, 500 ml and 2000 ml spinners are seeded with 3 x 10 5 cells/ml. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Patent No. 5,122,469, issued June 16. 1992 is actually used. 3L production spinner is seeded at 1.2 x I O 6 cells/ml. On day 0. the cell number and pH are determined. On day 1 , the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33"C.
  • the proteins are purified using a Ni 2+ -NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni :+ -NTA column equilibrated in 20 mM Hepes, pH 7.4. buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole.
  • the highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
  • lmmunoadhesin (Fc containing) constructs are purified from the conditioned media as follows.
  • the conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid. pH 3.5.
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ⁇ l of 1 M Tris buffer. pH 9.
  • the highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins.
  • the homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
  • PRQ7431 or PRQ7476 in Yeast The following method describes recombinant expression of PRO5800, PRO6000, PRO6 16. PRO6018.
  • PR06496 PR07154, PRO7170. PR07422. PR07431 or PR07476 in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of PRO5800.
  • PR07431 or PR07476 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO5800.
  • PR07431 or PR07476 for secretion.
  • PR07431 or PR07476 can be cloned into the selected plasmid. together with DNA encoding the ADH2/GAPDH promoter, a native PRO5800 PRO6000, PRO6016. PRO6018. PR06496. PR07154. PR07170 PR07422, PR07431 or PR07476 signal peptide or other mammalian signal peptide, or, for example, a veast alpha-factor or invertase secretory signal/leader sequence, and linker sequences of needed) tor expression ot PRO5800. PRO6000. PRO6016. PRO6018. PR06496. PR07154. PRO7170. PR07422. PR07431 or PR07476
  • 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 supematants can be analyzed by precipitation with 10% t ⁇ chloroacetic acid and separation by SDS-PAGE. followed by staining ot the gels with Coomassie Blue stain
  • Recombinant PRO5800, PRO6000. PRO6016. PRO6018, PR06496. PR07154, PRO7170, PR07422, PR07431 or PR07476 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 PRO5800, PRO6000. PRO6016. PRO6018. PR06496. PR07154, PRO7170. PR07422. PR07431 or PR07476 may further be purified using selected column chromatography resins
  • PRO6000 PRO6016 PRO601 . PRQ6496. PRQ7154. PRO7170 PRQ7422. PRQ7431 or PRQ7476 in Baculovirus-infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells The sequence coding for PRO5800. PRO6000, PRO6016, PRO6018. PR06496, PR07154, PRO7170,
  • PR07422, PR07431 or PR07476 is fused upstream of an epitope tag contained within a baculovirus expression vector
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions ot IgG)
  • plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO5800. PRO6000. PRO6016 PRO6018. PR06496. PR07154 PR07170, PR07422. PR07431 or PR07476 or the desired portion ot the coding sequence ot PRO5800. PRO6000. PRO6016. PRO6018.
  • PR07431 or PR07476 [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 w ith 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 bv co-transfectmg the above plasmid and BaculoGoldTM v ⁇ rus DNA
  • Expressed poly-His tagged PRO5800 PRO6000. PRO6016 PRO6018. PR06496. PR07154, PR07170 PR07422. PR07431 or PR07476 can then be purified, for example, by Nr + -chelate aftinitv chromatography as follows Extracts are prepared from recombinant virus-intected 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 ., 0 1 mM EDTA 10% glycerol, 0 1 % NP-40.
  • sonication buffer 25 ml Hepes. pH 7 9. 12 5 mM MgCl ., 0 1 mM EDTA 10% glycerol, 0 1 % NP-40.
  • N ⁇ 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml ot water and equilibrated ith 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- s0 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 A :8 Deutschenbasehne again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer One ml fractions are collected and anal zed by SDS-PAGE and silver staining or Western blot with N ⁇ 2+ -NTA-conjugated
  • PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
  • the proteins are expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region is fused to an IgGl 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
  • Baculogold® baculovirus DNA Puringen
  • Sf9 Spodopteta ftugipeida
  • pb PH IgG and pb PH His are modifications ot the commercial lv av ailable baculovirus expression vector pVLl 393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences
  • the cells are grown in Hink s TNM-FH medium supplemented with 10% FBS (Hyclone ) Cells are incubated for 5 days at 28 °C
  • the supernatant is harvested and subsequently used tor the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOD of 10 Cells are incubated for 3 days at 28 °C
  • the supernatant is harvested and the expression ot the constructs in the baculovirus expression vector is determined by batch binding ot 1 ml of supernatant to 25 ml of Ni + -NTA beads (QI
  • ESF-921 medium (Expression Systems LLC) at an approximate MOI ot 0 1 Cells are incubated tor 3 dav b at 28 °C The supernatant is harvested and filtered Batch binding and SDS-PAGE analysis are repeated, as necessary, until expression of the spinner culture is confirmed
  • the conditioned medium from the transfected cells (0 5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0 22 micron filters
  • the protein construct is purified using a Ni 2+ -NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni " NTA column equilibrated in 20 mM Hepes pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4°C After loading, the column is washed with additional equilib
  • the conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer. pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid. pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml ot 1 M T ⁇ s buffer, pH 9 The highly purified protein is subsequently desalted into storage buffer as described above tor the poly-His tagged proteins The homogeneity of the proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation
  • a modified baculovirus procedure may be used incorporating high 5 cells
  • the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) ot an epitope tag contained with a baculovirus expression vector
  • epitope tags include poly- His tags and immunoglobulin 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 ot recombinant proteins from the baculovirus le i promoter in stably- transformed insect cells
  • the plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIE
  • High 5 cells are grown to a confluency of 50% under the conditions of 27 °C. no C0 2 .
  • NO pen/strep For each 150 mm plate, 30 ⁇ g of pIE based vector containing the sequence is mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401 -78P (note this media is light sensitive)), and in a separate tube.
  • the conditioned media from the transfected cells (0 5 to 3 L) is harv ested by centrifugation to remove the cells and filtered through 0 22 micron filters
  • the protein comprising the sequence is purified using a Ni 2 "-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration ot 5 mM
  • the conditioned media is pumped onto a 6 ml Ni + -NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 48°C
  • the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole
  • the highly purified protein is then subsequently desalted into a storage buffer containing 1 O mM Hepes, 0 14 M NaCl and
  • the eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M T ⁇ s buffer, pH 9
  • the highly purified protein is subsequently desalted into storage buffer as described above tor the poly-His tagged proteins
  • the homogeneity of the sequence is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary
  • PRO6018, PR07154, PRO7170 and PR07476 were successfully expressed by the above modified baculovirus procedure inco ⁇ orating high 5 cells
  • PRO7170 PRO7170. PRQ7422. PRQ7431 or PRQ7476 This example illustrates preparation of monoclonal antibodies which can specifically bind PRO5800, PRO6000, PRO6016. PRO6018. PR06496, PR07154. PR07170, PR07422, PR07431 or PR07476
  • Immunogens that may be employed include purified PRO5800. PRO6000. PRO6016. PRO6018, PR06496. PR07154. PRO7170. PR07422, PR07431 or PR07476 fusion proteins containing PRO5800. PRO6000, PRO60I 6. PRO6018 PR06496, PR07154. PRO7170. PR07422. PR07431 or PR07476 and cells expressing recombinant PRO5800, PRO6000, PRO6016. PRO6018. PR06496, PR07154, PR07170, PR07422.
  • PR07431 or PR07476 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 w ith the PRO5800 PRO6000, PRO6016. PRO6018 PR06496.
  • PR07154 PR07170, PR07422, PR07431 or PR07476 immunogen emulsified in complete Freund s adjuvant and lniected subcutaneously or intraperitoneally in an amount from 1-100 micrograms Alternativ ely, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind toot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several vv eeks.
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, MT
  • mice may also be boosted with additional immunization injections
  • Serum samples may be pe ⁇ odicallv obtained from the mice by retro-orbital bleeding tor testing in ELISA assays to detect ant ⁇ -PRO5800, ant ⁇ -PRO6000. ant ⁇ -PRO6016. ant ⁇ -PRO6018. ant ⁇ -PR06496. anti- PRO? 154 ant ⁇ -PRO7170, ant ⁇ -PR07422, ant ⁇ -PR07431 or ant ⁇ -PR07476 antibodies After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO5800. PRO6000, PRO6016, PRO6018, PR06496.
  • mice Three to tour days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63 AgU 1 , available from ATCC, No CRL 1597 The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine. ammopterin. and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids
  • HAT hyperxanthine. ammopterin. and thymidine
  • hybridoma cells will be screened in an ELISA for reactivity against PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO5800, PRO6000. PRO6016, PRO6018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 is within the skill in the art
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the ant ⁇ -PRO5800, ant ⁇ -PRO6000, ant ⁇ -PRO6016, ant ⁇ -PRO6018, ant ⁇ -PR06496. ant ⁇ -PR07154, ant ⁇ -PRO7170.
  • 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 Altemativelv 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 upon 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 as compared to normal cells of the same tissue type 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 as predictors of the prognosis of tumor treatment. The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR
Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of tumor
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 an 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 variety of forms, characterized by different degrees of invasiveness and aggressiveness Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which 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 would normally function to prevent malignant cell growth, and/or overexpression of certain 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 ull neoplastic phenotvpe (Hunter. Cell. 64 1 129 [ 1991 ] and Bishop, Cell. 64 235-248 [19911)
A well known mechanism ot 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, ; e is proportionate to the number of copies made
Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of vaπous 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 glycoprotein receptor (p 185HER2, HER2) related to the epidermal growth factor receptor EGFR), is overexpressed in about 25% to 30% of human breast cancer (Slamon et al , Science. 235 177-182 [1987]. Slamon et al . Science 244 707-712 [1989])
It has been reported that gene amplification of a proto-oncogene is 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], Alitalo etal , sup ra) Thus, erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al , [1987] and [ 1989], supt a. Ravdin and Chamness, Gene. 159 19-27 [ 1995] , and Hynes and Stern, Biochim Biophys Acta, 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 anthracychnes (Baselga et al , Oncology. _H (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-negatι ve patients (Ibid) A recombinant humanized antι-ErbB2 (antι-HER2) monoclonal antibody (a humanized version of the murine antι-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 , J Clin Oncol 737-744 [1996]) In light of the above there is obvious interest in identifying novel methods and compositions which are useful for diagnosing and treating tumors which are associated with gene amplification
Summary of the Invention A Embodiments 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 contribute to tumoπgenesis 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 mav act as predictors of the prognosis of tumor treatment
In one embodiment the present invention concerns an isolated antibody which binds to a polypeptide designated herein as a PRO polypeptide In one aspect, the isolated antibody specifically binds to a PRO polypeptide In another aspect, the antibody induces the death of a cell which expresses a PRO polypeptide Often, the cell that expresses the PRO polypeptide is a tumor cell that overexpresses the polypeptide as compared to a normal cell of the same tissue type In yet another aspect, the antibody is a monoclonal antibody, which preferably has non-human complementaπtv determining region (CDR) residues and human framework region (FR) residues The antibody may be labeled and may be immobilized on a solid support In yet another aspect, the antibody is an antibody fragment a single-chain antibody, or a humanized antibody which binds, preferably specifically, to a PRO polypeptide In another embodiment the invention concerns a composition ot matter which comprises an antibody which binds, preferably specifically, to a PRO polypeptide in admixture with a pharmaceutically acceptable carrier In one aspect, the composition of matter compπses 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 Preferablv the composition is sterile
In a further embodiment, the invention concerns isolated nucleic acid molecules which encode anti-PRO antibodies and vectors and recombinant host cells comprising such nucleic acid molecules
In a still further embodiment, the invention concerns a method tor producing an anti-PRO antibody, wherein the method comprises culturing a host cell transformed with a nucleic acid molecule which encodes the antibody under conditions sufficient to allow expression of the antibody, and recovering the antibody from the cell culture
The invention further concerns antagonists of a PRO polypeptide that inhibit one or more of the biological and/or immunological functions or activities of a PRO polypeptide In a further embodiment, the invention concerns an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule encoding a PRO polypeptide or the complement thereof The isolated nucleic acid molecule is preferably DNA, and hybridization preferably occurs under stringent hybridization and wash 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 transcription and/or translation of the respective amplified genes, or as antisense primers in amplification reactions Furthermore, such sequences can be used as part of a ribozyme and/or a triple helix sequence which, in turn, may be used in regulation of the amplified genes
In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a sample suspected of containing a PRO polypeptide, wherein the method comprises exposing the sample to an anti-PRO antibody and determining binding of the antibody to a PRO polypeptide in the sample. In another embodiment, the invention provides a method for determining the presence of a PRO polypeptide in a cell, wherein the method comprises exposing the cell to an anti-PRO 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 PRO 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 as compared to the control sample, is indicative of 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 anti-PRO antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-PRO antibody and a PRO polypeptide in the test sample, wherein the formation of a complex is indicative of the presence ot a tumor in said mammal The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type A larger quantity of complexes formed in the test sample indicates the presence 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, tor 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-PRO antibody and a carrier (e g , a buffer) in suitable packaging The kit preferably contains instructions for using the antibody to detect the presence ot a PRO polypeptide in a sample suspected of containing the same
In yet another embodiment, the invention concerns a method for inhibiting the growth of tumor cells comprising exposing tumor eel Is which express a PRO polypeptide to an effective amount of an agent which inhibits a biological and or immunological activity and/or the expression of a PRO polypeptide, wherein growth of the tumor cells is thereby inhibited The agent preferably is an anti-PRO antibody, a small organic and inorganic molecule, peptide, phosphopeptide. antisense or ribozyme molecule, or a triple helix molecule In a specific aspect, the agent, e g , the anti-PRO 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 composing an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells and the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO polypeptide as compared to a normal cell of the same tissue type In particular aspects, the active agent in the composition is an agent which inhibits an activity and/or the expression of a PRO polypeptide In preferred aspects, the active agent is an anti-PRO antibody or an antisense oligonucleotide The invention also provides a method for identifying a compound that inhibits an activity of a PRO polypeptide, compnsmg contacting a candidate compound with a PRO polypeptide under conditions and for a time sufficient to allow these two components to interact and determining whether a biological and/or immunological activity of the PRO polypeptide is inhibited In a specific aspect, either the candidate compound or the PRO polypeptide is immobilized on a solid support In another aspect, the non-immobilized component carries a detectable label In a preferred aspect, this method comprises the steps ot (a) contacting cells and a candidate compound to be screened in the presence of the PRO polypeptide under conditions suitable for the induction of a cellular response normally induced by a PRO polypeptide and (b) determining the induction ot said cellular response to determine if the test compound is an effective antagonist
In another embodiment the invention provides a method for identifying a compound that inhibits the expression of a PRO polypeptide in cells that express the polypeptide, wherein the method comprises contacting the cells with a candidate compound and determining whether the expression of the PRO polypeptide is inhibited In a preferred aspect, this method comprises the steps of (a) contacting cells and a candidate compound to be screened under conditions suitable for allowing expression ot the PRO polypeptide and (b) determining the inhibition of expression ot said polypeptide
B Additional Embodiments
In other embodiments ot the present invention, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)
In other aspects, the isolated nucleic acid molecule compπses a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide. with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a) In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDN As deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a)
Another aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domaιn(s) ot such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein descπbed PRO polypeptides are contemplated
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, hybπdization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 1 10 nucleotides in length, altemativelv at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, altemativelv at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term about means the referenced nucleotide sequence length plus or minus 10% of that referenced length It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any ot a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence tragment(s) are novel All ot such PRO polypeptide-encoding nucleotide sequences are contemplated herein Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody In another embodiment, the invention provides an isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified
In a ceπain aspect, the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide. as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% am o acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, altemativelv at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein
In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence scoring at least about 80% positives, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83% positives, alternatively at least about 84% positives, alternatively at least about 85% positives, alternatively at least about 86% positives, altemativelv at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93% positives, alternatively at least about 94% positives, alternatively at least about 95% positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO polypeptide hav ing a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an ammo acid sequence as hereinbefore described Processes for producing the same are also herein described, wherein those processes compπse culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression ot the PRO polypeptide and recovering the PRO polypeptide from the cell culture
Another aspect of the present invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture
In yet another embodiment, the invention concerns antagonists of a native PRO polypeptide as defined herein In a particular embodiment, the antagonist is an anti-PRO antibody or a small molecule
In a further embodiment, the invention concerns a method of identifying antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide Preferably, the PRO polypeptide is a native PRO polypeptide
In a still further embodiment, the invention concerns a composition of matter comprising a PRO polypeptide. or an antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier Optionally, the carrier is a pharmaceutically acceptable carrier Another embodiment of the present invention is directed to the use of a PRO polypeptide. or an antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide. an antagonist thereof or an anti-PRO antibody
In additional embodiments of the present invention, the invention provides \ectors comprising DNA encoding any ot the herein described polypeptides Host cells comprising any such vector are also provided By way of example the host cells may be CHO cells. E coli, yeast, or Baculovirus-intected insect cells A process for producing anv ot the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides tused to a heterologous polypeptide or amino acid sequence Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin In yet another embodiment, the invention provides an antibody which specifically binds to any of the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody
In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences
Brief Description of the Figures Figure 1 shows the nucleotide sequence (SEQ ID NO 1 ) ot a cDNA containing a nucleotide sequence encoding native sequence PRO5800, wherein the nucleotide sequence (SEQ ID NO 1 ) is a clone designated herein as DNA 108912-2680 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 2 shows the amino acid sequence (SEQ ID NO 2) of a native sequence PRO5800 polypeptide as derived from the coding sequence of SEQ ID NO 1 shown in Figure 1
Figure 3 shows the nucleotide sequence (SEQ ID NO 3) of a cDNA containing a nucleotide sequence encoding native sequence PRO6000, wherein the nucleotide sequence (SEQ ID NO 3) is a clone designated herein as DNA102880-2689 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 4 shows the amino acid sequence (SEQ ID NO 4) of a native sequence PRO6000 polypeptide as derived from the coding sequence of SEQ ID NO 3 shown in Figure 3 Figure 5 shows the nucleotide sequence (SEQ ID NO 5) of a cDNA containing a nucleotide sequence encoding native sequence PRO6016 wherein the nucleotide sequence (SEQ ID NO 5) is a clone designated herein as DNA96881 -2699 Also presented in bold font and underlined are the positions of the respecm e staπ and stop codons
Figure 6 shows the amino acid sequence (SEQ ID NO 6) of a native sequence PRO6016 polypeptide as derived from the coding sequence of SEQ ID NO 5 shown in Figure 5
Figure 7 shows the nucleotide sequence (SEQ ID NO 7) of a cDNA containing a nucleotide sequence encoding native sequence PRO6018, wherein the nucleotide sequence (SEQ ID NO 7) is a clone designated herein as DNA98565-2701 Also presented in bold font and underlined are the positions of the respective start and stop codons Figure 8 shows the ammo acid sequence (SEQ ID NO 8) of a native sequence PRO6018 polypeptide as derived from the coding sequence of SEQ ID NO 7 shown in Figure 7
Figure 9 shows the nucleotide sequence (SEQ ID NO 9) of a cDNA containing a nucleotide sequence encoding native sequence PR06496 wherein the nucleotide sequence (SEQ ID NO 9) is a clone designated herein as DNA 1 19302-2737 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 10 shows the amino acid sequence (SEQ ID NO 10) of a native sequence PR06496 polypeptide as derived from the coding sequence of SEQ ID NO 9 shown in Figure 9 Figure 11 shows the nucleotide sequence (SEQ ID NO 1 1 ) of a cDNA containing a nucleotide sequence encoding native sequence PR07154, wherein the nucleotide sequence (SEQ ID NO 1 1 ) is a clone designated herein as DNA 108760-2740 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 12 shows the amino acid sequence (SEQ ID NO 12) of a native sequence PR07154 polypeptide as derived from the coding sequence of SEQ ID NO 1 1 shown in Figure 1 1
Figure 13 shows the nucleotide sequence (SEQ ID NO 13) of a cD A containing a nucleotide sequence encoding native sequence PR07170 wherein the nucleotide sequence (SEQ ID NO 13) is a clone designated herein as DNA 108722-2743 Also presented in bold font and underlined are the positions of the respective start and stop codons Figure 14 shows the amino acid sequence (SEQ ID NO 14) of a native sequence PRO7170 polypeptide as derived from the coding sequence of SEQ ID NO 13 shown in Figure 13
Figure 15 shows the nucleotide sequence (SEQ ID NO 15) of a cDNA containing a nucleotide sequence encoding native sequence PR07422, wherein the nucleotide sequence (SEQ ID NO 15) is a clone designated herein as DNA1 19536-2752 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 16 shows the amino acid sequence (SEQ ID NO 16) of a native sequence PR07422 polypeptide as derived from the coding sequence of SEQ ID NO 15 shown in Figure 15
Figure 17 shows the nucleotide sequence (SEQ ID NO 17) of a cDNA containing a nucleotide sequence encoding native sequence PR07431 , wherein the nucleotide sequence (SEQ ID NO 17) is a clone designated herein as DNA1 19542-2754 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 18 shows the amino acid sequence (SEQ ID NO 18) of a native sequence PR07431 polypeptide as derived from the coding sequence of SEQ ID NO 17 shown in Figure 17
Figure 19 shows the nucleotide sequence (SEQ ID NO 19) of a cDNA containing a nucleotide sequence encoding native sequence PR07476 wherein the nucleotide seq uence (SEQ ID NO 19) is a clone designated herein as DNA115253-2757 Also presented in bold font and underlined are the positions of the respective start and stop codons
Figure 20 shows the amino acid sequence (SEQ ID NO 20) of a native sequence PR07476 polypeptide as derived from the coding sequence of SEQ ID NO 19 shown in Figure 19
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. - e , the level of gene expression, also increases in the proportion of the number ot 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 by 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, gastrointestinal cancer, 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 preventati ve 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, cattle, pigs sheep, etc Preferably, the mammal is human
"Carπers as used herein include pharmaceutically acceptable carriers, excipients or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution Examples of physiologically acceptable carπers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins. hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosacchaπdes. disacchaπdes and other carbohydrates including glucose. mannose, or dextπns. chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol. salt-forming counterions such as sodium and/or nonionic surfactants such as TWEEN™ 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 ot cells The term is intended to include radioactive isotopes (e g , I l I12' Y9U 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 chemotherapeutic agents include adπamycin, doxorubicin, epirubicin, 5-fluorouracιl, cytosine arabinoside ("Ara- C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g , pac taxel (Taxol. Bπstol-Mvers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhόne-Poulenc Rorer, Antony, Rnace), toxotere methotrexate. cisplatin, melphalan, vinblastine bleomycin, etoposide, lfosfamide, mπomycin C, itoxantrone vincπstine, vinorelbme, carboplatin. teniposide, daunomycin, carminomycin, aminopteπn, dactinomycin mitomycins, esperamicins (see, U S Pat No 4,675,187), 5-FU, 6-thιoguanιne, 6-mercaptopuπne, actinomycin 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 in vitro or in vivo Thus. the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest Classical M-phase blockers include the v cas (vincπstine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds . Chapter 1 , entitled "Cell cycle regulation, oncogens, and antineoplastic drugs ' by Murakami etal , (WB Saunders Philadelphia, 1995), especially p 13
"Doxorubicin" is an anthracychne antibiotic The full chemical name of doxorubicin is (8S-cιs)-10-[(3- amιno-2.3.6-tπdeoxy-c--L-lyxo-hexapyranosyl)oxy]-7 8,9, 10-tetrahydro-6 8 1 l-tπhvdroxv-8-(hydroxyacetyl)-l methoxy 5, 12-naphthacenedιone
The term "cytokine" is a 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-methionvl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsu n relaxin, prorelaxin, glycoprotem hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteimzing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin. placental lactogen, tumor necrosis factor-α and -β, mulleπan-inhibiting substance, mouse gonadotropin-associated peptide. inhibin activin, vascular endothe al growth factor, integrin. thrombopoietin (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), osteoinductive factors, interterons such as interferon -α -β. and -γ, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF). granulocyte-macrophage-CSF (GM-CSF), and granulocvte-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 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 less 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, J4 375-382, 615th Meeting, Belfast ( 1986), and Stella et al 'Prodrugs A Chemical Approach to Targeted Drug Delivery ', Directed Drug Delivery Borchardt et al , (ed ), pp 147-267, Humana Press ( 1985) The prodrugs of this invention include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containingprodrugs, D-arτuno acid-modified prodrugs, glysocylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5- fluorocytosine and other 5-fluorouπdιne prodrugs which can be converted into the more active cytotoxic free drug Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above An "effective amount" of a polypeptide disclosed herein or an antagonist thereof, in reference to inhibition of neoplastic cell growth, tumor growth or cancer cell growth, is an amount capable of inhibiting, to some extent, the growth of target cells The term includes an amount capable of invoking a growth inhibitory, cytostatic and/or cytotoxic effect and/or apoptosis of the target cells An "effective amount ' of a PRO polypeptide antagonist for purposes of inhibiting neoplastic cell growth, tumor growth or cancer cell growth, may be determined empiπcally and in a routine manner
A "therapeutically effective amount", in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects ( 1 ) inhibition, to some extent, of tumor growth including, slowing down and complete growth arrest, (2) reduction in the number of tumor cells, (3) reduction in tumor size, (4) inhibition (/ e . reduction, slowing down or complete stopping) of tumor cell infiltration into peπpheral organs, (5) inhibition (. e reduction slowing down or complete stopping) of metastasis (6) enhancement of anti-tumor immune response which may but does not have to, result in the regression or rejection of the tumor, and/or (7) relief to some extent, of one or more symptoms associated with the disorder A "therapeutically effective amount" of a PRO polypeptide antagonist for purposes ot treatment of tumor may be determined empirically and in a routine manner A 'growth inhibitory amount of a PRO antagonist is an amount capable of inhibiting the growth of a cell, especially tumor, e g , cancer cell either ... vitro or in vivo A 'growth inhibitory amount ' of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
A "cvtotoxic amount of a PRO antagonist is an amount capable of causing the destruction of a cell, especially tumor, e g , cancer cell, either in vitro or in A cytotoxic amount ' of a PRO antagonist for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner
The terms PRO polypeptide and PRO as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (. e , PRO/number) refers to specific polypeptide sequences as described herein The terms 'PRO/number polypeptide and "PRO/number wherein the term number is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein) The PRO polypeptides descπbed herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods
A ' native sequence PRO polypeptide comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term ' native sequence PRO polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring alle c variants of the polypeptide In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the PRO polypeptides
The PRO polypeptide "extracellular domain' or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and or cytoplasmic domains and preferably, will have less than 0 5% of such domains It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention
The approximate location of the ' signal peptides" of the vaπous PRO polypeptides disclosed herein are shown in the present specification and/or the accompanying figures It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e g , Nielsen et al , Prot Eng , JO 1-6 (1997) and von Heinje et al , Nucl Acids Res , 144683-4690 (1986)) Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention
"PRO polypeptide variant' means an active PRO polypeptide as defined above or below having at least about 80% ammo acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without the signal peptide. as disclosed herein or anv other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted at the N- or C-terminus of the full-length nativ e amino acid sequence Ordinaπlv . a PRO polypeptide variant will hav e at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% ammo acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity. alternatively at least about 90% amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity , alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily, PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 am o acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more
"Percent (%) amino acid sequence identity' with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a PRO sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megahgn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Ine , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office. Washington D C , 20559. where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, lnc South San Francisco California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system preferably digital UNIX V4 OD All sequence comparison parameters are set b\ the ALIGN-2 program and do not vary
For purposes herein the % amino acid sequence identitv of a given amino acid sequence A to with or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to with, or against a given ammo acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of am o acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program s alignment of A and B and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B. the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A As examples of % amino acid sequence identity calculations. Tables 2A-2B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein' to the amino acid sequence designated "PRO"
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res . 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01 , constant for multi-pass = 25. dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % am o acid sequence identity to with or against a given amino acid sequence B) is calculated as follows
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B. the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A
In addition, % amino acid sequence identity may also be determined using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values. - e . the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125. word threshold (T) = 1 1, and scoring matrix = BLOSUM62 For purposes herein, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acids residues between the amino acid sequence of the PRO polypeptide of 5 interest having a sequence derived from the native PRO polypeptide and the comparison amino acid sequence ot interest (- e . the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest For example, in the statement "a polypeptide comprising an amino acid sequence A which has or having at least 80% amino acid sequence identity to the amino acid sequence B". the amino acid sequence
10 A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence ' means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as
15 disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide. as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinarily, a PRO vaπant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about
20 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least
25 about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence
30 PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein an extracellular domain ot a PRO polypeptide with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, altemativelv at least
35 about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more
"Percent (%) nucleic acid sequence identity with respect to the PRO polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a PRO polypeptide encoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN. ALIGN-2 or Mega gn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared For purposes herein, however, % nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 The ALIGN-2 sequence comparison computer program was authored by Genentech, Ine , and the source code shown in Table 1 has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Ine , South San Francisco. California or may be compiled from the source code provided in Table 1 The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4 0D All sequence comparison parameters are set by the ALIGN-2 program and do not vary
For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or compπses a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program s alignment of C and D and where Z is the total number ot nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C As examples of % nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO- DNA'
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program However, % nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes strand = all expected occurrences = 10 minimum low complexity length = 15/5 multi-pass e-value = 0 01 , constant for multi-pass = 25 dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62
In situations where NCBI-BLAST2 is employed for sequence compaπsons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or compπses a certain % nucleic acid sequence identity to with or against a given nucleic acid sequence D) is calculated as follows
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI- BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C
In addition, % nucleic acid sequence identity values may also be generated using the WU-BLAST-2 computer program (Altschul et al , Methods in Enzvmology, 266 460-480 (1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, ; e , the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 11, and scoring matrix = BLOSUM62 For purposes herein, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i e , the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide- encoding nucleic acid molecule of interest For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B" the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest
In other embodiments PRO variant polynucleotides are nucleic acid molecules that encode an active PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length PRO polypeptide shown in the accompanying figures PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide
The term positives' , in the context of the amino acid sequence identity compaπsons performed as described above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) of the amino acid residue of interest
For purposes herein the % value of positives ot a given amino acid sequence A to, with, or against a given am o acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to with or against a given amino acid sequence B) is calculated as follows 100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as defined above bv the sequence alignment program ALIGN-2 in that program s alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % positives ot A to B will not equal the % positives ot B to A
"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Preferably, the isolated polypeptide is free of association with all components with which it is naturally associated Contaminant components ot its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes In preferred embodiments, the polypeptide will be purified (1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator. or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step
An "isolated" nucleic acid molecule encoding a PRO polypeptide or an "isolated" nucleic acid encoding an anti-PRO antibody, 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 PRO-encoding nucleic acid or the anti-PRO-encoding nucleic acid Preferably, the isolated nucleic acid is free ot association with all components with which it is naturally associated An isolated PRO-encoding nucleic acid molecule or an anti- PRO-encoding 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 PRO-encoding nucleic acid molecule or the anti-PRO encoding nucleic acid molecule as it exists in natural cells However, an isolated nucleic acid molecule encoding a PRO polypeptide or an isolated nucleic acid molecule encoding an anti-PRO antibody includes PRO-nucleic acid molecules or anti-PRO-nucleic acid molecules contained in cells that ordinarily express PRO polypeptides or express anti-PRO antibodies where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The term "control sequences ' refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, tor example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukarvotic 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 tor a presequence or secretory leader is operably linked to DNA tor a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription ot the sequence, or a πbosome 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 hgation at convenient restriction sites If such sites do not exist the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-PRO monoclonal antibodies (including antagonist and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments ot anti-PRO antibodies (see below) 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 tor possible naturally-occurring mutations that may be present in minor amounts "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hvbridizable 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 stπngent. 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)
"Stπngent conditions" or ' high stπngency 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 chloπde/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% polyvιnylpyrrohdone/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 sultate 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 Spπng Harbor Press, 1989, and include the use ot washing solution and hybridization conditions (e g , temperature ionic strength and % SDS) less stringent 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 mM 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 35°C-50°C The skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like
The term "epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag polypeptide ' The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes Suitable tag polypeptides generally hav e at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)
"Active or activity for the purposes herein refers to form(s) of PRO polypeptides which retain a biological and/or an immunological activity/property of a native or naturally-occurring PRO polypeptide, wherein "biological ' activity refers to a function (either inhibitory or stimulatory) caused by a native or naturally occurring PRO polypeptide other than the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally-occurring PRO polypeptide and an immunological" activity refers to the ability to induce the production of an antibody against an antigemc epitope possessed by a native or naturally-occurring PRO polypeptide
"Biological activity" in the context of an antibody or another antagonist 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 or otherwise interfere with the transcription or translation of a PRO polypeptide 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 term "biological activity" in the context of a PRO polypeptide means the ability of a PRO polypeptide to induce neoplastic cell growth or uncontrolled cell growth The phrase "immunological activity" means immunological cross-reactivity with at least one epitope of a PRO polypeptide
"Immunological cross-reactivity" as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO polypeptide having this activity with polyclonal antisera raised against the known active PRO 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 (e g , antibody) identified, to the corresponding PRO polypeptide is significantly higher (preferably at least about 2-tιmes, more preferably at least about 4-tιmes, even more preferably at least about 8-tιmes, most preferably at least about 10-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 partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO polypeptide disclosed herein or the transcription or translation thereof Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments fragments, peptides. small organic molecules anti-sense nucleic acids etc Included are methods for identifying antagonists of a PRO polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide
A "small molecule is defined herein to have a molecular weight below about 500 Daltons "Antibodies" (Abs) and ' immunoglobulins (Igs) are glycoproteins having the same structural characteristics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity Polvpeptides 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 (<? g , 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 daltons, composed ot two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one co alent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes Each heavy and light chain also has regularly spaced intrachain 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 amino 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 vaπabihty 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) regions The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, 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, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al . NIH Publ No 91 -3242. Vol I. pages 647-669 (1991 )) The constant domains are not involved directly in binding an antibody to an antigen but exhibit various effector functions, such as participation ot the antibody in antibody-dependent cellular toxicity
The term "hypervaπable region ' 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 "complementaπty determining region ' or "CDR" (i e , residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI ), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain. Kabat et al , Sequences of Proteins ot Immunological Interest, 5th Ed Public Health Service National Institute of Health, Bethesda, MD [ 1991 ]) and/or those residues from a "hypervaπable loop" (; e , residues 26-32 (LI ), 50-52 (L2) and 91-96 (L3) in the light chain v aπable 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 vaπable 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 etal Protein Eng , 8(10) 1057- 1062 [ 1995]), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual 'Fc" fragment, whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab')-, fragment that has two antigen-combining sites and is still capable of cross-linking antigen "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site
This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site
The Fab fragment also contains the constant domain of the light chain and the first constant domain (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 1 domain including one or more cysteines 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')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , IgG2, IgG3, IgG4, IgA, and IgA2 The heavy-chain constant domains that correspond 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. i e the individual antibodies comprising the population are identical except for possible naturall v occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigemc site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybπdoma culture, uncontaminated by other immunoglobulins The modifier monoclonal indicates the character of the antibody as being obtained from a substantiallv 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 mav be made by the hybπdoma method first described by Kohler et al , Nature. 256 495 [1975], or may be made by recombinant DNA methods (see, e g , U S PatentNo 4,816,567) The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al , Nature. 352 624-628 [19911 and Marks et al I Mol Biol 222 581-597 ( 1991 ), tor example The monoclonal antibodies herein specificallv 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 ot the chaιn(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as tragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4,816,567 Morrison et al , Proc Natl Acad Sci USA. 81 6851 -6855 [1984])
"Humanized" forms ot non-human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived trom non-human immunoglobulin For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR ot 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 immunoglobulin 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 modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that ot a human immunoglobulin For further details, see, Jones et al Nature. 321 522-525 (1986), Reichmann et al Nature, 332 323-329 [1988], and Presta, Curr Op Struct Biol 2 593-596 (1992) The humanized antibody includes a PRIMATIZED™ antibody wherein the antigen-binding region of the antibody is deπved 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 Hol nger et al . Proc Natl Acad Sci USA, 90 6444-6448 ( 1993 )
An "isolated' antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components ot its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments the antibody will be purified ( 1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal am o 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, 1-131, 1-123, 1-125, Y-90, Re- 188, Re- 186, At-211 , Cu-67, Bι-212, and Pd- 109 The label may also be a non-detectable entity such as a toxin
By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere Examples of solid phases encompassed herein include those formed partially or entirely of glass (e g , controlled pore glass), polysaccharides (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e g., an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those descπbed in U S Patent No. 4,275,149
A "liposome" is a small vesicle composed of various types of lipids, phospho pids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid 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 immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i e , is ' heterologous '), and an immunoglobulin constant domain sequence The adhesin part of an lmmunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The immunoglobulin constant domain sequence in the lmmunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM
As shown below. Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program This source code may be routinely compiled for use on a UNIX operating system to provide the ALIGN-2 sequence comparison computer program
In addition. Tables 2A-2D show hypothetical exemplifications tor using the below described method to determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid sequence identity (Tables 2C-2D) using the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the amino acid sequence of a hypothetical PRO polypeptide of interest. "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide ot interest is being compared, "PRO-DNA" represents a hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X", "Y", and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides.
Table 1
/*
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0 */
#define _M -8 /* value of a match with a stop */ int day [26] [26] = {
/* A" B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
/* A */ 2,0,-2,0,0,-4, 1, -1,-1, 0,-1,-2,-1.0,_M, 1, 0,-2, 1, 1,0, 0,-6, 0,-3, 0},
/*B*/ 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0.-3,-2.2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
/*C*/ •2,-4,15,-5,-5,-4,-3 ,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5.-4, 0,-2, 0,-2,-8, 0, 0,-5}, l*O*l 0,3,-5,4, 3,-6, 1, 1.-2.0, 0,-4.-3.2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2}, l*E*l 0, 2,-5, 3, 4,-5, 0, 1,-2.0, 0,-3,-2.1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, l*F*l -4,-5,-4,-6,-5, 9,-5, -2, 1, 0,-5, 2, 0.-4._M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, l*G*l 1,0,-3, 1,0,-5,5,- 2,-3, 0.-2,-4,-3.0,_M,-l,-l,-3, 1, 0, 0,-1,-7, 0,-5, 0}, ι*n*ι -1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2._M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2}, ι*ι *ι -1,-2,-2,-2,-2, 1,-3, -2, 5, 0,-2.2, 2,-2,_M,-2,-2,-2,-l, 0, 0, 4,-5, 0,-1,-2}, ι*ι*ι 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, t*κ*ι -1,0,-5,0,0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0}, t* *l -2,-3,-6,-4,-3, 2,-4, -2, 2, 0,-3, 6, 4.-3,_M,-3,-2,-3,-3,-l, 0, 2,-2, 0,-1,-2}, I* M */ -1,-2,-5,-3,-2,0,-3, -2, 2.0, 0, 4, 6,-2,_M,-2,-l, 0,-2,-1, 0, 2,-4, 0,-2,-1}, /*N*/ 0,2,-4,2, 1,-4,0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1},
/*o*/ JV1,_M, M, M, M. M,_M,_M,_M._M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M._M,_M,_M._M,_M},
/*P*/ 1,-1,-3,-1,-1,-5,-1, 0,-2, 0,-l,-3,-2,-l,_M, 6, 0 , 0, 1,0,0,-1,-6,0,-5,0}, /*Q*/ 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1,0,-2,-5,0,-4, 3}, /*R*/ 2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, /*S*/ 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1,_M, 1,-1, 0,2, 1,0,-1,-2,0,-3,0}, 1*1*1 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1, -1, 1, 3,0,0,-5,0,-3,0}, t*υ*t 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
I* \ *l 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2._M,-l,-2 ,-2,-1,0,0,4,-6, 0,-2,-2},
I* w*/ -6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4,_M,-6,-5 ,2,-2,-5,0,-6,17,0,0,-6}, /*x*/ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
/* Y*/ -3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-l,-2,-2,_M,-5,-4 ,-4,-3,-3,0,-2,0,0,10,-4}, ι*z*ι 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1.1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} >;
Table 1 (conf)
/* */
^include < stdιo.h > ^include < ctype.h >
#define MAXJMP 16 /* max jumps in a diag */
#define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
#defme MX 4 /* save if there's at least MX-1 bases since last jmp */
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
^define DINSO 8 /* penalty for a gap */
^define DINS1 1 /* penalty per base */
#defiπe PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP], /* size of jmp (neg for dely) unsigned short x[MAXJMP] ; /* base no. of jmp in seq x *
}; /* limits seq to 2*16 -1 */ struct diag { int score; /* score at last jmp */ long offset; /* offset of prev block */ short ljmp; /* current jmp index */ struct jmp JP; /* list of jmps */
}; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last elem before gap) */
}; char *ofιle; output file name */ char *namex[2]; seq names: getseqs() */ char *prog; prog name for err msgs */ char *seqx[2]; seqs- getseqs() */ int dmax; best diag' nw() */ int dmaxO; final diag */ int dna; /* set if dna maιn() */ int endgaps; /* set it penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl ; /* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw() */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path PP[2]; /* holds path for seqs */ char *calloc(), *malloc(), *mdex(), *strcpy(), char *getseq(), *g_calloc(), Table 1 (cont')
/* Needleman-Wunsch alignment program
*
* usage: progs filel file2
* where filel and file2 are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ';', '>' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more ot its elements ACGTU is assumed to be DNA
* Output is in the file "align. out" *
* The program may create a imp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
#include "nw.h" ^include "day.h" static dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
}; static _pbval[26] = {
1,2|(1<<('D'-'A'))|(1<<('N'-'A')), 4, 8, 16, 32,64,
128, 256, OxFFFFFFF, 1< < 10, 1< < 11, 1< < 12, 1< < 13.1< < 14,
1< < 15.1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1 < <21.1< <22,
1<<23, 1<<24, 1<<25|(1<<('E'-'A'))|(K<('Q'-'A'))
}; mam(ac, av) main int ac; char *av[]; prog = av[0]; if(ac!= 3){ fpπntf(stderr, "usage: %s filel file2\n", prog); fpπntf(stderr, "where filel and file2 are two dna or two protein sequencesΛn"); fpπntf(stderr,"The sequences can be in upper- or lower-case\n"); fpπntf(stderr,"Any lines beginning with ';' or ' < ' are ιgnored\n"); fpπntf(stderr, "Output is in the file \"ahgn.out\"\n"); exιt(l);
} namex[0] = av[l]; namex[l] = av[2]; seqx[0] = getseq(namex[0], &len0); seqx[l] = getseq(namex[l], &lenl); xbm = (dna)? dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile = "align. out"; /* output file */ nw(); /* fill in the matrix, get the possible jmps */ readjmpsO; /* get the actual jmps */ pπnt(); /* print stats, alignment */ cleanup(O); /* unlink any tmp files */ Table 1 (cont')
/* do the alignment, return best score maιn()
* dna: values m Fitch and Smith. PNAS, 80, 1382-1386, 1983
* pro- PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y
*/ nw() nw { char *px, *py, /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx, /* keep track of delx */ int *tmp; /* for swapping rowO, rowl */ int mis; /* score for each type */ int lnsO, insi , /* insertion penalties */ register id, /* diagonal index */ register u; /* jmp index */ register *col0, *coll, /* score for curr, last row */ register xx, yy; /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", lenO + lenl + 1, sizeoftstruct diag)); ndely = (int *)g_calloc("to get ndely" , lenl + 1 , sizeof(int)); dely = (int *)g_calloc("to get dely", lenl + 1 , sizeof(int)); colO = (int *)g_calloc("to get colO", lenl + 1, sizeof(int)); col l = (int *)g_calloc("to get coll ", lenl + 1, sizeof(int)); insO = (dna)? DINSO . PINSO; insi = (dna)? DINS 1 : PINS1 ; smax = -10000, if (endgaps) { for (col0[0] = dely[0] = -msO, yy = 1; yy < = lenl ; yy+ +) { col0[yy] = dely[yy] = col0[yy-l] - insi; ndely[yy] = yy;
} col0[0] = 0, /* Waterman Bull Math Biol 84 */
} else for (yy = 1 , yy < = lenl ; yy + + ) dely[yy] = -msO,
/* fill in match matrix
*/ for (px = seqx[0] , xx = 1 ; xx < = lenO; px+ + , xx+ +) {
/* initialize first entry in col
* // if (endgaps) { if (xx = = 1) coll [0] = delx = -(insO + insI); else coll [0] = delx = co!0[0] - insi , ndelx = xx,
} else { ndelx = 0, Table 1 (cont')
...nw seqx[l], yy = 1; yy < = lenl; py+ + , yy+ +) {
else mis += _day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* tavor new del over ongong del
* ignore MAXGAP if weighting endgaps
*/" if (endgaps | | ndely[yy] < MAXGAP) { if (colOfyy] - insO > = dely[yy]) { dely[yy] = col0[yy] - (insO + insl); ndely[yy] = 1; } else { dely[yy] -= insi; ndelyfyy] + + ; }
} else { if (col0[yy] - (insO + insl) > = dely[yy]) { dely[yy] = col0[yy] - (insO + insl), ndely [yy] = 1;
} else ndely [yy] + + ,
/* update penalty for del in y seq; * favor new del over ongong del */ if (endgaps | | ndelx < MAXGAP) { if (coll[yy-l] - msO > = delx) { delx = coll[yy-l] - (insO + insl); ndelx = 1; } else { delx -= insi; ndelx + + ; }
} else { if (coll[yy-l] - (insO + insl) > = delx) { delx = coll [yy-1] - (insO + insl); ndelx = 1;
} else ndelx+ + ,
/* pick the maximum score; we re favoring * mis over any del and delx over deiy
*/
-5 - Table 1 (cont')
...nw id = xx - yy + lenl - 1 , if (mis > = delx && mis > = dely[yy]) else if (delx > = dely[yy]) { coll [yy] = delx; lj = dx[ιd].ιjmp; if (dx[ιd].jp.n[0] && ('dna 1 1 (ndelx > = MAXJMP && xx > dx[ιd] jp.x[ιj] +MX) | | mis > dx[ιd] score+DINSO)) { if (+ + ιj > = MAXJMP) { wπtejmps(ιd), dx[ιd]. offset = offset; offset + = sizeof(struct jmp) + sizeof(offset), } } dx[ιd].jp n[ιj] = ndelx, dx[ιd] jp x[ιj] = xx; dx[ιd]. score = delx,
} else { collfyy] = dely[yy], if (dx[ιd] jp.n[0] && (!dna 1 1 (ndely[yy] > = MAXJMP
&& xx > dx[ιd].jp.x[ιj] +MX) | [ mis > dx[ιd]. score+DINSO)) { if (+ +ιj > = MAXJMP) { wπtejmps(ιd), dx[ιd]. offset = offset, offset + = sizeof(struct jmp) + sizeof(offset),
} } dx[ιd] jp.n[ιj] = -ndely [yy], dx[ιd]. score = dely[yy],
} if (xx = = lenO && yy < lenl) { /* last col */ if (endgaps) coll [yy] -= ιnsO + ιnsl *(lenl-yy), if (col 1 [yy] > smax) { smax = coll[yy], dmax = id, } } } if (endgaps && xx < lenO) coll [yy-l] - = ιnsO + ιnsl *(lenO-xxj, if (coll [yy 1] > smax) {
} tmp = colO colO = col l, col l = tmp,
}
(void) free((char *)ndely),
(void) freeftchar *)delv),
(void) free((char *)col0),
(void) free((char *)coll), } Table 1 (cont')
/* *
* pπntO -- only routine visible outside this module
* static.
* getmat() - trace back best path, count matches: pπnt()
* pr_ahgn() — print alignment of described in array p[] : pπnt()
* dumpblockO - dump a block of lines with numbers, stars: pr_alιgn()
* nums() — put out a number line. dumpblockO
* putline() -- put out a line (name, [num], seq. [num]). dumpblockO
* stars() - -put a line ot stars: dumpblockO
* stπpnameO - strip any path and prefix from a seqname */
^include "nw.h"
#define SPC 3
#define P LINE 256 /* maximum output line */
#define P SPC 3 /* space between name or num and seq */ extern _day[26][26]; int olen; /* set output line length */
FILE *fx; /* output file */ pπnto print
{ int lx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofιle, " ")) = = 0) { fpπntf(stderr," %s: can't write %s\n", prog, ofile), cleanup(l);
} fpπntf(fx, " < first sequence %s (length = d)\n", namexfO], lenO); fpπntf(fx, " < second sequence: %s (length = %d)\n", namex[l], lenl); olen = 60; lx = lenO; ly = lenl; firstgap = lastgap = 0, if (dmax < lenl - 1) { /* leading gap in x */ pp[0].spc = firstgap = lenl - dmax - 1 , ly -= pp[0].spc,
} else if (dmax > lenl - 1) { /* leading gap in y */ pp[l].spc = firstgap = dmax - (lenl - 1), lx -= pp[l].spc;
} if (dmaxO < lenO - 1) { '" trailing gap in x */ lastgap = lenO - dmaxO - 1 , lx -= lastgap,
} else if dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1), ly -= lastgap,
} getmatdx, ly, firstgap. lastgap), pr_ahgn(); Table 1 (conf)
/*
* trace back the best path, count matches
*/ static getmatUx, ly, firstgap, lastgap) getmat int lx, ly; /* "core" (minus endgaps) *' int firstgap. lastgap; /* leading trailing overlap *'
{ int nm, iO, il, sizO. siz char outx[32]; double pet; register nO. nl; register char *p0, *pl;
/* get total matches, score
*/ p0 = seqx[0] + pp[l].spc; pi = seqx[l] + pp[0].spc; nO = pp[l].spc + 1; nl = PP[0].spc + 1; nm = = 0; while ( *p0 && *pi){ if (sizO) { pl + + ; nl + + ;
if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ; if(nO++ == pp[0].x[ιO]) if (nl + + == pp[l].x[ιl])
}
/* pet homology:
* if penalizing endgaps. base is the shorter seq
* else, knock off overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl)? lenO : lenl; else lx = (lx < ly)? lx : I . pet = 100.*(double)nm/(double)lx; fpπntf(fx, "\n"); fpπntf(fx, " < %d match%s in an overlap of %d: %.2f percent sιmιlaπty\n" nm, (nm == 1)? "" . "es", lx, pet): Table 1 (cont') fprintf(fx, " < gaps in first sequence: %d", gapx); .getmat if (gapx) {
(void) sprintffoutx, " ( %d %s%s)", ngapx, (dna)? "base" : "residue", (ngapx = = 1 )? " ": "s"); fprintf(fx, " %s", outx); fprintf(fx, " , gaps in second sequence: %d", gapy); if (gapy) {
(void) sprintftoutx, " (%d %s%s)", ngapy, (dna)? "base": "residue" , (ngapy = = 1)? " " : "s"); fprintf(fx, " %s" , outx);
} if (dna) fprintf(fx,
"\n< score: %d (match = d, mismatch = %d, gap penalty = %ά + %d per base)\n", smax, DMAT, DMIS, DINSO, DINS1); else fprintf(fx,
"\n< score: %ά (Dayhoff PAM 250 matrix, gap penalty = Ψc d + %d per residue)\n", smax, PINSO. PINsi); if (endgaps) fprintf(fx,
" < endgaps penalized, left endgap: %d %s%s, right endgap: %d %s%s\n", firstgap, (dna)? "base" : "residue", (firstgap = = 1)? "" : "s", lastgap, (dna)? "base" : "residue", (lastgap = = 1)? "" : "s"); else fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core — for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number — for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char oouutt[[22]][[IP LINE] ; / * output line */ static char starfP 1 ]; /* set by stars() */
print alignment of described in struct path pp[] static pr alignO pr align int nn: /* char count */ int more; register i: for (i = 0, lmax = 0: i < 2: i + +) { nn = stripname(namex[i]); if (nn > lmax) lmax = nn: nc[i] = 1 : ni[i] = 1 : siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; Table 1 (cont')
for (nn = nm = 0, more = 1 , more, ) { ...pr align for (ι = more = 0 1 < 2 ι + +) { /*
* do we have more of this sequence ? */ if ('*ps[ι]) continue more + + , if (pp[ι] spc) { /* leading space */ *po[ι] + + PP[ι] spc- ,
} else if (sιz[ι]) { /* in a gap */
*po[ι] + + = '-', sιz[ι]~,
} else { /* we re putting a seq element */ *po[ι] = *ps[ι], if (ιslower(*ps[ι]))
*ps[ι] = toupper(*ps[ι]), po[ι] + + , ps[ι] + + ,
/*
* are we at next gap for this seq''
*/ if (m[ι] = = pp[ι] x[ιj[ι]]) { /*
* we need to merge all gaps
* at this location */ sιz[ι] = pp[ι] n[ιj[ι] + +], while (nι[ι] = = pp[ι] x[ιj[ι]]) sιz[ι] + = pp[ι] n[ιj[ι] + +]
} m[ι] + -
}
} if (+ +nn = = olen | | 'more && nn) { dumpblockO, for (i = 0, i < 2, ι + +) po[ι] = OUt[l], nn = 0 }
* dump a block of lines, includine numbers stars pr_ahgn()
*/ static dumpblockO dumpblock
{ register I, for d = 0 i < 2, 1+ -"- ) *po[ι]~ = '0 Table 1 (cont')
...dumpblock
(void) putc('.n', fx); for (i = 0; l < 2, ι++) { if (*out[ι] && (*out[ι] ! = *(po[ι]) ')){ if 0 ==0) nums(ι); if 0 == 0&&*out[l]) stars(), puthne(i); if (l == 0&&*out[l]) fpπntt(fx, star); if(ι == 1) nums(ι);
}
/*
* put out a number line: dumpblockO */ static nums(ιx) nums int ιχ; /* index in out[] holding seq line */ { char nhne[P _LINE]; register i. j; register char *pn, *px, *py; for (pn = nlme, 1 = 0: 1 < Imax+P SPC; ι+ + , pn++) for (1 = nc[ix], py = out[ιx]; *py; py+ + , pn+ + ) { if(*py == " II *py == '-')
*pn = ' '; else { if(i%10 ==0 || 0 == 1 &&nc[ιx] != 1)) { j = (i < 0)? -i : I; for (px = pn; j; j /= 10, px-)
*px = j%10 + '0'; if (l < 0)
*px
} else
*pn = ι + + ,
*pn = '\0', nc[ιx] = I; for (pn = nline; *pn; pn++) (void) putc(*pn, fx); (void) putc('\n'. fx);
/* * put out a line (name, [num], seq, |num]): dumpblockO
*/ static putlιne(ix) putline int Table 1 (conf)
.putline int 1; register char *px, for (px = namex[ιx], 1 = 0; *px && *px != '■': px+ + , ι++)
(void) putc(*px, fx); for(; l < lmax + P SPC: ι++)
(void) putcf ', tx);
/* these count from 1:
* nι[] is current element (from 1)
* nc[] is number at start of current line */ for (px = out[ιx]; *px; px+ +)
(void) putc(*px&0x7F, fx); (void)putc('.n', fx);
* put a line ot stars (seqs alwavs in out[0], out[l]): dumpblockO */ static s rso stars
{ int 1; register char *p0, *pl, ex, *px; if (!*out[0] I I (*out[0] == ' '&& *(po[0]) == ' ') 11 !*out[l] I I (*out[l] == ' '&& *(po[l]) == ' ')) return; px = star; for(ι = lmax + P_SPC;ι; ι-)
*px+ + = ' '; for(p0 = out[0], pi = out[l]; *p0 && *pl; p0+ + , pl + +) { if (isaipha(*pO) && ιsalpha(*pl)) { if (xbm[*pO-'A'l&xbm[*pl-'A']) { ex = '*', nm+ + ;
} elseif (!dna && _day[*pO-'A'][*pl-'A'] > 0) ex = ' ', else ex = ' ';
} else ex = ' ', + = ex:
Table 1 (cont')
/* strip path or prefix from pn, return len: pr_align()
*/ static stπpname(pn) stripname char *pn; /* file name (may be path) */
{ register char *px, *py; py = 0; for (px = pn; *px; px+ +) if (*px = = V) py = px + 1 ; if (py)
(void) strcpy(pn, py); return! strlen(pn));
Table 1 (cont')
/*
* cleanupO - cleanup any tmp file
* getseqO — read in seq, set dna, len, maxlen
* g_calloc() - callocO with error checkin
* readjmpsO ~ get the good jmps, from tmp file it necessary
* writejmpsO - write a filled array of jmps to a tmp file nw() */
^include nw h
#include < sys/file h > char *jname = /tmp/homgXXXXXX" , /* tmp file for jmps */
FILE *fj, int cleanupO, /* cleanup tmp file */ long lseek(),
/*
* remove anv tmp file if we blow
*/ cleanup(ι) cleanup { if (fj)
(void) unlink name), exιt(ι),
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines staπmg with ',' , < , or ' >
* seq in upper or lower case */ char * getseq(file, len) getseq char *file, /* file name */ char hne[1024], *pseq, register char *px, *py, int natgc, tlen,
FILE *tp if ((fp = fopen(file, r )) = = 0) { fpπntf(stderr s can t read %s\n , prog, file), exιt(l),
} tlen = natgc = 0, while (fgetsdine, 1024, fp)) { if (*hne = = , ' | | *lιne = = < ' | | *l-ne = = > ') continue, for (px = line, *px ' = '\n , px+ +) if (ιsupper(*px) | | ιslower(*px)) tlen+ +
} if ((pseq = malloc((unsιgned)(tlen+6))) = = 0) { fpπntf(stderr %s mallocO failed to get %d bytes for %s\n , prog, tlen+6 file) exιt(l),
} pseq[0] = pseq[l] = pseq[2] = pseq[3] = \0 , Table 1 (conf)
...getseq py = pseq + 4; *len = tlen: rewιnd(fp); while (fgetsdine.1024, fp)) { if (*lιne = = ';' | | *hne = = ' < ' | | *hne = = ' > ') continue; for (px = line; *px ! = '\n'; px++) { if (isupper(*px))
*py+ + = *px; else if (ιslower(*px))
*py+ + = toupper(*px); if (ιndex("ATGCU",*(py-l))) natgc + + ; } }
*py++ = '\0'; *py = '\0'; (void) fclose(fp); dna = natgc > (tlen3); return(pseq+4); } char * g_calloc(msg, nx, sz) g_CallθC char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */
{ char *px, *calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) { fprintffstderr, " s: g_calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz); exιt(l); } } return(px);
}
I"
* get final jmps from dx[] or tmp file, set pp[], reset dmax: maιn()
"7 readjmpsO readjmps
{ int fd = -1, register i. j, xx; if(fj){
(void) fclose(fj), if ((fd = open(jname, O RDONLY, 0)) < 0) { fpπntflstderr, "%s: can t open() %s\n", prog, jname); cleanupO); } } for (i = iO = il =0, dmaxϋ = dmax, xx = lenO, , ι + +) { while (1){ for (j = dx[dmax].ιjmp; j > = 0 && dx[dmax].jp.x|j] > = xx, j— ) Table 1 (conf)
...readjmps if 0 < 0 && dxfdmax]. offset && fj) {
(void) lseek(fd. dx[dmax] .offset, 0);
(void) read.fd. (char *)&dx[dmax].jp, sizeof(struct jmp)),
(void) readffd, (char *)&dx[dmax]. offset, sizeof(dx[dmax]. offset)); dx[dmax].ιjmp = MAXJMP- 1;
} else break:
} if (i > = JMPS) { fpπntt(stderr, "7 : too many gaps in alignment. n", prog); cleanup(l); } if 0 > = 0){ siz = dxfdmax] .jp.n[j]; xx = dx[dmax].jp.x[j], if (siz < 0) { /* gap in second seq */
/* id = xx - yy + lenl - 1 */ pp[l].x[ιl] = xx - dmax + lenl - 1: gapy+ + . ngapy -= siz: /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)? -siz : MAXGAP; ιl + + ;
} else if (siz > 0) { /* gap in first seq */ gapx+ + ; ngapx + = siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps)? siz : MAXGAP; ι0+ + ; } } else break; }
/* reverse the order of jmps */ for 0 =0, iO-; j < iO; j + + , ι0~) { i = pp[0].nQ]; pp[0].nD] = pp[0].n[ι0]; pp[0].n[ι0] = ι,
. = pp[0].xDl; pp[0].xDl = Pp[0].x[ι0]; pp[0].x[ι0] = ., } for(j = 0, ιl-;j < ιl;j + +, il-) {
. = pp[l].n[j]; Pp[l].n ] = pp[l].n[ιl]; pp[l].n[ιl] = i, i = pp[l].x|j]; pp[l].xϋl = pp[l].x[ιl], pp[l].x[ιl] = i. } if (fd > = 0)
(void) close(fd), if(fj){
(void) unhnk(jname). fj =0; offset = 0. } } Table 1 (cont')
/*
* write a filled jmp struct offset of the prev one (if any): nw() wπtejmps(iλ) writejmps int IX; char *mktemp(), if ('fj) { if (mktemptjname) < 0) { fprιntt(stderr, " %s: can't mktempO %s\n", prog, jna e); cleanup(l),
} if ((fj = fopen(jname, "w")) = = 0) { tpπntf(stderr, " %s: can't write %s\n", prog, jname); exιt(l); } }
(void) fwπte((char *)&dx[ιx] .jp, sizeoftstruct jmp), 1, fj); (void) f πte((char *)&dx[ιx]. offset, sιzeof(dx[ιx]. offset), 1 , fj);
Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 ammo acids)
% ammo acid sequence identity =
(the number ot identically matching amino acid residues between the two polypeptide sequences as determmed by ALIGN-2) divided by (the total number of amino acid residues ot the PRO polypeptide) =
5 divided by 15 = 33.3 %
Table 2B
PRO XXXXXXXXXX (Length = 10 ammo acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 ammo acids)
% ammo acid sequence identity =
(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of ammo acid residues of the PRO polypeptide) =
5 divided by 10 = 50%
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides ot the PRO-DNA nucleic acid sequence) =
6 divided by 14 = 42.9%
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number ot identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides ot the PRO-DNA nucleic acid sequence) =
4 divided by 12 = 33.3 %
U_ Compositions and Methods of the Invention
A Full-length PRO Polypeptides
The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides In particular. cDNA encoding PRO polypeptides has been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed However for sake ot simplicity, in the present specification the proteins encoded by the herein disclosed nucleic acid sequences as well as all further native homologues and variants included in the foregoing definition of PRO polypeptides will be referred to as "PRO" regardless of their origin or mode of preparation
As disclosed in the Examples below. cDNA clones have been deposited with the ATCC The actual nucleotide sequence ot the clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art The predicted amino acid sequences can be determined from the nucleotide sequences using routine skill For the PRO polypeptides and encoding nucleic acid described herein, Applicants have identified what are believed to be the reading frames best identifiable with the sequence information available
B PRO Polypeptide Variants
In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO polypeptide variants can be prepared. PRO polypeptide variants can be prepared by introducing appropπate nucleotide changes into the PRO DNA and/or by synthesis of the desired PRO polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO polypeptide. such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics
Variations in the native full-length sequence PRO polypeptide or in various domains of the PRO polypeptide described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U S Patent No 5.364.934 Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO polypeptide that results in a change in the amino acid sequence of the PRO polypeptide as compared with the native sequence PRO polypeptide Optionally the \aπatιon is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the PRO polypeptide Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the number ot amino acid sequence changes made in regions of high homology Amino acid substitutions can be the result ot replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine. . e . conservative amino acid replacements Insertions or deletions may optionally be in the range ot about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence PRO polypeptide fragments are provided herein Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, tor example, when compared with a full-length native protein Certain fragments lack amino acid residues that are not essential tor a desired biological activity of the PRO polypeptide
PRO polypeptide fragments may be prepared by any ot a number of conventional techniques Desired peptide fragments may be chemically synthesized An alternative approach involves generating PRO polypeptide fragments by enzymatic digestion e g , by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or bv digesting the DNA with suitable restriction enzymes and isolating the desired fragment Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR) Ohgonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR Preferably, PRO polypeptide fragments share at least one biological and or immunological activity with the native PRO polypeptide
In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened
Table 3
Original Exemplary Preferred Residue Substitutions Substitutions Arg (R) lys, gin, asn lys Asn (N) gin, his, lys, arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro. aid ala His (H) asn. gin lys, arg arg He (I) leu. val met. ala. phe. norleucine leu Leu (L) norleucine lie. val
Phe (F) leu. val, lie. ala tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr, phe tyr Tyr (Y) tip, phe thr. ser phe Val (V) lie, leu met, phe. ala, norleucine leu
Substantial modifications in function or immunological identity of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, tor example, as a sheet or helical conformation (b) the charge or hydrophobicity ot the molecule at the target site or (c) the bulk of the side chain Naturally occurring residues are divided into groups based on common side chain properties (1) hydrophobic norleucine, met, ala. val. leu, lie (2) neutral hydrophilic cys, ser, thr,
(3) acidic asp, glu,
(4) basic asn, gin, his, lys, arg
(5) residues that influence chain orientation gly, pro. and
(6) aromatic trp, tyr, phe Non-conservative substitutions will entail exchanging a member ot one of these classes tor another class
Such substituted residues also may be introduced into the conservative substitution sites or. more preferably, into the remaining (non-conserved) sites
The variations can be made using methods known in the art such as ohgonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al , Nucl Acids Res . 13 4331 (1986), Zoller et al , Nucl Acids Res K) 6487 ( 1987)], cassette mutagenesis [Wells et al . Gene. 34 315 (1985)], restriction selection mutagenesis [Wells etal , Philos Trans R Soc. London SerA.317 415 (1986)] or other known techniques can be pei formed on the cloned DNA to produce the PRO variant DNA
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence Among the preferred scanning amino acids are relatively small, neutral amino acids Such amino acids include alanine, glycine, serine, and cysteine Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science 244 1081-1085 ( 1989)] Alanine is also typically preferred because it is the most common amino acid Further, it is frequently found in both buried and exposed positions [Creighton. The Proteins, (W H Freeman & Co . N Y ), Chothia, J Mol Biol 150 1 ( 1976)] If alanine substitution does not yield adequate amounts of \ aπant. an lsoteπc amino acid can be used
C Modifications of PRO Polypeptides
Covalent modifications ot the PRO polypeptide are included within the scope of this invention One type of covalent modification includes reacting targeted amino acid residues ot a PRO polypeptide with an organic deπvatizing agent that is capable ot reacting with selected side chains or the N- or C- terminal residues ot the PRO polypeptide Derivatization with bitunctional agents is useful for instance, tor crosslinking the PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies and vice versa Commonly used crosslinking agents include e g , ] ] -bιs(dιazoacetyl)-2-phenylethane. glutaraldehyde. N- hydroxysuccinimide esters, for example, esters with 4-azιdosahcylιc acid, homobif unctional lmidoesters including disuccimmidyl esters such as 3 3'-dιthιobιs(succιnιmιdylpropιonate) bitunctional maleimides such as bis-N- maleιmιdo-l ,8-octane and agents such as methyl-3-[(ρ-azιdophenyl)dιthιo]propιoιmιdate
Other modifications include deamidation of glutaminyl and asparaginyi residues to the corresponding glutamyl and aspartyl residues respectively, hydroxylation of proline and Ksine. phosphorylation ot hydroxyl groups ot sen 1 or threonyl residues, methylation ot the α-amino groups ot lysine. arginine. and histidine side chains [T E Creighton. Proteins Structure and Molecular Properties W H Freeman & Co , San Francisco, pp 79-86 (1983)]. acetv lation of the N-terminal amine. and amidation ot any C-terminal carboxyl group
Another type of covalent modification of the PRO polypeptide included within the scope ot this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended tor purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO polypeptides (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions ot the various carbohydrate moieties present
Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence The alteration may be made, for example, by the addition of, or substitution by, one or more seπne or threonine residues to the native sequence PRO polypeptide (for O-linked glycosylation sites) The PRO amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids
Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e.g , in WO 87/05330 published 11 September 1987, and in Aplin andWπston. CRC Cπt Rev Biochem . pp 259-306 (1981) Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and descπbed, for instance, by Hakimuddin. et al , Arch Biochem Biophys , 259 52 (1987) and by Edge et al , Anal Biochem . JJ_8 131 (1981) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al Meth Enzvmol 138 350 (1987)
Another type ot covalent modification of PRO polypeptides comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes in the manner set forth in U S Patent Nos 4 640,835, 4.496,689, 4,301 , 144, 4,670,417, 4,791 ,192 or 4.179,337 The PRO polypeptides of the present invention may also be modified in a way to form a chimeric molecule comprising the PRO polypeptide fused to another, heterologous polypeptide or amino acid sequence
In one embodiment, such a chimeric molecule comprises a fusion of the PRO polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino- or carboxyl-terminus ot the PRO polypeptide The presence of such epitope-tagged forms of the PRO polypeptide can be detected using an antibody against the tag polypeptide Also provision of the epitope tag enables the PRO polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type ot affinity matrix that binds to the epitope tag Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidme (poly-His) or poly-histidine-glycine (poly- His-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol 8 2159-2165 ( 1988)], the c-myc tag and the 8F9, 3C7, 6E10 G4, B7 and 9E10 antibodies thereto [Evan et al . Molecular and Cellular Biology. 5 3610-3616 (1985)]. and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborskyerα/ . Protein Engineering, 3(6) 547-553 ( 1990)] Other tag polypeptides include the Flag-peptide [Hopp e a/ , BιoTechnology. 6 1204- 1210 (1988)1, the KT3 epitope peptide [Martin et al , Science, 255 192-194 (1992)], an -tubulin epitope peptide [Skinner et al . J Biol Chem . 266 15163-15166 ( 1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA 87 6393-6397 (1990)]
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO polypeptide with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an "lmmunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region within an Ig molecule In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI , CH2 and CH3 regions of an IgGl molecule For the production of immunoglobulin fusions see also, U S Patent No 5,428, 130 issued June 27, 1995
D Preparation of PRO Polypeptides
The description below relates primarily to production of PRO polypeptides by culturing cells transformed or transfected with a vector containing the PRO nucleic acid It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare PRO polypeptides For instance, the PRO 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, CA (1969), Merπfield, J Am Chem Soc . 85 2149-2154 (1963)] In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer s instructions Various portions of the PRO polypeptide may be chemically synthesized separately and combined using chemical or enzvmatic methods to produce the full-length PRO polypeptide
a Isolation of DNA Encoding a PRO Polypeptide
DNA encoding the PRO polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue such as described in the Examples The PRO-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis
Libraries can be screened with probes (such as antibodies to the PRO 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 the PRO polypeptide 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 tor screening a cDNA library The oligonucleotide sequences selected as probes should be ot 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 ot labeling are well known in the art. and include the use of radiolabels like ^P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supia
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 amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA
b Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors described herein for PRO polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach. M Butler, ed (IRL Press, 1991) and Sambrook et al , supra
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinaπly skilled artisan, for example. CaCl , CaP04 liposome-mediated and electroporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supra, or electroporation is generally used for prokaryotes Infection with Agiobacτerium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al , Gene 23 315 ( 1983) and WO 89/05859 published 29 June 1989 For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb Virology. 52 456- 457 (1978) can be employed General aspects of mammalian cell host system transfections have been described in U S Patent No 4.399.216 Transformations into yeast are typically carried out according to the method of Van Sohngen et al , J Bact . J_30 946 ( 1977) and Hsiao et al . Proc Natl Acad Sci (USA) 76 3829 (1979) However, other methods tor introducing DNA into cells, such as by nuclear microinjection electroporation bacteπal protoplast fusion with intact cells or poivcations e g , polybrene. polyormthine. may also be used For various techniques tor transforming mammalian cells, see, Keown et al . Methods in Enzymology, 185 527-537 ( 1990) and Mansour et al . Nature. 336 348-352 ( 1988) Suitable host cells for cloning or expressing the DNA in the vectors herein include prokarvote veast or higher eukarvote cells Suitable prokaryotes include but are not limited to eubacteπa, such as Gram-negative or Gram-positn e organisms for example Enterobacteπaceae such as E coli Various £ coli strains are pubhclv available such as £ coh YΛ 2 strain MM294 (ATCC 31 446), £ coli XI 776 (ATCC 31 ,537) £ co/. strain W31 10 (ATCC 27,325) and £ coli strain K5 772 (ATCC 53,635) Other suitable prokarvotic host cells include Enterobacteπaceae such as £-.c/7£/-c/--<2 e g , E coli Enterobacter Etwinia Klebsiella Pioteus Salmonella, e g Salmonella nphtmutium, Serratia, e g , Sen alia marcescans, and Shigella as well as Bacilli such as B subtilis and -5 licheniformis (e g , B hcheniformis 41 P disclosed in DD 266 710 published 12 April 1989), Pseuaomonas such as P aeruginosa, and Sti eptom ces These examples are illustrative rather than limiting Strain W 31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations Preferably, the host cell secretes minimal amounts of proteolytic enzymes For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including £ coli W31 10 strain 1A2, which has the complete genotype tonA , E coli W31 10 strain 9E4, which has the complete genotype tonA ptr3 E colt W31 10 strain 27C7 (ATCC 55 244) w hich has the complete genotype tonA ptr3 phoA El 5 (argF lac) J 69 degP o pT kan , E colt W31 10 strain 37D6 which has the complete genotype tonA ptr3 phoA El 5 (atgF-lac)169 degP ompT rbs7 ιl\G kan' , E coli W31 10 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation, and an £ coli strain having mutant peπplasmic protease disclosed in U S Patent No 4,946 783 issued 7 August 1990 Alternatively in vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions, are suitable In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors Saccharom\ ces cerevisiae is a commonly used lower eukaryotic host microorganism Others include Schι?.osaccharom\ces pombe (Beach and Nurse, Nature 290 140 [1981 ] EP 139,383 published 2 May 1985), Kluyverom\ces hosts (U S Patent No 4 943,529, Fleer et al Bio/Technology. 9 968-975 (1991)) such as e g , K lactis (MW98-8C, CBS683 CBS4574, Louvencourt et al J Bacteπol 737 [1983]), K fiagi s (ATCC 12 424) K bu ancus (ATCC 16 045) K \s ιckeramιι (ATCC 24 178) k naltu (ATCC 56,500), K drosophύanim (ATCC 36 906 Vanden Berg et al Bio/Technology 8 135 ( 1990)), K thermotolerans, and K marxianus (EP 402 226), Pιchιapastorιs (EP ] %3 070 Sreekπshna erα/ J Basic Microbiol .28 265-278 [1988]) Candida Trιchoderma reesιa (EP 244 234) Neuιospora crassa (Ca$>e etal Proc Natl Acad Sci USA 76 5259-5263 [1979]), Schwannιom\ces such as Schwanmonnces occidentals (EP 394 538 published 31 October 1990) and filamentous fungi such as e g Neurospora Penicillium Tohpocladium (WO 91/00357 published 10 January 1991 ) and Aspergillus hosts such as A mdulans (Ballance et al Biochem Biophys Res Commun , 1 12 284-289 [ 1983], Tilburn et al , Gene 26 205 221 [ 1983] Yelton er α/ Proc Natl Acad Sci USA 81 1470 1474 [ 1984]) and A mger (Kelly and Hvnes EMBO J 4 475-479 [ 1985]) Methylotropic yeasts are suitable herein and include but are not limited to yeast capable of growth on methanol selected from the genera consisting ot Hansenula Candida Kloeckera Pichia Sacchawi ces Toπdopsis and Rhodotoiula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony The Biochemistry of Methylotrophs. 269 ( 1982)
Suitable host cells tor the expression of glycosylated PRO polypeptides are derived from multicellular organisms Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9 as well as plant cells Examples ot useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV 1 line transformed by S V40 (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 Natl Acad Sci USA. 77 4216 ( 1980)) mouse sertoh cells (TM4, Mather Biol Reprod 23 243-251 (1980)), human lung cells (W 138, 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 the PRO polypeptide may be inserted into a replicable vector for cloning (amplification ot the DNA) or for expression Various vectors are publicly available The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage The appropriate nucleic acid sequence may be inserted into the vector by a variety ot 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 transcπption termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
The PRO 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 PRO-encoding DNA that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast invertase leader, alpha factor leader (including Saccharonnces and Kluw erom\ces α-factor leaders the latter descπbed in U S Patent No 5,010,182), or acid phosphatase leader, the C albicans 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 polypeptides of the same or related species as well as viral secretory leaders Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Such sequences are well known tor a variety of bacteria, yeast and viruses The origin ot replication from the plasmid pBR322 is suitable for most Gram negative bacteria, the 2μ plasmid origin is suitable tor yeast and various viral origins (SV40 polyoma adenovirus VSV or BPV) are useful for cloning vectors in mammalian cells Expression and cloning vectors will typically contain a selection gene also termed a selectable marker
Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicilhn neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e g , the gene encoding D-alanine racemase tor Bacilli
An example ot suitable selectable markers tor mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid such as DHFR or thymidine kinase An appropπate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Proc Natl Acad Sci USA. 77 4216 ( 1980) A suitable selection gene for use in yeast is the tip] gene present in the yeast plasmid YRp7 [Stinchcomb et al Nature. 282 39 (1979), Kings an et al , Gene. 7 141 (1979) Tschemper et al , Gene 10 157 ( 1 80)] The tip] gene provides a selection marker for a mutant strain ot yeast lacking the ability to grow in tryptophan, for example ATCC No 44076 or PEP4- 1 [Jones, Genetics 85 12 (1977)] Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis Promoters recognized by a variety ot potential host cells are well known Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al , Nature, 275 615 (1978), Goeddel et al , Nature, 281 544 (1979)], alkaline phosphatase, a tryptophan (tip) promoter system [Goeddel. Nucleic Acids Res . 8 4057 (1980), EP 36,776], and hybrid promoters such as the tac promoter [deBoerefα/ . Proc Natl Acad Sci USA, 80 21-25 (1983)] Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S D ) sequence operably linked to the DNA encoding the PRO polypeptide
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 (1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg .7 149 (1968), Holland, Biochemistry, H 4900 (1978)], such as enolase, glyceraldehyde- 3-phosphate dehydrogenase, hexokmase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tπosephosphate isomerase, phosphoglucose isomerase, and glucok ase
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2. lsocytochrome C. acid phosphatase. degradative enzymes associated with nitrogen metabolism metallothionein. glyceraldehyde-3- phosphate dehydrogenase. and enzymes responsible tor maltose and galactose utilization Suitable vectors and promoters tor use in yeast expression are further described in EP 73,657
PRO polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (S V40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
Transcription of a DNA encoding the PRO polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector Enhancers are cis-acting elements of DNA. usually about from 10 to 300 bp, that act on a promoter to increase its transcription Many enhancer sequences are now known from mammalian genes (globin. elastase albumin α-fetoprotein, and insulin) Typically, however, one will use an enhancer from a eukaryotic cell virus Examples include the S V40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus earh promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers The enhancer may be spliced into the vector at a position 5' or 3' to the PRO coding sequence, but is preferably located at a site 5' from the promoter
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3'. untranslated regions of eukaryotic or viral DN As or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion ot the mRNA encoding the PRO polypeptide
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture are described in Gething et al , Nature. 293 620-625 (1981 ), Mantei et al , Nature 281 40-46 ( 1979), EP 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 ot mRNA [Thomas. Proc Natl Acad Sci USA. 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against an exogenous sequence fused to PRO DNA and encoding a specific antibody epitope
e Purification of Polypeptide
Forms of PRO polypeptides may be recovered from culture medium or from host cell lvsates If membrane-bound it can be released from the membrane using a suitable detergent solution (e g , Tπton-X 100) or by enzymatic cleavage Cells employed in expression of the PRO polypeptide can be disrupted by vaπous physical or chemical means, such as freeze thaw cycling, sonication. mechanical disruption, or cell lysing agents
It may be desired to purify the PRO polypeptide from recombinant cell proteins or polypeptides The following procedures are exemplary ot suitable purification procedures bv 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 PRO polypeptide Various methods of protein purification may be employed and such methods are known in the art and described tor example in Deutscher, Methods in Enzvmology. 182 (1990). Scopes. Protein Purification Principles and Practice Spπnger-Verlag New York ( 1982) The purification step(s) selected will depend, tor example, on the nature ot the production process used and the particular PRO polypeptide produced
E Amplification of Genes Encoding PRO Polypeptides in Tumor Tissues and Cell Lines
The present invention is based on the identification and characterization of genes that 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 original 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 quantitative modifications of the genetic mateπal Qualitative modifications include DNA mutations, in which coding sequences are altered resulting in a structurally and/or functionally different protein Gene amplification is a quantitative modification, whereby the actual number of complete coding sequence, i 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 amplification and its underlying mechanisms have been investigated in \ itro 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 drug methotrexate (MTX) MTX is a tolic acid analogue and interferes with DNA synthesis by blocking the enzyme dihydrofolate reductase (DHFR). Duπng the initial exposure to low concentrations of MTX most cells (>99 9%) will 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 ot the single DHFR gene The additional copies ot the gene are found as extrachromosomal copies in the form ot small. supernumerar> 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 chemotherapeutic agents 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 ot the most common genetic changes observed in human malignancies are mutations ot the p53 protein p53 controls the transition of cells from the stationary (Gl ) to the rephcative (S) phase and prevents this transition in the presence of DNA damage In other words, one of the main consequences ot disabling p53 mutations is the accumulation and propagation of DNA damage, i e , genetic changes Common types of genetic changes in neoplastic cells are. in addition to point mutations, amplifications and gross structural alterations, such as translocations
The amplification of DNA sequences may indicate a specific functional requirement as illustrated in the DHFR experimental system Therefore, the amplification of certain oncogenes in malignancies points toward a causative role ot these genes in the process ot 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 ot neoplastic cells Members of the gene family of growth factor receptors have been found to be amplified in various types ot 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 therapy and the amplification of the grow th factor receptor homologue ERB2 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 ot 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 transformation The case of ERB2 also demonstrates the feasibility from a therapeutic standpoint, since transforming proteins may represent novel and specific targets tor tumor therapy Several different techniques can be used to demonstrate amplified genomic sequences Classical cytogenetic analysis of chromosome spreads prepared from cancer cells is adequate to identify gross structural 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 mateπal While cytogenetics was 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 illustrated the widespread phenomenon of genomic amplification in neoplasms Tumor and normal DNA are hybridized simultaneously onto 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 etal . Radiation Res , 137 275-289 [ 1994]) As a screening method this type of analysis has revealed a large number ot recurring amplicons (a stretch ot 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 w ithin the amphcon 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, prov ide 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 survev 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, thy us, testis, ovary, uterus, etc , tumor, or tumor cell lines with pooled DNA from healthy donors Quantitative PCR was performed using a TaqMan™ instrument (ABI) Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs
Human lung carcinoma cell lines include A549 (SRCC768). Calu- 1 (SRCC769), Calu-6 (SRCC770), HI 57 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832),and H810 (SRCC833), all available from ATCC Primary human lung 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, SRCC724 (adenocarcinoma. abbreviated as "AdenoCa")(LTl), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa)(LTla), SRCC726 (adenocarcιnoma)(LT2), SRCC727 (adenocarcιnoma)(LT3), SRCC728 (adenocarcιnoma)(LT4), SRCC729 (squamous cell carcιnoma)(LT6), SRCC730 (adeno/squamous cell carcιnoma)(LT7), SRCC731 (adenocarcιnoma)(LT9), SRCC732 (squamous cell carcιnoma)(LT10), SRCC733 (squamous cell carcιnoma)(LTl l), SRCC734 (adenocarcιnoma)(LT12), SRCC735 (adeno/squamous cell carcιnoma)(LT13), SRCC736 (squamous cell carcιnoma)(LT15), SRCC737 (squamous cell carcιnoma)(LT16), SRCC738 (squamous cell carcιnoma)(LT17), SRCC739 (squamous cell carcιnoma)(LTl 8), SRCC740 (squamous cell carcιnoma)(LT19), SRCC741 (lung cell carcinoma, abbreviated as "LCCa")(LT21 ), SRCC81 1 (adenocarcιnoma)(LT22), SRCC825 (adenocarcιnoma)(LT8), SRCC886 (adenocarcιnoma)(LT25), SRCC887 (squamous cell carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) (LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma) (LT29), SRCC891 (adenocarcinoma) (LT30), SRCC892 (squamous cell carcinoma) (LT31), SRCC894 (adenocarcinoma) (LT33) Also included are human lung tumors designated SRCC1125 [HF-000631], SRCC1127 [HF-000641], SRCC1 129 [HF-000643], SRCC1133 [HF-000840], SRCC1 135 [HF-000842], SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230 [HF-001294], SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233 [HF-001297], SRCC1235 [HF-001299], SRCC1236 [HF-001300], SRCC1296[HF-001640], SRCC 1299 [HF-001643], SRCC1300 [HF001644], SRCC1301 [HF-001645], SRCC1302 [HF-001646], SRCC1303 [HF-001647], and SRCC1304 [HF-001648] Colon cancer cell lines include, for example, ATCC cell lines SW480 (adenocarcinoma. SRCC776),
SW620 (lymph node metastasis of colon adenocarcinoma. SRCC777), Colo320 (carcinoma, SRCC778), HT29 (adenocarcinoma. SRCC779), HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr Robert Warren, UCSF), CaWiDr (adenocarcinoma, SRCC781 ), HCT1 16 (carcinoma, SRCC782), SKCOl (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), Colo205 (carcinoma, SRCC828), HCT15 (carcinoma, SRCC829), HCC2998 (carcinoma, SRCC830), and KM12 (carcinoma, SRCC831 ) Primary colon tumors include colon adenocarcinomas designated CT2 (SRCC742). CT3 (SRCC743) ,CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17 (SRCC750), CT1 (SRCC751 ). CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT1 1 (SRCC757), CTI 8 (SRCC758), CT19 (adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21 (adenocarcinoma. SRCC908). CT22 (adenocarcinoma, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (adenocarcinoma. SRCC91 1 ), CT25 (adenocarcinoma. SRCC912), CT26 (adenocarcinoma, SRCC913), CT27 (adenocarcinoma, SRCC914).CT28 (adenocarcinoma, SRCC915), CT29 (adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31 (adenocarcinoma, SRCC918), CT32 (adenocarcinoma. SRCC919), CT33 (adenocarcinoma. SRCC920). CT35 (adenocarcinoma. SRCC921 ), and CT36 (adenocarcinoma. SRCC922) Also included are human colon tumor centers designated SRCC1051 [HF-000499]. SRCC1052 [HF-000539], SRCC1053 [HF-000575], SRCC1054 [HF-000698], SRCC1059 [HF-000755], SRCC1060 [HF-000756], SRCC1 142 [HF-000762], SRCC1 144 [HF-000789], SRCC1 146 [HF-000795] and SRCC1 148[HF-00081 1 ]
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), and SKBR3 (SRCC767), and human breast tumor center designated SRCC1057 [HF-000545] Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1 100, SRCC1 101 , and human breast-met-lung-NS tumor designated SRCC893 [LT 32] Human rectum tumors include SRCC981 [HF-000550] and SRCC982 [HF-000551] Human kidney tumor centers include SRCC989 [HF-00061 1 ] and SRCC1014 [HF-000613] Human testis tumor center include SRCC1001 [HF-000733] and testis tumor margin SRCC999 [HF- 000716] Human parathyroid tumors include SRCC1002 [HF-000831 ] and SRCC1003 [HF-000832]
Human lymph node tumors include SRCC1004 [HF-000854], SRCC1005 [HF-000855], and SRCC1006 [HF-000856]
F Tissue Distribution
The results of the gene amplification assays herein can be veπfied 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 Southern blotting. Northern blotting to quantitate the transcription of mRNA (Thomas Proc Natl Acad Sci USA.77 5201 -5205 [ 1980] ), dot blotting (DNA analysis), or in situ hybridization, using an appropπately 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 v aπous tissues, alternatively, may be measured by immunological methods such as immunohistochemical staining ot 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 ot sample fluids may be either monoclonal or polyclonal. and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to sequence PRO DNA and encoding a specific antibody epitope General techniques tor generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided hereinbelow
G Chromosome Mapping
If the amplification ot a given gene is tunctionall v relevant then that gene should be amplified more than neighboring genomic regions which are not important tor 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 the neighboring genomic region Selective or preferential amplification at the genomic region to which the 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)
H Antibody Binding Studies
The results of the gene amplification study can be further verified by antibody binding studies, in which the ability of anti-PRO antibodies to inhibit the expression of PRO polypeptides on 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, Ine . 1987) Competitive binding assays rely on the ability ot 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 facilitate determining the amount of standard that becomes bound, the antibodies preferably are msolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound
Sandwich assays 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, e g , U S Patent No 4 376 1 10 The second antibodv mav 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 ELISA assay, in which case the detectable moiety is an enzyme
For immunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, tor example
I Cell-Based Tumor Assays
Cell-based assays and animal models tor tumors (e g , cancers) can be used to verify the findings ot the gene amplification assay, and further understand the relationship between the genes identified herein and the development and pathogenesis ot 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, tor example, the breast, colon and lung cancer cells and cell lines listed above In a different approach, cells of a cell type known to be involved in a particular tumor are transfected with the cDNAs herein, and the ability of these cDNAs to induce excessive growth is analyzed Suitable cells include, for example, stable tumor cell lines such as, the B 104-1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene) and ras-transtected NIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumoπgenic growth Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumoπgenic cell growth by exerting cytostatic or cytotoxic activity on the growth ot the transformed cells, or by mediating antibody-dependent cellular cytotoxicity (ADCC) Cells transfected w ith 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-based assays herein, although stable cell lines are preferred Techniques to deπve 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])
J 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 ot 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-recombinant animal models include, for example, rodent, e g , murine models Such models can be generated by introducing tumor cells into syngeneic mice using standard techniques, e g subcutaneous injection, tail vein injection, spleen implantation, lntrapeπtoneal 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)
Probablv the most often used animal species in 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 v ery large number of distinct congenic strains of nude mouse, including, for example, AS W, A/He, AKR, BALB/c B 10 LP, C17. C3H, 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 in Oncology Research E Boven and B Winograd, eds , CRC Press, Ine . 1991
The cells introduced into such animals 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) . αs-transf ected NIH 3T3 cells, Caco-2 (ATCC HTB-37), a moderately well- differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmah 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 transplanted s c as solid blocks as needle biopsies by use ot a trochar, or as cell suspensions For solid block or trochar implantation, tumor tissue fragments ot suitable size are introduced into the s c space Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously 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 neuroblastoma cells (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 et al , 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, Ine . (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 drug 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 vaπous agents (Palladino et al , J Immunol , 138 4023-4032
[1987]) Briefly, tumor cells are propagated in vitro in cell culture Prior to injection into the animals, the cell lines are washed and suspended in buffer, at a cell density of about lOx lO6 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 survive For further information about this tumor model see Zacharski Haemostasis 16 300-320 [1986])
One wav ot evaluating the efficacy of a test compound in an animal model on an implanted tumor is to measure the size of the tumor 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 drug candidate can be better described as treatment-induced growth delay and specific growth delay Another important v aπable in the description ot tumor growth is the tumor volume doubling time Computer programs for the calculation and description ot 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
Recombinant (transgenic) animal 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 Animals that 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 microinjection (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 Natl Acad Sci USA. 82 6148-615 [1985]) gene targeting in embryonic stem cells (Thompson et al , Cell, 56 313-321 [ 1989]), electroporation of embryos (Lo, Mol Cell Biol , 3 1803-1814 [ 1983]), sperm-mediated gene transfer (Lavitrano et al , Cell, 57 717-73 [ 1989]) For review, see, for example, U S Patent No 4,736,866
For the purpose of the present invention, transgenic animals include those that carry the transgene only in part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA. 89 6232- 6236 (1992)
The expression of the transgene in transgenic animals can be monitored by standard techniques For example, Southern 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 immunocvtochemistrv The animals are further examined for signs of tumor or cancer development
Alternatively, 'knock out animals can be constructed which have a defectiv e or altered gene encoding a PRO 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 PRO polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques A portion of the genomic DNA encoding a particular PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration Typicallv 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 ot 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 by standard techniques and used to breed animals in which all cells ot 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 pathological conditions due to absence of the PRO polypeptide
The efficacy ot antibodies specifically binding the polypeptides identified herein and other drug candidates, can be tested also in the treatment of spontaneous animal 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 sublingual 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 However, the use of this model is limited by the rare occurrence of this type ot tumor in animals
K Screening Assays for Drug Candidates
Screening assays for drug 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 drug candidates Small molecules contemplated include synthetic organic or inorganic compounds, including peptides preferably soluble peptides, (poly)peptιde-ιmmunoglobuhn fusions, and. in particular, antibodies including, without limitation poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments 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 tor 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 bv the gene identified herein or the drug candidate is immobilized on a solid phase, e g , on a microtiter plate by covalent or non-covalent attachments Non- covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying Alternatively, an immobilized antibody, e , 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, bv using a labeled antibody specifically binding the immobilized complex
If the candidate compound interacts with but does not bind to a particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions Such assays include traditional approaches, such as, cross-linking, co- lmmunoprecipitation, and co-purification through gradients or chromatographic columns In addition, protein- protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature. 340 245-246 (1989), Chien et al , Proc Natl Acad Sci USA 88 9578-9582 ( 1991)] as disclosed by Chevray and Nathans. Proc Natl Acad Sci USA. 89 5789-5793 (1991)] Many transcπptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain The yeast expression system described in the foregoing publications (generally referred to as the ' two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4. and another in which candidate activating proteins are fused to the activation domain The expression of a GAL1 -lacZ reporter gene under control of a GAL4-actι vated promoter depends on reconstitution ot G AL4 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 crucial for these interactions
Compounds that interfere with the interaction of a PRO-encoding gene identified herein and other mtra- or extracellular components can be tested as follows usually a reaction mixture is prepared containing the product of the amplified gene and the intra- or extracellular component under conditions and tor a time allowing tor the interaction and binding ot the two products To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound In addition, a placebo may be added to a third reaction mixture to serve as positive control The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove The formation ot 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
To assay for antagonists, the PRO polypeptide mav be added to a cell along w ith the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide Alternatively, antagonists may be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions tor a competitive inhibition assay The PRO polypeptide can be labeled, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist The gene encoding the receptor can be identified by numerous methods known to those of skill in the art. for example, ligand panning and FACS sorting Cohgan et al . Current Protocols in Immun . 1 (2) Chapter 5 (1991 ) Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide and a cDNA library created from this RNA is divided into pools and used to transtect COS cells or other cells that are not responsive to the PRO polypeptide Transfected cells that are grown on glass slides are exposed to labeled PRO polypeptide The PRO polypeptide can be labeled by a variety of means including lodination or inclusion of a recognition site for a site-specific protein kinase Following fixation and incubation, the slides are subjected to autoradiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor. As an alternative approach for receptor identification, labeled PRO polypeptide can be photoaffinity-hnked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved by PAGE and exposed to X-ray film The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing The amino acid sequence obtained from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor
In another assay tor antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound The ability of the compound to enhance or block this interaction could then be measured
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with the PRO polypeptide, and. in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the PRO polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide
Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e g , an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation Antisense technology can be used to control gene expression through tπple-hehx formation or antisense DNA or RNA. both ot which methods are based on binding ot a polynucleotide to DNA or RNA For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptide herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see, Lee etal , Nucl Acids Res . 6 3073 (1979), Cooney etal . Science. 241 456 (1988), Dervan et al Science. 251 1360 (1991 )), thereby preventing transcription and the production of the PRO polypeptide The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem . 56 560 (1991 ), Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press Boca Raton, FL, 1988) The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide When antisense DNA is used, oligodeoxyπbonucleotides derived from the translation-initiation site, e g , between about -10 and +10 positions of the target gene nucleotide sequence, are preferred
Antisense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-hke molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA followed bv endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g , Rossi, Current Biology. 4 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)
Nucleic acid molecules in tπple-hehx formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides The base composition ot these oligonucleotides is designed such that it promotes tπple-hehx 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 97/33551 , supra
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known tor those skilled in the art
L Compositions and Methods for the Treatment of Tumors
The compositions useful in the treatment ot tumors associated with the amplification of the genes identified herein include, without limitation antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc , that inhibit the expression and/or activ uy ot the target gene product
For example, antisense RNA and RNA molecules act to directly block the translation ot mRNA by hybridizing to targeted mRNA and preventing protein translation When antisense DNA is used, oligodeoxyπbonucleotides 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 Biology, 4 469-471 ( 1994), and PCT publication No WO 97/33551 (published
September 18, 1997)
Nucleic acid molecules in triple helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides The base composition of these oligonucleotides is designed such that it promotes 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 97/33551, 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
M Antibodies
Some of the most promising drug 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
1 Polyclonal Antibodies
Methods ot 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 lntrapeπtoneal injections The immunizing agent may include the PRO polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include but are not limited to keyhole limpet hemocyanin. serum albumin, bovine thyroglobuhn, and soybean trypsin inhibitor Examples ot adjuvants which may be employed include Freund s complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A. synthetic trehalose dicorynomycolate) The immunization protocol may be selected by one skilled in the art without undue experimentation
2 Monoclonal Antibodies
The anti-PRO antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybπdoma methods, such as those described by Kohler and Milstein Nature. 256 495 (1975) In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the PRO polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell line* are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection (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 etal., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO polypeptides. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. 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 heav v and light chains of murine antibodies) The hybπdoma cells ot the invention serv e as a preferred soui ce ot 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 ov ai v (CHO) cells 01 myeloma cells that do not otherwise pioduce immunoglobulin protein to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U S Patent No 4,816,567, Morrison et al , supia] or bv covalently joining to the immunoglobulin coding sequence all or part of the coding sequence tor a non-immunoglobulin polypeptide Such a non-immunoglobuhn polypeptide can be substituted for the constant domains ot an antibody of the invention, or can be substituted tor the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric biv alent antibody
The antibodies may be monovalent antibodies Methods tor preparing monovalent antibodies are well known in the art For example, one method inv olves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking
In viti o methods are also suitable for preparing monovalent antibodies Digestion ot antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art
3 Human and Humanized Antibodies
The anti-PRO antibodies may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins. immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) ot the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will comprise substantially all of at least one. and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those ot a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al Nature. 321 522-525 ( 1986) Riechmann et al . Nature, 332 323-329 ( 1988), and Presta. Curr Op Stiuct Biol , 2 593-596 (1992)]
Methods tor humanizing non-human antibodies aie well know n in the art Generall) . 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 Natui e 321 522 525 ( 1986) Riechmann et al Nature 332 323 327 ( 1988) Verhoeyen et al Science 239 1534- 1536 (1988)] by substituting iodent CDRs or CDR sequences tor the corresponding sequences ot a human antibody Accordingly such humanized antibodies are chimenc antibodies (U S Patent No 4 816,567) wheiein substantially less than an intact human v ariable domain has been substituted by the corresponding sequence from a non human species In practice humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991 ), Marks et al J Mol Biol , 222 581 (1991 )] The techniques of Cole et al , and Boerner et al , are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss p 77 (1985) and Boerner et al J Immunol , 147(1 ) 86-95 (1991 )] Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed which closely resembles that seen in humans in all respects, including gene rearrangement assembly, and antibody repertoire This approach is described, for example, in U S 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, JO 779-783 (1992), Lonberg ef al , Nature 368 856 859 (1994), Morrison. Nature 368 812-1 (1994), Fishwild et al , Nature Biotechnology. 14 845-51 (1996), Neuberger. Nature Biotechnology, 14 826 ( 1996) Lonberg and Huszar, Intern Rev Immunol . 13 65-93 (1995)
4 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 enzv, me which converts a prodrug (e g a peptidyl chemotherapeutic agent see WO 81/01 145) to an active anti-cancer drug See, for example WO 88/07378 and U S Patent No 4 975,278 The enzyme component of the immunoconj ugate useful for ADEPT includes any enzyme capable ot 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 lysozyme, human glucuronidase, alkaline phosphatase useful tor converting phosphate-containing prodrugs into free drugs arylsulfatase useful for converting sulfate containing prodrugs into free drugs cytosine deaminase useful for converting non toxic 5-fluorocytosιne into the anti cancerdrug5 fluorouracil, proteases, such as serratia protease, thermolysin, subtihsin, carboxypeptidases (e g carboxypeptidase G2 and carbox peptidase A) and cathepsins (such as cathepsms B and L), that are useful tor conv erting peptide containing prodrugs into free drugs, D-alanylcarboxvpeptidases useful for converting prodrugs that contain D amino acid substituents carbohydrate-cleaving enzymes such as β galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs β lactamase useful for converting drugs deriv atized ith β lactams into free drugs, and penicillin amidases, such as penicillin Vamidase or penicillin G amidase useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups respectively into free drugs Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes'" can be used to convert the prodrugs of the invention into free active drugs (see. e.g., Massey, Nature. 328:457-458 ( 1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
The enzymes of this invention can be covaiently bound to the anti-PRO antibodies by techniques well known in the art such as the use of the heterobifunctional cross-linking agents 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 et al., Nature. 312:604-608 ( 1984)).
5. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO polypeptide the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 [1983]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829. published 13 May 1993. and in Traunecker etal.. EMBO J.. 10:3655-3659 ( 1991 ). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy- chain constant domain, comprising at least part of the hinge, CH2. and CH3 regions. It is prefeπed to have the first heavy-chain constant region (CH I ) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh etal.. Methods in Enzvmology. 121 :210 ( 1986).
According to another approach described in WO 96/2701 1. the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products uch 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 bispecific antibodies can be prepared using chemical linkage Brennan etal Science 229 81 ( 1985) describe a procedure wherein intact antibodies are proteolvtically 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) deπvativ es One ot the Fab -TNB derivatives is then reconverted to the Fab - thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ot the other Fab'-TNB derivative to form the bispecific antibody The bispecific antibodies produced can be used as agents tor the selective immobilization ot enzymes
Fab' fragments may be directly recovered from £ coli and chemically coupled to form bispecific antibodies Shalaby et al . J Exp Med , J_75 217 225 (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 ot human cytotoxic lymphocytes against human breast tumor targets Various techniques tor making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol , 148(5) 1547- 1553 ( 1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody" technology described by Hollinger et al , Proc Natl Acad Sci USA.90 6444-6448 ( 1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly the VH and VL domains of one tragment are forced to pair with the complementary VL and VH domains ot another fragment, thereby forming two antigen binding sites Another strategy tor making bispecific antibody fragments by the use ot single-chain Fv (sFv ) di ers has also been reported See Gruber et al , J Immunol 152 5368 (1994)
Antibodies with more than two valencies are contemplated For example tπspecif ie antibodies can be prepared Tutt et al , J Immunol , 147 60 ( 1991 ) Exemplary bispecific antibodies may bind to two different epitopes on a given polypeptide herein
Alternatively an anti-polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e g , CD2 CD3. CD28 or B7) or Fc receptors tor IgG (FcγR) such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular polypeptide Bispecific antibodies may also be used to localize cvtotoxic agents to cells which express a particular polypeptide These antibodies possess a polypeptide-binding arm and an arm w hich binds a cytotoxic agent or a radionuchde chelator such as EOTUBE DPTA DOTA or TETA Another bispecific antibody ot interest binds the polypeptide and further binds tissue factor (TF) 6 Heterocontugate Antibodies
Heteroconjugate antibodies are composed ot two covalently joined 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 ot HIV infection [WO 91/00360, WO 92/200373, EP 03089] It is contemplated that the antibodies may be prepared ... x itro using known methods in synthetic protein chemistry, including those involving crosslinking agents For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples ot suitable reagents tor this purpose include lminothiolate and methyl-4- mercaptobutyπmidate and those disclosed, tor example in U S Patent No 4.676,980
7 Effector function engineering It may be desirable to modify the antibody ot the invention with respect to effector function, so as to enhance the effectiveness ot 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 homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See, Caron et al . J Exp Med . 176 1 191 -1 195 ( 1992) and Shopes. J Immunol , 148 2918-2922 (1992) Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al , Cancer Research.53 2560- 2565 (1993) Alternatively, an antibody can be engineered 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)
8 Immu nocon I u gates
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 ot such immunoconjugates have been described above Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbinding 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. Aleut ites foidu proteins dianthin proteins. Plntolaca amencana proteins (PAPI, PAPII. and PAP-S), momordica charantia inhibitor curcin, crotin. sapaonaπa officina s inhibitor, gelonin sapoπn mitogelhn restπctocin phenomycin, enomycin and the tπcothecenes Small molecule toxins include, tor example calicheamicins. maytansinoids, palytoxin and CC 1065
A variety of radionuclides are av ailable for the production of radioconjugated antibodies Examples include 21 Bι
'"I. ' "In "Y and IS6Re
Conjugates of the antibody and cvtotoxic agent are made using a variety of bitunctional protein coupling agents such as N-succιnιmιdyl-3-(2-pyπdvldιthιol ) propionate (SPDP) lminothiolane (IT), bitunctional derivatives of imidoesters (such as dimethvl adipimidate HCL) active esters (such as disucci midvl suberate). aldehydes (such as glutaraldehyde). bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine) bis-diazonium derivatives (such as bιs-(p-dιazonιumbenzovl)-ethvlenedιamιne), dnsocvanates (such as tolyene 2.6-dιιsocyanate). and bis- active fluorine compounds (such as 1 5 dιfluoro-2 4-dιnιtrobenzene) For example, a πcin immunotoxin can be prepared as described in Vitetta et al Science 238 1098 ( 1987) Carbon- 14-Iabeled l -ιsothιocyanatobenzyl-3- methyidiethylene tπaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation ot radionucleotide to the antibody See, WO94/1 1026
In another embodiment, the antibody may be conjugated to a receptor ' (such as streptav idin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient totϊowed by removal ot unbound conjugate from the circulation using a clearing agent and then administration ot A gand (e g , avidin) which is conjugated to a cytotoxic agent (e g , a radionucleotide)
9 Immunohposomes
The antibodies disclosed herein may also be formulated as immunohposomes Liposomes containing the antibody are prepared bv methods known in the art, such as described in Epstein et al , Proc Natl Acad Set USA. 82 3688 (1985), Hwang et al Proc Natl Acad Sci USA 77 4030 ( 1980). and U S Patent 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 phosphatidylcholine. cholesterol and PEG-deπvatized phosphatidylethanolaπune (PEG- PE) Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al J Biol Chem . 257 286-288 (1982) via a disulfide interchange reaction A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome See. Gabizon etal . J National Cancer Inst 81 19).1484 ( 1989)
N Pharmaceutical Compositions
Antibodies specifically binding the product ot an amplified gene identified herein as well as other molecules identified bv the screening assays disclosed hereinbefore can be administered tor the treatment ot tumors, including cancers, in the form of pharmaceutical compositions
If the protein encoded bv the amplified gene is intracellular and whole antibodies are used as inhibitors internalizing antibodies are preferred However, hpofections 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 upon the ariable region sequences ot an antibody peptide molecules can be designed which retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technologv (see, e g . Marasco et al Proc Natl Acad Sci USA 90 7889-7893 [ 1993])
Therapeutic formulations ot the antibody are prepared tor storage bv mixing the antibody having the desired degree ot puπtv with optional pharmaceutically acceptable carriers excipients or stabilizers (Remington s Pharmaceutical Sciences. 16th edition Osol. A ed [ 1980]) in the form ot lyophihzed formulations or aqueous solutions Acceptable carriers excipients. or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids: antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethomum chloride, benzalkonium chloride, benzetho um 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 polyvinylpyrrolidone. amino acids such as glycine, glutamine. asparagine. histidine arginine. or lysine. monosacchaπdes. disacchaπdes and other carbohydrates including glucose, mannose. or dextπns. chelating agents such as EDTA, sugars such as sucrose, mannitol. trehalose or sorbitol. salt-forming counter-ions such as sodium, metal complexes (e g . Zn-protein complexes), and or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG)
Non-antibody compounds identified by the screening assays ot 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 tor the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent cytokine or growth inhibitory agent Such molecules are suitably present in combination 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 mtertacial polymeπzation. tor example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules. respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions. nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences. 16th edition, Osol. A ed (1980)
The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matπces are in the form ot shaped articles, e g , films or microcapsules Examples ot sustained-release matrices include polyesters. hydrogels (for example, poly(2-hydroxyethyl-methacrylate). or poly(vιnylalcohol)), polylactides ( U S Pat No 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate. non-degradable ethylene-v in l acetate, degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate), and poly-D-(-)-3-hydro\ybut\πc acid While polymers such as ethylene-v my 1 acetate and lactic acid-glycohc acid enable release of molecules tor over 100 days, certain hydrogels release proteins tor shorter time periods When encapsulated antibodies remain in the body tor a long time, they may denature or aggregate as a result ot exposure to moisture at 37°C. resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised tor stabilization depending on the mechanism involved For example, it the aggregation mechanism is discovered to be lntermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions O Methods ot Treatment
It is contemplated that the antibodies and other anti-tumor compounds ot the present invention mav be used to treat v aπous conditions, including those characterized by ov erexpression 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 malignant tumors (e ? , renal, liv er kidney bladder breast gastric, ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic carcinomas, sarcomas, ghoblastomas. and vaπous head and neck tumors), leukemias and lymphoid malignancies, other disorders such as neuronal, glial, astrocytal. hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoehc disorders, and inflammatory, angiogenic and lmmunologic disorders
The anti-tumor agents ot 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, lntrapeπtoneal, intracerobrospinal. subcutaneous lntra-articular, intrasynovial. intrathecal, oral, topical or inhalation routes Intravenous administration ot the antibody is preferred Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e g , 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' instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed , M C. Perry, Williams & Wilkins. Baltimore. MD
(1992) The chemotherapeutic agent may precede, or follow administration of the 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 as onapπstone (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 the ErbB2. EGFR. ErbB3. ErbB4 or vascular endothehal factor (VEGF) Alternativ ely, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein mav be co- administered to the patient Sometimes it may be beneficial to 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 ot the present invention However. simultaneous administration or administration of the antibody of the present invention first is also contemplated Suitable dosages tor 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 appropriate dosage ot 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 ot the disease whether the agent is administered tor preventive or therapeutic purposes previous therapy the patient s clinical history and response to the agent, and the discretion ot the attending phvsician The agent is suitably administered to the patient at one time or over a series ot treatments
For example, depending on the type and seventy ot the disease, about 1 /.g/kg to 15 mg/kg (e ? 0 1-20 mg/kg ) ot antibody is an initial candidate dosage for administration to the patient, whether, tor example bv one or more separate administrations, or by continuous infusion A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more depending on the factors mentioned above For repeated administrations over several days or longer depending on the condition, the treatment is sustained until a desired suppression of disease svmptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays
P Articles of Manufacture
In another embodiment of the invention an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided The article of manufacture comprises a container and a label Suitable containers include, tor 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 ot 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 mav further compπse a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer s solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters needles, syringes, and package inserts with instructions tor use
Q Diagnosis and Prognosis of Tumors
While cell surface proteins, such as growth receptors overexpressed in certain tumors are excellent targets for drug 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 ot tumors For example, antibodies directed against the protein 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 qualitatively or quantitativ eh 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 microscopv flow cytometrv. fluoπmetry, or other techniques known in 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 ot the marker gene product in the tissue examined It will be apparent for those skilled in the art that a wide variety ot histological methods are readily available for in situ detection
The following examples are ottered tor illustrative purposes only, and are not intended to limit the scope of the present invention in any way
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety
EXAMPLES Commercially available reagents referred to in the examples were used according to manufacturer s instructions unless otherwise indicated The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection. 10801 University Blvd , Manassas, VA 201 10-2209 All original deposits referred to in the present application were made under the provisions ot 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. Ine . and ATCC. which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U S patent or upon laying open to the public ot any U S or foreign patent application, whichever comes first, and assures availability ot the progeny to one determined by the U S Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC 122 and the Commissioner" s rules pursuant thereto (including 37 CFR § 1 14 with particular reference to 886 OG 638) Unless otherwise noted, the present invention uses standard procedures ot 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 etal , Current Protocols in Molecular Biology. Green Publishing Associates and Wiley Interscience. N Y , 1989. Innis et al , PCR Protocols A Guide to Methods and Applications. Academic Press, Ine , N Y . 1990 Hariow etal Antibodies A Laboratory Manual Cold Spring Harbor Press. Cold Spring Harbor. 1988 Gait. Oligonucleotide Synthesis. IRL Press. Oxford. 1984. R I Freshney, Animal Cell Culture. 1987, Cohgan et al , Current Protocols in Immunology, 1991
EXAMPLE 1
Isolation of cDNA Clones Encoding a Human PRO5800
The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included public EST databases (e g , GenBank) The search was performed using the computer program
BLAST or BLAST2 [Altschul et al . Methods in Enzvmologv 266 460-480 ( 1996)] as a comparison of the ECD protein sequences to a 6 frame translation ot the EST sequences Those compaπsons 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 ot Washington. Seattle. Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA 102836 In some cases, the consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles ot BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above Based on the DNA 102836 consensus sequence, oligonucleotides vv ere synthesized 1 ) to identify by PCR a cDNA library that contained the sequence ot interest, and 2) for use as probes to isolate a clone of the full-length coding sequence tor PRO5800 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product ot about 100-1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized w hen the consensus sequence is greater than about 1-1 5kbp In order to screen several libraries for a full-length clone. DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, supra, with the PCR primer pair A positive hbrarv was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs
PCR primers (forward and reverse) were synthesized forward PCR primer 1
5'-CAGCGAACCGGGTGCCGGGTC-3' (SEQ ID NO 21 ) forward PCR primer 2
5'-GAGCGACGAGCGCGCAGCGAAC-3' (SEQ ID NO 22) forward PCR primer 3 5'-ATACTGCGATCGCTAAACCACCATGCGCCGCCGCCTGTGGCTG-3' (SEQ ID NO 23) reverse PCR primer 1
5'-GCCGGCCTCTCAGGGCCTCAG-3' (SEQ ID NO 24) reverse PCR primer 2
5'-CCCACGTGTACAGAGCGGATCTC-3' (SEQ ID NO 25) reverse PCR primer 3
5'-GAGACCAGGACGGGCAGGAAGTG-3 (SEQ ID NO 26) reverse PCR primer 4
5'-CAGGCACCTTGGGGAGCCGCC-3 (SEQ ID NO 27) reverse PCR primer 5 5'-CCCACGTGTACAGAGCGGATCTC-3' (SEQ ID NO 28) reverse PCR primer 6
5'-GAGACCAGGACGGGCAGGAAGTG-3' (SEQ ID NO 29)
Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA 102836 sequence which had the following nucleotide sequence hybridization probe
5'-CTCTACGGGTACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGG-3 (SEQ ID NO 30)
RNA tor construction ot the cDNA libraries was isolated from human fetal liver tissue The cDNA libraries used to isolate the cDNA clones were constructed bv standard methods using commercially available reagents such as those from Invitrogen, San Diego. C A The cDNA w s primed with ohgo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl. sized appropriately by gel electrophoresis. and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD. pRK5B is a precursor of pRK5D that does not contain the Sfil site. see. Holmes et al Science. 253 1278-1280 ( 1991 )) in the unique Xhol and Notl sites
DNA sequencing ot the clones isolated as described above gave the full-length DNA sequence for a full-length PRO5800 polypeptide (designated herein as DNA 108912-2680 [Figure 1. SEQ ID NO 1 ]) and the derived protein sequence for that PRO5800 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 7-9 and a stop signal at nucleotide positions 517-519 (Figure 1 SEQ ID NO 1 ) The predicted polypeptide precursor is 170 amino acids long, has a calculated molecular weight of approximately 19.663 daltons and an estimated pi of approximately 1 1 81 Analysis of the full-length PRO5800 sequence shown in Figure 2 (SEQ ID NO 2) evidences the presence ot a variety of important polypeptide domains as shown in Figure 2, wherein the locations given for those important polypeptide domains are approximate as described above Clone DNA 108912-2680 has been deposited with ATCC on May 25. 1999 and is assigned ATCC deposit no PTA- 124
An analysis ot the Dayhoff database (v ersion 35 45 SwissProt 35). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 2 (SEQ ID NO 2), evidenced sequence identity between the PRO5800 amino acid sequence and the following Dayhoff sequences P_W52595, P_W57313, FGFA_HUMAN, P_W57264, FGFA_RAT, P_W52597, MMU94517_1 , FGFA_MOUSE. P_W57306 and D86333
EXAMPLE 2 Isolation of cDNA Clones Encoding a Human PRO6000 A cDNA clone (DNA 102880-2689) encoding a native human PRO6000 polypeptide was identified using a yeast screen, in a human uterine cDNA library that preferentially represents the 5 ends ot the primary cDNA clones
Clone DNA 102880-2689 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 28-30 and ending at the stop codon at nucleotide positions 580-582 (Figure 3. SEQ ID NO 3) The predicted polypeptide precursor is 184 am o acids long (Figure 4. SEQ ID NO 4) The full-length PRO6000 protein shown in Figure 4 has an estimated molecular weight of about 21.052 daltons and a pi of about 5 01 Analysis of the full-length PRO6000 sequence shown in Figure 4 (SEQ ID NO 4) evidences the presence ot a variety of important polypeptide domains as shown in Figure 4, wherein the locations given tor those important polvpeptide domains are approximate as described above Clone DNA 102880-2689 has been deposited w ith ATCC on July 20. 1999 and is assigned ATCC Deposit No PTA-383
An analysis ot the Dayhoff database ( v ersion 35 45 S w issProt 35). using the ALIGN-2 sequence alignment analysis ot the full-length sequence shown in Figure 4 (SEQ ID NO 4) ev idenced sequence identity between the PRO6000 amino acid sequence and the following Dayhotf sequences SPS_VICFA, ADU85448 G64635 AE001516_3. P_W20328, P_W20747. and SPS_SPIOL EXAMPLE 3 Isolation ot cDNA Clones Encoding a Human PRO6016 DNA96881-2699 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech. Ine . (South San Francisco. CA) upon ESTs as well as clustered and assembled EST fragments from public (e g , Genbank) and/or private (LIFESEQ*, Incyte Pharmaceuticals, Ine , 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 tor 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 v, hether 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 ot the above described signal sequence algorithm allowed identification of an EST sequence from the LIFESEQ* database, Incyte Pharmaceuticals. Palo Alto C A, designated herein as 3035248H 1 This EST sequence 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 etal , Methods in Enzvmology, 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 with the program "phrap" (Phil Green, University of Washington, Seattle, Washington) The consensus sequence obtained therefrom is herein designated DNA82389
In hght ot an observed sequence homology between the DNA82389 sequence and an EST sequence encompassed within clone no 3035248H1 from the LIFESEQ*1 database. Incyte Pharmaceuticals. Palo Alto. CA, clone no 3035248H1 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 5 and is herein designated as DNA96881 -2699
Clone DNA96881 -2699 contains a single open reading frame ith an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 1005-1007 (Figure 5 SEQ ID NO 5) The predicted polypeptide precursor is 315 amino acids long (Figure 6 SEQ ID NO 6) The full-length PRO6016 protein shown in Figure 6 has an estimated molecular weight of about 35,963 daltons and a pi of about 5 38 Analysis ot the full-length PRO6016 sequence shown in Figure 6 (SEQ ID NO 6) evidences the presence ot a variety of important polypeptide domains as shown in Figure 6. wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA96881 -2699 has been deposited with ATCC on August 17 1999 and is assigned ATCC Deposit No PTA-553
An analysis ot the Dayhoff database (version 35 45 SwissProt 35). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 6 (SEQ ID NO 6) evidenced sequence identity between the PRO6016 amino acid sequence and the following Dayhoff sequences P_W88499. HGS_RE347, P_W88647, Y087_CAEEL. S44095, P_W03626, P_W03627 IE68_HSVSA, PN0009 RNU51583_1
EXAMPLE 4
Isolation ot cDNA Clones Encoding a Human PRO6018
DNA98565-2701 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech. Ine (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e g , Genbank) and/or private (LIFESEQ*1, Incyte Pharmaceuticals, Ine , Palo Alto. CA) databases The signal sequence algorithm computes a secretion signal score based on the character ot 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 am o 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 ot an EST sequence from the
LIFESEQ* (Incyte Pharmaceuticals, Palo Alto, CA) database, designated herein as 745575H1 This EST sequence 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 etal , Methods in Enzymology.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 with the program "phrap" (Phil Green, University of Washington, Seattle. Washington) The consensus sequence obtained therefrom is herein designated DNA8241 1
In light of an observed sequence homology between the DNA8241 1 sequence and an EST sequence encompassed w ithin clone no 745575H 1 from the LIFESEQ" (Incyte Pharmaceuticals. Palo Alto CA) database clone no 745575H1 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence ot this cDNA insert is shown in Figure 7 and is herein designated as DNA98565-2701
Clone DNA98565-2701 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 352-357 and ending at the stop codon at nucleotide positions 3085-3087 (Figure 7 SEQ ID NO 7). The predicted polypeptide precursor is 91 1 amino acids long (Figure 8. SEQ ID NO 8) The full-length PRO6018 protein shown in Figure 8 has an estimated molecular weight ot about 99,1 17 daltons and a pl of about 4.62. Analysis ot the full-length PRO6018 sequence shown in Figure 8 (SEQ ID NO 8) evidences the presence of a variety of important polypeptide domains as shown in Figure 8 wherein the locations given for those important polypeptide domains are approximate as described above Clone DNA98565 2701 has been deposited with ATCC on August 3. 1999 and is assigned ATCC Deposit No PTA-481
An analvsis of the Dayhoff database (version 35 45 S wissProt 35), using the ALIGN-2 sequence alignment analysis ot the full-length sequence shown in Figure 8 (SEQ ID NO 8). ev idenced sequence identity between the PRO6018 amino acid sequence and the following Dayhoff sequences PGCB_BOVIN, P_R85442. P_R77034. P_R12609. AC0031 10_2. PGCV_HUMAN, AF1 16856J P_W75099. HGS_A 176, A098460J
EXAMPLE 5 Isolation ot cDNA Clones Encoding a Human PRQ6496
The extracellular domain (ECD) sequences ( including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included a proprietary EST 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 comparison of the ECD protein sequences to a 6 frame translation of the EST sequences 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 w ith the program "phrap" (Phil Green, University ot Washington. Seattle. Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA43048 In some cases, the DNA43048 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of
BLAST and phrap to extend that intermediate consensus sequence as far as possible using the sources of EST sequences discussed above
Based on the DNA43048 consensus sequence, and in light of an observed sequence homology between the DNA43048 sequence and an EST sequence encompassed within clone no 1636952 from the LIFESEQ® database, Incyte Pharmaceuticals. Palo Alto, CA, clone no 1636952 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 9 and is herein designated as DNA 1 19302-2737
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 63-65 and a stop signal at nucleotide positions 2328-2330 (Figure 9. SEQ ID NO 9) The predicted polypeptide precursor is 755 amino acids long, has a calculated molecular weight ot approximately 82.785 daltons and an estimated pi ot approximately 8 71 Analysis of the full-length PR06496 sequence shown in Figure 10 (SEQ ID NO 10) evidences the presence of a variety ot important polypeptide domains as shown in Figure 10. wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA 1 19302-2737 has been deposited with ATCC on August 10. 1999 and is assigned ATCC Deposit No PTA-520
An analysis of the Dayhoff database ( version 35 45 S wissProt 35). using the ALIGN-2 sequence alignment analvsis ot the full-length sequence shown in Figure 10 (SEQ ID NO 10) evidenced sequence identity between the PR06496 amino acid sequence and the following Davhotf sequences P_W81365. NEC3JV10USE, MUSPRCON 14J . FURI_HUMAN P_R77540 S71340 P_W73932 DROFUR1 ISO . GEN 12660. and
P_R59784 EXAMPLE 6 Isolation ot cDNA Clones Encoding a Human PRQ7154 The extracellular domain (ECD) sequences (including the secretion signal sequence if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases The EST databases included ( 1 ) public EST databases ( g , Merck/Washington University), and (2) a proprietary EST 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 comparison ot the ECD protein sequences to a 6 frame translation ot the EST sequences Those comparisons resulting in a BLAST score ot 70 (or in some cases, 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences w ith the program "phrap" (Phil Green. University of Washington, Seattle. Washington)
A consensus DNA sequence was assembled relative to other EST sequences using phrap as described above This consensus sequence is herein designated DNA38237 In some cases, the DNA38237 consensus sequence derives from an intermediate consensus DNA sequence which was extended using repeated cycles of BLAST and phrap to extend that intermediate consensus sequence as tar as possible using the sources ot EST sequences discussed above.
Based on the DNA38237 consensus sequence, and in light ot an observed sequence homology between the DNA38237 sequence and an EST sequence encompassed within clone no 1855755 from the LIFESEQ® database. Incyte Pharmaceuticals. Palo Alto. CA. clone no 1855755 was purchased and the cDNA insert was obtained and sequenced. It was found herein that that cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 1 1 and is herein designated as DNA 108760-2740
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 102- 104 and a stop signal at nucleotide positions 1083- 1085 (Figure 1 1. SEQ ID NO 1 1) The predicted polypeptide precursor is 327 amino acids long, has a calculated molecular weight of approximately 34.348 daltons and an estimated pi ot approximately 7 88 Analysis ot the full-length PR07154 sequence shown in Figure 12 (SEQ ID NO 12) evidences the presence ot a variety ot important polypeptide domains as shown in Figure 12. wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA 108760-2740 has been deposited w ith ATCC on August 17. 1999 and is assigned ATCC Deposit No PTA-548 An analysis of the Dayhoff database ( ersion 35 45 S wissProt 35). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 12 (SEQ ID NO 12 ) evidenced sequence identity between the PR07154 amino acid sequence and the following Dayhoff sequences AF061022_1 AF061024_1 HS889N15_1 , GGY14064J GGY 14063J AF061023 , XLU43330_ 1 GEN 14531 , MMCARHJ . and MMU90715
EXAMPLE 7
Isolation of cDNA Clones Encoding a Human PRO7170 DNA 108722-2743 was identified bv applying a proprietary signal sequence finding algorithm developed by Genentech. Ine . (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e g Genbank) and/or private (LIFESEQ*. Incvte Pharmaceuticals. Ine . Palo Alto, CA) databases The signal sequence algorithm computes a secretion signal score based on the character ot the DNA nucleotides surrounding the tirst and optionally the second methionine codon(s) (ATG) at the 5'-end ot 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 ammo 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 ot seven sensors (evaluation parameters) known to be associated with secretion signals
Use ot the above described signal sequence algorithm allowed identification ot an EST cluster sequence from the LIFESEQ® database, Incyte Pharmaceuticals. Palo Alto, designated herein as CLU57836 This EST cluster sequence 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 Enzymology, 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 with the program "phrap ' (Phil Green, University of Washington. Seattle. Washington) The consensus sequence obtained therefrom is herein designated DNA58756 In light of an observed sequence homology between the DNA58756 sequence and an EST sequence encompassed within clone no 2251462 from the LIFESEQ® database. Incyte Pharmaceuticals, Palo Alto. CA, clone no 2251462 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence of this cDNA insert is shown in Figure 13 and is herein designated as DNA 108722-2743 Clone DNA 108722-2743 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 60-62 and ending at the stop codon at nucleotide positions 1506- 1508 (Figure 13. SEQ ID NO 13) The predicted polypeptide precursor is 482 amino acids long (Figure 14. SEQ ID NO 14) The full-length PR07170 protein shown in Figure 14 has an estimated molecular weight of about 49,060 daltons and a pi ot about 4 74 Analysis of the full-length PR07170 sequence shown in Figure 14 (SEQ ID NO 14) evidences the presence of a variety of important polypeptide domains as shown in Figure 14 wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA108722-2743 has been deposited with ATCC on August 17, 1999 and is assigned ATCC Deposit No PTA-552
An analysis of the Dayhott database (version 35 45 S wissProt 35). using the ALIGN-2 sequence alignment analysis ot the full-length sequence shown in Figure 14 (SEQ ID NO 14). evidenced sequence identity between the PRO7170 amino acid sequence and the following Dayhott sequences P_Y 12291. 147141. D88733 , DMC56G7_1 , P_Y 1 1606. HWPl_CANAL. HSMUC5BEX_l . HSU78550_l , HSU70I 36_l . and SGS3_DROME EXAMPLE 8 Isolation ot cDNA Clones Encoding a Human PR07422 DNA 1 19536-2752 was identified by applying a proprietary signal sequence finding algorithm developed by Genentech. Ine , (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e g , GenBank) and/or private (LIFESEQ0, Incyte Pharmaceuticals. Ine . Palo Alto, CA) databases The signal sequence algorithm computes a secretion signal score based on the character ot the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end ot 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 ot the above described signal sequence algorithm allowed identification of an EST cluster sequence from the Incyte database, designated herein as 81575 This EST cluster sequence 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 Enzymology. 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 with the program "phrap" (Phil Green, University of Washington. Seattle, Washington) The consensus sequence obtained therefrom is herein designated DNA 104391
In light of an observed sequence homology between the DNA 104391 sequence and an EST sequence encompassed within clone no 1922888 from the Incyte database, clone no 1922888 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence ot this cDNA insert is shown in Figure 15 and is herein designated as DNA 1 19536-2752
Clone DNA 1 19536-2752 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 47-49 and ending at the stop codon at nucleotide positions 31 1 -313 (Figure 15. SEQ ID NO 15) The predicted polypeptide precursor is 88 amino acids long (Figure 16) The full-length PR07422 protein shown in Figure 16 has an estimated molecular weight of about 9.645 daltons and a pi of about 5 45 Analysis ot the full-length PR07422 sequence shown in Figure 16 (SEQ ID NO 16) evidences the presence ot a variety of important polypeptide domains as shown in Figure 16. wherein the locations given tor those important polypeptide domains are approximate as described above Clone DNA1 19536-2752 has been deposited with ATCC on August 17. 1999 and is assigned ATCC deposit no PTA-551
EXAMPLE 9
Isolation ot cDNA Clones Encoding Human PRQ74Η DNA 1 19542-2754 was identified by apply mg a proprietary signal sequence finding algorithm developed by Genentech. Ine . ( South San Francisco C A) upon ESTs as well as clustered and assembled EST fragments from public (e g . GenBank) and/or priv ate (LIFESEQ0, Incyte Pharmaceuticals. Ine . Palo Alto. CA) databases The signal sequence algorithm computes a secretion signal score based on the character ot the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end ot the sequence or sequence fragment under consideiation 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 It 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 coπesponding amino acid sequences surrounding the ATG codon are scored using a set ot seven sensors (evaluation parameters) known to be associated with secretion signals
Use of the above descπbed signal sequence algorithm allowed identification of an EST sequence from the LIFESEQ®, Incyte Pharmaceuticals, Ine , Palo Alto. CA database, designated herein as clone No 2201182. This EST sequence was then compared to a variety ot 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 identity existing homologies The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al . Methods in Enzymology. 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 with the program "phrap" (Phil Green. University of Washington, Seattle, Washington) The consensus sequence obtained therefrom is herein designated DNA 104392 In light of an observed sequence homology between the DNA 104392 sequence and an EST sequence encompassed within clone no 2201 182 from the Incyte database, clone no 2201 182 was purchased and the cDNA insert was obtained and sequenced It was found herein that that cDNA insert encoded a full-length protein The sequence of this cDNA insert is herein designated as DNA1 19542-2754
Clone DNA 1 19542-2754 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 247-249 and ending at the stop codon at nucleotide positions 838-840 (Figure 17 SEQ ID NO 17) The predicted polypeptide precursor is 197 amino acids long (Figure 18) The full-length PR07431 protein shown in Figure 18 has an estimated molecular weight ot about 21.992 daltons and a pi ot about 12 18 Analysis ot the full-length PR07431 sequence shown in Figure 18 (SEQ ID NO 18) evidences the presence of a variety ot important polypeptide domains as shown in Figure 18. wherein the locations given for those important polypeptide domains are approximate as described above Clone DNA 1 19542-2754 has been deposited with ATCC on August 31 , 1999 and is assigned ATCC deposit no PTA-619
An analysis of the Davhotf database ( version 35 45 S wissProt 35), using the ALIGN-2 sequence alignment analysis ot the full-length sequence shown in Figure 18 (SEQ ID NO 18). evidenced sequence identity between the PR07431 amino acid sequence and the following Davhotf sequences AF061943J . RNU08136_1 , MAVOH 838J . HXAA_HUMAN Y653_HUMAN P_R51263, P_R74041 . AF10J 057 , AF101058. AF101059J EXAMPLE 1 Isolation ot cDNA Clones Encoding a Human PRQ7476 A search was performed using the computer program BLAST or BLAST2 [Altschul et al . Methods in
Enzymology. 266 460-480 ( 1996)] tor cytokine/growth factor homologs A 94 5 KB piece was found to contain exons encoding growth factor homologs. however, this piece was broken up by large introns The introns were removed by a computer algorithm Based on the DNA 102863 consensus sequence, oligonucleotides were synthesized 1 ) to identity 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 PR07476 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100- 1000 bp in length The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 -1 5kbp In order to screen several libraries tor a full-length clone.
DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular
Biology, supra, with the PCR primer pair A positive library was then used to isolate clones encoding the gene ot interest using the probe oligonucleotide and one ot the primer pairs PCR primers (forward and reverse) were synthesized forward PCR primer:
5'-ATGCAGCTCCCACTGGCCCTG-3' (SEQ ID NO 31 ) reverse PCR primer
5'-CTAGTAGGCGTTCTCCAGCTCGGCCTG-3' (SEQ ID NO 32) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA 102863 sequence which had the following nucleotide sequence hybridization probe.
5'-CTTCCGCTGCATCCCCGACCGCTACCGCGCGCAGCGCGTG-3' (SEQ ID NO 33)
DNA sequencing ot the clones isolated as described above gav e the full-length DNA sequence tor a full-length PR07476 polypeptide (designated herein as DNA 1 15253-2757 [Figure 19. SEQ ID NO 19] ) and the derived protein sequence for that PR07476 polypeptide
The full length clone identified above contained a single open reading frame with an apparent translational initiation site at nucleotide positions 62-64 and a stop signal at nucleotide positions 701 -703 (Figure 19. SEQ ID NO 19) The predicted polypeptide precursor is 213 amino acids long, has a calculated molecular weight ot approximately 24.031 daltons and an estimated pi ot approximately 9 59 Analysis ot the full-length PR07476 sequence shown in Figure 20 (SEQ ID NO 20) evidences the presence of a variety of important polypeptide domains as shown in Figure 20 wherein the locations given for those important polypeptide domains are approximate as descπbed above Clone DNA 1 15253-2757 has been deposited with ATCC on August 31 1999 and is assigned ATCC Deposit No PTA-612
An analysis of the Dayhott database ( version 35 45 S wissProt 35 ). using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in Figure 20 (SEQ ID NO 20), evidenced sequence identity between the PR07476 amino acid sequence and the following Dayhoff sequences P_W58704 P W9571 1 , P_W09408. P_Y 12009 T08710, P_W44090 P^W27654 P_Y03225. LSHB_MELGA. AB01 1030J
EXAMPLE 1 1
Gene Amplification This example shows that the PRO5800-. PRO6000-. PRO6016-. PRO6018-. PR06496-. PR07154-, PR07170-. PR07422-, PR07431 - or PR07476-encodιng genes are amplified in the genome ot certain human lung, colon and/or breast cancers and/or cell lines Amplification is associated with overexpression ot the gene product, indicating that the polypeptides are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast and other cancers Therapeutic agents may take the form ot antagonists of PRO5800, PRO6000. PRO6016, PRO6018 PR06496, PR07154. PR07170. PR07422. PR07431 or PR07476 polypeptides, for example, muπne- human chimeric. humanized or human antibodies against a PRO5800. PRO6000. PRO6016, PRO6018. PR06496, PR07154. PRO7170, PR07422. PR07431 or PR07476 polypeptide
The starting mateπal tor the screen was genomic DNA isolated from a variety of cancers The DNA is quantitated precisely, e g , fluorometπcally As a negative control, DNA was isolated from the cells of ten 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 whether the DNA encoding PRO5800, PRO6000. PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 is over-represented in 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 individuals with tumors of the type and stage as indicated in Table 4 An explanation ot the abbreviations used for the designation of the primary tumors listed in Table 4 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore
The results of the TaqMan r are reported in delta (Δ) Ct units One unit corresponds to 1 PCR cycle or approximately a 2-told amplification relative to normal, two units corresponds to 4-fold 3 units to 8-fold amplification and so on Quantitation was obtained using primers and a TaqMan™ fluorescent probe derived from the PRO5800-. PRO6000-, PRO6016-, PRO6018-. PR06496-. PR07154-, PR07170-, PR07422-, PR07431 - or PR07476-encodιng gene Regions of PRO5800. PRO6000. PRO6016. PRO6018. PR06496, PR07154. PR07170, PR07422, PR07431 or PR07476 which are most likely to contain unique nucleic acid sequences and which are least likely to have spliced out introns are preferred for the primer and probe deπv ation. e g .3'-untranslated regions The sequences tor the primers and probes (forward, reverse and probe) used tor the PRO5800. PRO6000, PRO6016. PRO6018. PR06496. PR07154. PRO7170. PR07422. PR07431 or PR07476 gene amplification analysis were as follows
PRO5800 (DNA 108912-2680) 108912.tm t l
5'-GCGTCGTGGTCATCAAAG-3 (SEQ ID NO 34 ) 108912 tmrl '-TGCAGTCCACGGTGTAGAG-3 (SEQ ID NO 35)
108912 tmpl
5'-CTTCTACGTGGCCATGAACCGC-3 (SEQ ID NO 36) 108912 tmt2
5'-CCTGGAGATCCGCTCTGTA-3' (SEQ ID NO 37)
108912 tmp2
5'-CTTTGATGACCACGACGCCCA-3' (SEQ ID NO 38) 108912 tmr2 5 -ACGTAGAAGCCTGAGGACACT-3' (SEQ ID NO 39)
PRO6000 (DNA 102880-2689) 102880 tmfl
5'-GATGCTCCAGCTGAAATCC-3 (SEQ ID NO 40)
102880 tmrl 5'-CACATGGCTGGAAATGATG-3 (SEQ ID NO 41 )
102880 tmpl 5'-AAGCTAAGCTCCCAACTGACAGCCA-3 (SEQ ID NO 42)
PRO6016 (DNA96881 -2699) 96881 tmfl 5'-TGGCCTACATGTGTCTTCATC-3 (SEQ ID NO 43) 96881 tmrl
5'-CACAACTTTCTGGTCATATTCCAT-3 (SEQ ID NO 44)
96881 tmpl 5'-CCTGCCCCAAGACGGCATTAG-3 (SEQ ID NO 45)
PRO6018 (DNA98565-2701)
98565 tmtl
5'-CCTGGGCACCAGATCTTC-3 (SEQ ID NO 46)
98565 t rl
5'-AGGGCAGTTGAGGCACTT 3 (SEQ ID NO 47) 98565 tmpl
5'-CATCAGGGCCGGAGTAAATCCCT 3 (SEQ ID NO 48)
PR06496 (DNA 1193022737)
119302 tmtl
5'-TCCATGGACCTCCCACTATAC-3 (SEQ ID NO 49) 119302.tm.rl:
5'-GCTGACAACTTCAGGTTCCA-3' (SEQ ID NO:50) 119302.tm.pl: 5'-ACCCCCACCAAACCCCAGGT-3' (SEQIDNO:51)
PR07154 (DNA 108760-2740):
108760.tm.fl:
5'-GATCTCTGAGCACACTTGTATGAG-3' (SEQ ID NO:52)
108760.tm.rl:
5'-GGCAGACGAGGGTCTTTC-3' (SEQ ID NO:53)
108760.tm.pl:
5'-CAGGAACCCCTTGCTAGAATCAGCC-3' (SEQ ID NO:54)
PRO7170 (DNA 108722-2743): 108722.tm.fl:
5'-CCCAGAAGGTTCCCATGA-3' (SEQ ID NO:55) 108722.tm.rl:
5'-GGGTCCTGTTGCCACATC-3' (SEQ ID NO:56)
108722.tm.pl:
5'-CAGCATGTCCAAGCCCCTAACCC-3' (SEQ ID NO:57)
PR07422 (DNA119536-2752): 119536.tm.fl:
5'-TCTCCCCGATTCTCATCTG-3' (SEQ ID NO:58)
119536.tm.rl:
5'-CCCTGAGAGTCCTGCACAT-3' (SEQ ID NO:59)
119536.tm.pl: 5'-CCCATAATCATGGACACAGCCCC-3' (SEQ ID NO:60)
PR07431 (DNA 119542-2754): 119542.tm.fl:
5'-AGTGAAGTTTCTCCAGTCCCTAGT-3' (SEQIDNO:61) 119542.tm.rl: 5'-CCTGGGGTAAGTGAGCAAA-3' (SEQ ID NO:62) 119542.tm.pl: 5'-CCTCTCTTTTCACCCACCTTCCTCAG-3' (SEQ ID NO:63) PR07476 (DNA 1 15253-2757) 1 15253 tm tl
5'-GGGACTGGTTAAGAAAGTTGGAT-3 (SEQ ID NO 64)
1 15253 tm.rl 5'-CGCCTCAGGCTTTCTGAT-3 (SEQ ID NO 65)
1 15253 tm p l 5'-AGATTCCCCCTTGCACCTCGC-3 (SEQ ID NO 66)
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 oligonucleotide primers are used to generate an amplicon typical ot 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 the released reporter dye is free from the quenching effect of the second fluorophore One molecule ot reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis tor 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 ot 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 running the instrument and for analyzing the data
5' Nuclease assay data are initially expressed as Ct. or the threshold cycle This 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 ot 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 4 describes the stage T stage and N stage of various primary tumors which were used to screen the PRO5800, PRO6000, PRO6016, PRO6018 PR06496. PR07154, PRO7170, PR07422 PR07431 or PR07476 compounds ot the invention Table 4 Primary Lung and Colon Tumor Profiles
Primary Tumor Stage Other Stage Dukes Stage T Stage N Stage
Human lung tumor AdenoCa (SRCC724) [LT 1 ] II A Tl N l Human lung tumor SqCCa (SRCC725) [LTI ] IIB T3 NO
Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 NO
Human lung tumor AdenoCa (SRCC727) [LT3] IIIA Tl N2
Human lung tumor AdenoCa (SRCC728) [LT4] IB T2 NO
Human lung tumor SqCCa (SRCC729) [LT6] IB T2 NO Human lung tumor Aden/SqCCa (SRCC730) [LT7] I A Tl NO
Human lung tumor AdenoCa (SRCC731 ) [LT9] IB T2 NO
Human lung tumor SqCCa (SRCC732) [LT10] IIB T2 Nl
Human lung tumor SqCCa (SRCC733) [LTI 1 ] IIA Tl Nl
Human lung tumor AdenoCa (SRCC734) [LT 12] IV T2 NO Human lung tumor AdenoSqCCa (SRCC735)[LT13] IB T2 NO
Human lung tumor SqCCa (SRCC736) [LTI 5] IB T2 NO
Human lung tumor SqCCa (SRCC737) [LTI 6] IB T2 NO
Human lung tumor SqCCa (SRCC738) [LTI 7] IIB T2 Nl
Human lung tumor SqCCa (SRCC739) [LTI 8] IB T2 NO Human lung tumor SqCCa (SRCC740) [LT 19] IB T2 NO
Human lung tumor LCCa (SRCC741 ) [LT21] IIB T3 Nl
Human lung AdenoCa (SRCC81 1 ) [LT22] 1 A Tl NO
Human colon AdenoCa (SRCC742) [CT2] Ml D pT4 NO
Human colon AdenoCa (SRCC743) [CT3] B pT3 NO Human colon AdenoCa (SRCC 744) [CT8] B T3 NO
Human colon AdenoCa (SRCC745) [CT10] A pT2 NO
Human colon AdenoCa (SRCC746) [CT12] MO, Rl B T3 NO
Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pNO
Human colon AdenoCa (SRCC748) [CT15] M1 , R2 D T4 N2 Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pNO
Human colon AdenoCa (SRCC750) [CT17] Cl pT3 pNl
Human colon AdenoCa (SRCC751 ) [CT1 ] MO, Rl B pT3 NO
Human colon AdenoCa (SRCC752) [CT4] B pT3 M0
Human colon AdenoCa (SRCC753) [CT5] G2 Cl pT3 pNO Human colon AdenoCa (SRCC754) [CT6] pMO. RO B pT3 pNO
Human colon AdenoCa (SRCC755) [CT7] Gl A pT2 pNO
Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2
Human colon AdenoCa (SRCC757) [CT1 1 ] B T3 NO
Human colon AdenoCa (SRCC758) [CT1 ] MO, RO B pT3 pNO
DNA Preparation
DNA was prepared from cultured cell lines, primary tumors, and normal human blood The isolation was performed using purification kit, buffer set and protease and all from Qiagen. according to the manufacturer s instructions and the description below
Cell cultuie sis Cells were washed and trypsinized at a concentration ot 7 5 x 10 per tip and pelleted by centπtuging at
1000 rpm for 5 minutes at 4"C followed by washing again with 1/2 volume ot PBS and 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 Butfer C l was equilibrated at 4 'C Qiagen protease #19155 was diluted into 6 25 ml cold ddH .O to a tinal concentration ot 20 mg/ml and equilibrated at 4"C 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse A stock ( 100 mg/ml) to a final concentration ot 200 μg/ml
Buffer Cl ( 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 tor 10 minutes The cell nuclei were pelleted by centπfuging in a Beckman swinging bucket rotor at 2500 rpm at 4"C tor 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 bv a second 4"C centrifugation at 2500 rpm for 1 ^ 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 was applied Upon completion ot buffer addition vigorous vortexing was applied tor 30 seconds Qiagen protease (200 μl, prepared as indicated above) was added and incubated at 50°C for 60 minutes The incubation and centrifugation were repeated until the lysates were clear (e g , incubating additional 30-60 minutes, pelleting at 3000 x g tor 10 min 4"C)
Solid human tumor sample preparation and hsis
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 ml cold ddH^O 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 ot the polvtron in a laminar-flow TC hood in order to avoid inhalation ot 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 Qiagen protease (prepared as indicated above 1 0 ml) was added, followed by vortexing and incubation at 50°C for 3 hours The incubation and centrifugation were repeated until the lysates were clear (e g , incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min , 4°C)
Human blood preparation and hsis
Blood was drawn from healthy volunteers using standard infectious agent protocols and citrated into 10 ml samples per tip Qiagen protease was freshly prepared bv dilution into 6 25 ml cold ddH^O to a final concentration ot 20 mg/ml and stored at 4"C G2 butter was prepared bv 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 lO ml C l buffer and 30 ml ddH O (both previously equilibrated to 4"C) were added and the components mixed by inverting and held on ice tor 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 Cl 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 butter ( 10 ml) was added to the suspended nuclei while gentlv vortexing, followed by v igorous vortexing tor 30 seconds Qiagen protease was added (200 \) and incubated at 50 C for 60 minutes The incubation and centrifugation were repeated until the lysates were clear (e ? incubating additional 30-60 minutes pelleting at 3000 x g for 10 m 4 C)
Purification of cleared sates
(1 ) Isolation of genomic DNA
Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) w ith 10 ml QBT butter QF elution butter was equilibrated at 50 C The samples were vortexed for 30 seconds then loaded onto equilibrated tips and drained by gravity The tips w ere washed with 2 x 15 ml QC buffer The DNA was eluted into 30 ml silanized, autoclaved 30 ml Corex tubes w ith 15 ml QF buffer (50°C) Isopropanol ( 10 5 ml) was added to each sample, the tubes covered with paratin and mixed by repeated inversion until the DNA precipitated Samples were pelleted by centrifugation in the SS-34 rotor at 15.000 rpm tor 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 tor 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 overnight at 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 ot genomic DNA and preparation for gene amplification assay The DNA levels in each tube were quantified by standard A^w A:sπ spectrophotometry on a I 20 dilution
(5 μl DNA + 95 μl ddH^O) using the 0 1 ml quartz cuvettes in the Beckman DU640 spectrophotometer A-60/A28n ratios were in the range of 1 8-1 9 Each DNA sample was then diluted further to approximately 200 ng ml in TE (pH 8 5) If the original mateπal was highly concentrated (about 700 ng/μl) the material was placed at 50°C for several hours until resuspended Fluorometπc DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelines as modified below This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometer to warm-up tor about 15 mιnutes The Hoechst dye working solution (#H33258. 10μl, prepared within 12 hours ot 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 used as the quantification value
The fluorometπcly determined concentration was then used to dilute each sample to 10 ng/μl in ddH^O 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 used prov ided that the CT value ot normal human DNA subtracted from test DNA was +/- 1 Ct The diluted, lot qualified genomic DNA was stored in 1 0 ml ahquots at -80 C Ahquots which were subsequently to be used in the gene amplification assay were stored at 4"C Each 1 ml aliquot is enough for 8-9 plates or 64 tests Gene amplification
The PRO5800 PRO6000 PRO6016 PRO6018 PR06496 PR07154 PRO7170 PR07422 PR07431 or PR07476 compounds of the invention were screened in the following primary tumors and the resulting ΔCt values are reported in Table 5 Table 5 ΔCt Values in Lung, Colon and Other Primary Tumoi and Cell Line Models
10
15
DISCUSSION AND CONCLUSION PRO5800 (DNA 108912-2680)
The ΔCt values tor DNA 108912-2680 in a vaπetv ot tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling ot gene copv Table 5 indicates that significant amplification of nucleic acid DNA 108912-2680 encoding PRO5800 occurred in pπmary lung tumors HF-001644 and HF-001647
Because amplification ot DNA 108912-2680 occurs in various lung tumors, 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 DNA 108912-2680 (PRO5800) would be expected to have utility in cancer therapy
PRO6000 (DNA 102880-2689)
The ΔCt values tor DNA 102880-2689 in a variety of tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold value tor amplification scoring, as this represents a doubling of gene copy Table 5 indicates that significant amplification of nucleic acid DNA 102880-2689 encoding PRO6000 occurred in pπmary lung tumor HF-001295 Because amplification of DNA102880-2689 occurs in lung tumor, 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 DNA102880-2689 (PRO6000) would be expected to have utility in cancer therapy
PRO6016 (DNA96881 -2699)
The ΔCt values tor DNA96881-2699 in a variety of tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold value tor amplification scoπng, as this represents a doubling of gene copy Table
5 indicates that significant amplification of nucleic acid DNA96881-2699 encoding PRO6016 occurred (1 ) in pπmary lung tumor HF-000641 , and (2) in primary colon tumor centers HF-000762, HF-000789 and HF-00081 1
Because amplification of DNA96881 -2699 occurs in various tumors 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 DNA96881-2699 (PRO6016) would be expected to have utility in cancer therapy
PRO6018 (DNA98565-2701 )
The ΔCt values for DNA98565-2701 in a variety ot tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold value tor amplification scoring, as this represents a doubling of gene copy Table 5 indicates that significant amplification of nucleic acid DNA98565-2701 encoding PRO6018 occurred ( 1 ) in pπmary lung tumor HF-000840. and (2) in primary colon tumor center HF-00081 1
Because amplification ot DNA98565 2701 occurs in various tumors 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 DNA98565-2701 (PRO6018) would be expected to hav e utility in cancer therapy PR06496 (DNA 1 19302-2737)
The ΔCt values tor DNA 1 19302-2737 in a variety ot tumors are reported in Table 5 A ΔCt ot > 1 was typically used as the threshold value for amplification scoπng. as this represents a doubling of gene copv Table 5 indicates that significant amplification of nucleic acid DNA 1 19302-2737 encoding PR06496 occurred in primary lung tumors HF-000840, HF-000842. HF-001294 and HF-001296
Because amplification of DNA1 19302-2737 occurs in various lung tumors, 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 DNA1 19302-2737 (PR06496) would be expected to have utility in cancer therapy
PRQ7154 (DNA 108760-2740) The ΔCt values for DNA 108760-2740 in a variety ot tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table
5 indicates that significant amplification of nucleic acid DNA 108760-2740 encoding PR07154 occurred in pπmary lung tumors HF-001296 and HF-001299
Because amplification of DNA 108760-2740 occurs in various lung tumors, 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 DNA 108760-2740 (PR07154) would be expected to have utility in cancer therapy
PRQ7170 (DNA 108722-2743)
The ΔCt values for DNA 108722-2743 in a variety of tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5 indicates that significant amplification of nucleic acid DNA 108722-2743 encoding PR07170 occurred in primary lung tumor HF-001296
Because amplification of DNA 108722-2743 occurs in lung tumor, it is highly probable to play a significant role in tumor formation or growth As a result, antagonists (e s> , antibodies) directed against the protein encoded by DNA 108722-2743 (PRO7170) would be expected to have utility in cancer therapy
PRQ7422 (DNA1 19536-2752)
The ΔCt values for DNA1 19536-2752 in a variety of tumors are reported in Table 5 A ΔCt ot >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 5 indicates that significant amplification of nucleic acid DNA 1 19536-2752 encoding PR07422 occuπed in primary lung tumor HF-001647 Because amplification of DNA 1 19536 2752 occurs in lung tumor 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 DNA1 19536-2752 (PR07422) would be expected to have utility in cancer therapy
PRQ7431 (DNA 1 19542-2754)
The ΔCt values for DNA1 19542-2754 in a variety of tumors are reported in Table 5 A ΔCt of >1 was typically used as the threshold v alue tor amplification scoring, as this represents a doubling of gene copv Table 5 indicates that significant amplification ot nucleic acid DNA 1 19542-2754 encoding PR07431 occurred ( 1 ) in testis tumor center HF-000733 (2) in primary colon tumor center HF-000539 and (3) in primary lung tumors HF- 000842 and HF-001296 Because amplification ot DNA 1 19542-2754 occurs in various tumors, 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 DNA1 19542-2754 (PR07431 ) would be expected to have utility in cancer therapy
PRQ7476 (DNA 1 15253-2757)
The ΔCt values for DNA 1 15253-2757 in a variety of tumors are reported in Table 5 A ΔCt of > 1 was typically used as the threshold v alue for amplification scoring, as this represents a doubling of gene copy Table
5 indicates that significant amplification ot nucleic acid DNA1 15253-2757 encoding PR07476 occurred (1) in testis tumor center HF-000733. (2) in primary colon tumor centers HF-000539 and HF-000575, and (3) in primary lung tumors HF-001294 and HF-001296
Because amplification ot DNA1 15253-2757 occurs in various tumors 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 DNA115253-2757 (PR07476) would be expected to have utility in cancer therapy
EXAMPLE 12 Use of PRO5800. PRO6000 PRO6016. PRO6018. PRQ6496 PRQ7154 PRO7170. PRQ7422. PRQ7431 or
PRQ7476 as a hybridization probe The following method describes use of a nucleotide sequence encoding a PRO5800. PRO6000. PRO6016,
PRO6018, PR06496, PR07154. PRO7170, PR07422, PR07431 or PR07476 polypeptide as a hybridization probe
DNA comprising the coding sequence of a full-length or mature PRO5800", "PRO6000' PRO6016 '
"PRO6018", -'PR06496". "PR07154 , "PRO7170" "PR07422". "PR07431 or -'PR07476" polypeptide as disclosed herein and/or fragments thereof may be employed as a probe to screen tor homologous DNAs (such as those encoding naturally-occurring variants of PRO5800, PRO6000. PRO6016. PRO6018 PR06496, PR07154,
PRO7170, PR07422. PR07431 or PR07476) 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 ot radiolabeled PRO5800-, PRO6000- PRO6016-, PRO6018- PR06496- PR07154-, PR07170-, PR07422- PR07431 - or PR07476-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 tor 20 hours Washing of the filters is performed in an aqueous solution of 0 1 x SSC and 0 1 % SDS at 42"C
DNAs having a desired sequence identity with the DNA encoding full-length nativ e sequence PRO5800, PRO6000, PRO6016. PRO6018. PR06496. PR07154 PR07170. PR07422. PR07431 or PR07476 can then be identified using standard techniques known in the art EXAMPLE 13 Expression of PRO5800 PRO6000 PRO6016 PRO601 8 PRQ6496 PRQ7154 PRO7170 PRQ7422
PRQ7431 or PRQ7476 Polypeptides in £ coli This example illustrates preparation ot an unglycosylated form ot PRO5800 PRO6000, PRO6016, PRO6018 PR06496, PR07154 PRO7170 PR07422 PR07431 or PR07476 bv recombinant expression in E
The DNA sequence encoding the PRO polypeptide of interest 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 ot expression vectors may be employed An example of a suitable vector is pBR322 (derived from E coli see Bolivar et al Gene 2 95 (1977)) which contains genes tor ampicillin 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 trp promoter a poly-His leader (including the first six STII codons, polv-His sequence, and enterokinase cleavage site) the PRO5800 PRO6000, PRO6016, PRO6018, PRO6496, PRO7154 PRO7170, PR07422, PR07431 or PR07476 coding region lambda transcriptional terminator and an argU gene
The ligation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on
After culturing the cells for several more hours, the cells can be harvested by centrifugation The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art and the solubilized PRO5800 PRO6000 PRO6016 PRO6018 PR06496 PR07154 PRO7170 PR07422 PR07431 or PR07476 protein can then be purified using a metal chelating column under conditions that allow tight binding ot the protein
PRO5800, PRO6000 PRO6016 PRO6018, PR06496 PR07154 PRO7170 PR07422 PR07431 or
PR07476 may be expressed in E co/. in a poly His tagged form using the following procedure The DNA encoding
PRO5800, PRO6000, PRO6016 PRO6018 PR06496 PR07154, PRO7170 PR07422 PR07431 or PR07476 is initially amplified using selected PCR primers The primers contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector and other useful sequences providing tor efficient and reliable translation initiation rapid purification on a metal chelation column and proteolvtic removal with enterokinase The PCR-amphfied, polv-His tagged sequences are then hgated into an expression vector which is used to transform an E coli host based on strain 52 (W31 10 tuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq) Transformants are first grown in LB containing 50 mg ml carbenicilhn at 30°C with shaking until an O D ot 3-5 at 600 nm is reached Cultures are then diluted 50-100 fold into CRAP media (prepared bv mixing 3 57 g
(NH4),S0 , 0 71 g sodium cιtrate«2H .0 1 07 g KC1 5 36 g Difco veast extract 5 36g Sheffield hvcase SF in 500 ml water, as well as 1 10 mM MPOS pH 7 3 0 55% (w/v) glucose and 7 mM MgS0 ) and grown for approximately 20-30 hours at 30°C with shaking Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centπfuged to pellet the cells Cell pellets are frozen until purification and refolding
E coli paste from 0 5 to 1 L fermentations (6 10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tπs, pH 8 buffer Solid sodium sultite and sodium tetrathionate are added to make final concentrations ot 0 I M and 0 02 M. respectively, and the solution is stirred overnight at 4°C This step results in a denatured protein with all cysteine residues blocked by sulfitolization The solution is centπfuged at 40.000 rpm in a Beckman Ultracentifuge for 30 mm The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tπs, pH 7 4) and filtered through 0 22 micron filters to clarify The clarified extract is loaded onto a 5 ml Qiagen Ni +-NTA metal chelate column equilibrated in the metal chelate column buffer The column is washed with additional buffer containing 50 mM imidazole (Calbiochem. Utrol grade), pH 7 4 The proteins are eluted with buffer containing 250 mM imidazole Fractions containing the desired protein are pooled and stored at 4°C Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence
The protein is refolded by diluting sample slowly into freshly prepared refolding buffer consisting of 20 M Tπs, pH 8 6, 0 3 M NaCl, 2 5 M urea. 5 mM cysteine, 20 mM glycine and 1 mM EDTA Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml The refolding solution is stirred gently at 4°C for 12-36 hours The refolding reaction is quenched by the addition of TFA to a final concentration of 04% (pH of approximately 3) Before further purification of the protein, the solution is filtered through a 0 22 micron filter and acetonitrile is added to 2-10% final concentration The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0 1 % TFA with elution with a gradient of acetonitrile from 10 to 80% Aliquots of fractions with A,so absorbance are analyzed on SDS polyacryl amide gels and fractions containing homogeneous refolded protein are pooled Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin Aggregated species are usually eluted at higher acetonitrile concentrations In addition to resolving misfolded forms ot proteins from the desired form, the reversed phase step also removes endotoxin from the samples
Fractions containing the desired folded PR05800, PRO6000, PRO6016 PRO6018 , PR06496, PR07154, PRO7170. PR07422. PR07431 or PR07476 protein are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution Proteins are formulated into 20 mM Hepes, pH 6 8 with 0 14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered
EXAMPLE 14 Expression of PRO5800 PRO6000 PRO6016 PRO6018 PRQ6496 PRQ7154 PRO7170 PRQ7422
PRQ74 1 or PRQ7476 in mammalian cells This example illustrates preparation ot a potentially glycosylated form ot PRO5800. PRO6000 PRO6016
PRO6018. PR06496 PR07154. PRO7170, PR07422. PR07431 or PR07476 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 PRO5800, PRO6000. PRO6016, PRO6018 PR06496, PR07154, PR07170, PR07422 PR07431 or PR07476 DNA is hgated into pRK5 with selected restriction enzymes to allow insertion ot the PRO5800, PRO6000, PRO6016, PRO6018, PR06496. PR07154 PR07170, PR07422, PR07431 or PR07476 DNA using hgation methods such as described in Sambrook et al , supra The resulting vector is called pRK5-[PRO5800, PRO6000 PRO6016. PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476]
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-[PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476] DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al , CeM, 31 543 (1982)] and dissolved in 500 μl of 1 mM Tris- HCl, 0 1 mM EDTA, 0 227 M CaCl2 To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7 35), 280 mM NaCl, 1 5 mM 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 μCi/ml 35S-cysteιne and 200 μCi/ml 35S-methιonιne After a 12 hour incubation, the conditioned medium is collected concentrated on a spin filter, and loaded onto a 15% SDS gel The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of the PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422. PR07431 or PR07476 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, PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154 PR07170, PR07422, PR07431 or PR07476 DNA may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al . Proc Natl Acad Sci J_2 7575 (1981 ) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-[PRO5800, PRO6000, PRO6016 PRO6018. PR06496. PR07154, PRO7170, PR07422. PR07431 or PR07476] 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 tor four hours The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium and re- lntroduced into the spinner flask containing tissue culture medium, 5 μg/ml bov ine insulin and 0 1 μg/ml bovine transferπn After about four days, the conditioned media is centπfuged and filtered to remove cells and debris The sample containing expressed PR05800 PRO6000 PRO6016, PRO6018 PR06496, PR07154 PRO7170 PR07422 PR07431 or PR07476 can then be concentrated and purified by any selected method such as dialysis and/or column chromatography
In another embodiment PR05800, PRO6000, PRO6016 PRO6018 PR06496 PR07154 PRO7170, PR07422. PR07431 or PR07476 can be expressed in CHO cells The pRK5-[PRO5800 PRO6000 PRO6016, PRO6018, PR06496 PR07154, PR07170 PR07422, PR07431 or PR07476] v ector can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S- methiomne After determining the presence ot the PRO5800 PRO6000. PRO6016. PRO6018. PR06496. PR07 I 54. PRO7170. PR07422. PR07431 or PR07476 polypeptide the culture medium may be replaced with serum free medium Preferably, the cultures are incubated for about 6 days and then the conditioned medium is harvested The medium containing the expressed PRO5800. PRO6000. PRO6016. PRO6018, PR06496. PR07154, PRO7170, PR07422, PR07431 or PR07476 can then be concentrated and purified by any selected method
Epitope-tagged PRO5800. PRO6000. PRO6016. PRO6018. PR06496, PR07154. PR07170. PR07422, PR07431 or PR07476 may also be expressed in host CHO cells The PRO5800. PRO6000, PRO6016. PRO6018, PR06496. PR07154, PR07170. PR07422, PR07431 or PR07476 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 poiy-His tag into a Baculovirus expression vector The poly-His tagged PRO5800. PRO6000. PRO6016, PRO6018. PR06496, PR07154, PRO7170. PR07422. PR07431 or PR07476 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 PRO5800. PRO6000, PRO6016, PRO6018, PR06496. PR07154, PRO7170, PR07422, PR07431 or PR07476 can then be concentrated and purified by any selected method, such as by Nι2+-chelate affinity chromatography Expression in CHO and/or COS cells may also be accomplished by a transient expression procedure PRO5800. PRO6000. PRO6016, PRO6018, PR06496, PR07154. PR07170, PR07422. PR07431 or
PR07476 may be expressed in CHO cells by a transient procedure Stable expression in CHO cells can be performed using the following procedure The proteins are expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e g , extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or in a poly-His tagged form Following PCR amplification the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al . Current Protocols ot Molecular Biology, Unit 3 16. lohn Wiley and Sons ( 1997) CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA ot interest to allow the convenient shuttling of cDNA' s The vector used tor expression in CHO cells is as described in Lucas et al , Nucl Acids Res . 24 9 (1774-1779 ( 1996), and uses the S V40 early promoter/enhancer to drive expression ot the cDNA of interest and dihydrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance ot the plasmid following transfection
Twelve micrograms ot the desired plasmid DNA are introduced into approximately 10 million CHO cells using commercially available transfection reagents Supertect11 (Qiagen) Dosper" or Fugene (Boehringer Mannheim) The cells are grown as described in Lucas et al , s pia Approximately 3 x 107 cells are frozen in an ampule tor further growth and production as described below
The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing The contents are pipetted into a centrifuge tube containing 10 mis ot media and centπtuged at 1000 rpm tor 5 minutes The supernatant is aspirated and the cells are resuspended in 10 ml ot selective media (0 2 μm filtered PS20 with 57c 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 ml spinner containing 90 ml of selective media. After I -2 days, the cells are transferred into a 250 ml spinner filled with 150 ml selective growth medium and incubated at 37"C. After another 2-3 days. 250 ml, 500 ml and 2000 ml spinners are seeded with 3 x 105 cells/ml. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Patent No. 5,122,469, issued June 16. 1992 is actually used. 3L production spinner is seeded at 1.2 x I O6 cells/ml. On day 0. the cell number and pH are determined. On day 1 , the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33"C. and 30 ml of 500 g/L glucose and 0.6 ml of 10% anti foam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) added. Throughout the production, the pH is adjusted as necessary to keep at around 7.2. After 10 days, or until viability drops below 70%. the cell culture is harvested by centrifugation and filtered through a 0.22 m filter. The filtrate is either stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni 2+-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni :+-NTA column equilibrated in 20 mM Hepes, pH 7.4. buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C. lmmunoadhesin (Fc containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid. pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μl of 1 M Tris buffer. pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
EXAMPLE 15 Expression of PRO5800. PRO6000. PRO6016. PRO6018. PRQ6496. PRQ7154. PRO7170. PRQ7422.
PRQ7431 or PRQ7476 in Yeast The following method describes recombinant expression of PRO5800, PRO6000, PRO6 16. PRO6018.
PR06496. PR07154, PRO7170. PR07422. PR07431 or PR07476 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PRO5800. PRO6000. PRO6016. PRO6018. PR06496. PR07154, PRO7170. PR07422, PR07431 or PR07476 from the ADH2/GAPDH promoter. DNA encoding PRO5800, PRO6000. PRO6016. PRO6018. PR06496. PR07154. PR07170. PR07422. PR07431 or PR07476 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO5800. PRO6000. PRO6016. PRO6018. PR06496. PR07154. PRO7170. PR07422. PR07431 or PR07476. For secretion. DNA encoding PRO5800. PRO6000. PRO6016. PRO6018. PR06496. PR07154 PR07170. PR07422. PR07431 or PR07476 can be cloned into the selected plasmid. together with DNA encoding the ADH2/GAPDH promoter, a native PRO5800 PRO6000, PRO6016. PRO6018. PR06496. PR07154. PR07170 PR07422, PR07431 or PR07476 signal peptide or other mammalian signal peptide, or, for example, a veast alpha-factor or invertase secretory signal/leader sequence, and linker sequences of needed) tor expression ot PRO5800. PRO6000. PRO6016. PRO6018. PR06496. PR07154. PRO7170. PR07422. PR07431 or PR07476
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 supematants can be analyzed by precipitation with 10% tπchloroacetic acid and separation by SDS-PAGE. followed by staining ot the gels with Coomassie Blue stain
Recombinant PRO5800, PRO6000. PRO6016. PRO6018, PR06496. PR07154, PRO7170, PR07422, PR07431 or PR07476 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 PRO5800, PRO6000. PRO6016. PRO6018. PR06496. PR07154, PRO7170. PR07422. PR07431 or PR07476 may further be purified using selected column chromatography resins
EXAMPLE 16 Expression of PRQ5800. PRO6000 PRO6016 PRO601 . PRQ6496. PRQ7154. PRO7170 PRQ7422. PRQ7431 or PRQ7476 in Baculovirus-infected Insect Cells The following method describes recombinant expression in Baculovirus-infected insect cells The sequence coding for PRO5800. PRO6000, PRO6016, PRO6018. PR06496, PR07154, PRO7170,
PR07422, PR07431 or PR07476 is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly-His tags and immunoglobulin tags (like Fc regions ot IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly, the sequence encoding PRO5800. PRO6000. PRO6016 PRO6018. PR06496. PR07154 PR07170, PR07422. PR07431 or PR07476 or the desired portion ot the coding sequence ot PRO5800. PRO6000. PRO6016. PRO6018. PR06496, PR07154 PRO7170. PR07422. PR07431 or PR07476 [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 w ith 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 bv co-transfectmg the above plasmid and BaculoGold™ v ιrus DNA
(Pharmingen ) into Spodopteiafi ugψeida ("Sf 9 ) cells (ATCC CRL 171 1 ) using potectin ( commerciallv av ailable from GIBCO-BRL) After 4 - 5 days ot 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 PRO5800 PRO6000. PRO6016 PRO6018. PR06496. PR07154, PR07170 PR07422. PR07431 or PR07476 can then be purified, for example, by Nr+-chelate aftinitv chromatography as follows Extracts are prepared from recombinant virus-intected 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 ., 0 1 mM EDTA 10% glycerol, 0 1 % NP-40. 0 4 M KC1), and sonicated twice tor 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 ot water and equilibrated ith 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-s0 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 A:8„basehne again, the column is developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer One ml fractions are collected and anal zed by SDS-PAGE and silver staining or Western blot with Nι2+-NTA-conjugated to alkaline phosphatase (Qiagen) Fractions containing the eluted Hιsl(rtagged PRO5800, PRO6000, PRO6016, PRO6018. PR06496 PR07154, PRO7170. PR07422. PR07431 or PR07476, respectively, are pooled and dialyzed against loading buffer Alternatively, purification of the IgG tagged (or Fc tagged) PRO5800. PRO6000. PRO6016 PRO6018.
PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography
While expression is actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e g , 8 L) preparations The proteins are expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region is fused to an IgGl constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly- His tagged forms
Following PCR amplification, the respective coding sequences are 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) are co-transfected into 105 Spodopteta ftugipeida ("Sf9") cells (ATCC CRL 171 1 ). using Lipotecun (Gibco BRL) pb PH IgG and pb PH His are modifications ot the commercial lv av ailable baculovirus expression vector pVLl 393 (Pharmingen), with modified polyhnker regions to include the His or Fc tag sequences The cells are grown in Hink s TNM-FH medium supplemented with 10% FBS (Hyclone ) Cells are incubated for 5 days at 28 °C The supernatant is harvested and subsequently used tor the first viral amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOD of 10 Cells are incubated for 3 days at 28 °C The supernatant is harvested and the expression ot the constructs in the baculovirus expression vector is determined by batch binding ot 1 ml of supernatant to 25 ml of Ni +-NTA beads (QIAGEN) tor 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 ot protein standard by Coomassie blue staining The first viral amplification supernatant is used to infect a spinner culture (500 ml) of Sf9 celN grown in
ESF-921 medium (Expression Systems LLC) at an approximate MOI ot 0 1 Cells are incubated tor 3 dav b at 28 °C The supernatant is harvested and filtered Batch binding and SDS-PAGE analysis are repeated, as necessary, until expression of the spinner culture is confirmed The conditioned medium from the transfected cells (0 5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0 22 micron filters For the poly-His tagged constructs, the protein construct is purified using a Ni 2+-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni " NTA column equilibrated in 20 mM Hepes pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4°C After loading, the column is washed with additional equilibration butter and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes.0 14 M NaCl and 47c mannitol. pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) constructs of proteins are purified from the conditioned media as follows
The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer. pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid. pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml ot 1 M Tπs buffer, pH 9 The highly purified protein is subsequently desalted into storage buffer as described above tor the poly-His tagged proteins The homogeneity of the proteins is verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation
Alternatively, a modified baculovirus procedure may be used incorporating high 5 cells In this procedure, the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) ot an epitope tag contained with a baculovirus expression vector Such epitope tags include poly- His tags and immunoglobulin tags (like Fc regions of IgG) A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pIEl-1 (Novagen) The pIEl-1 and pIEl -2 vectors are designed for constitutive expression ot recombinant proteins from the baculovirus le i promoter in stably- transformed insect cells The plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lei -mediated gene expression in uninfected insect cells as well as the hr5 enhancer element pIEl - 1 and pIEl-2 include the 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 is then digested with those selected restriction enzymes and subcloned into the expression vector For example, derivatives ofpIEl - 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
High 5 cells are grown to a confluency of 50% under the conditions of 27 °C. no C02. NO pen/strep For each 150 mm plate, 30 μg of pIE based vector containing the sequence is mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401 -78P (note this media is light sensitive)), and in a separate tube. 100 μl ofCellFectιn (CellFECTIN (GιbcoBRL#10362-010) (vortexed to mιx)) ιs mιxed wιth 1 ml of Ex-Cell medium The two solutions are combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex Cell media is added to the 2 ml ot DNA/CellFECTIN mix and this is layered on high 5 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 ot fresh Ex-Cell media is added and the cells are incubated tor 3 days at 28"C The supernatant is harvested and the expression ot the sequence in the baculovirus expression v ector is determined by batch binding of 1 ml ot supernatant to 25 ml ot Ni :"-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) tor IgG tagged proteins followed by SDS-PAGE analysis comparing to a known concentration ot protein standard by Coomassie blue staining
The conditioned media from the transfected cells (0 5 to 3 L) is harv ested by 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 2"-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration ot 5 mM The conditioned media is pumped onto a 6 ml Ni +-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 48°C After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein is then subsequently desalted into a storage buffer containing 1 O 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 are purified from the conditioned media as follows The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid. pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tπs buffer, pH 9 The highly purified protein is subsequently desalted into storage buffer as described above tor the poly-His tagged proteins The homogeneity of the sequence is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation and other analytical procedures as desired or necessary
PRO6018, PR07154, PRO7170 and PR07476 were successfully expressed by the above modified baculovirus procedure incoφorating high 5 cells
EXAMPLE 17 Preparation ot Antibodies that Bind PRO5800 PRO6000. PRO6016 PRO6018. PRQ6496. PRQ7154.
PRO7170. PRQ7422. PRQ7431 or PRQ7476 This example illustrates preparation of monoclonal antibodies which can specifically bind PRO5800, PRO6000, PRO6016. PRO6018. PR06496, PR07154. PR07170, PR07422, PR07431 or PR07476
Techniques tor producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra Immunogens that may be employed include purified PRO5800. PRO6000. PRO6016. PRO6018, PR06496. PR07154. PRO7170. PR07422, PR07431 or PR07476 fusion proteins containing PRO5800. PRO6000, PRO60I 6. PRO6018 PR06496, PR07154. PRO7170. PR07422. PR07431 or PR07476 and cells expressing recombinant PRO5800, PRO6000, PRO6016. PRO6018. PR06496, PR07154, PR07170, PR07422. PR07431 or PR07476 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 w ith the PRO5800 PRO6000, PRO6016. PRO6018 PR06496. PR07154 PR07170, PR07422, PR07431 or PR07476 immunogen emulsified in complete Freund s adjuvant and lniected subcutaneously or intraperitoneally in an amount from 1-100 micrograms Alternativ ely, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind toot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, for several vv eeks. the mice may also be boosted with additional immunization injections Serum samples may be peπodicallv obtained from the mice by retro-orbital bleeding tor testing in ELISA assays to detect antι-PRO5800, antι-PRO6000. antι-PRO6016. antι-PRO6018. antι-PR06496. anti- PRO? 154 antι-PRO7170, antι-PR07422, antι-PR07431 or antι-PR07476 antibodies After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO5800. PRO6000, PRO6016, PRO6018, PR06496. PR07154, PRO7170, PR07422, PR07431 or PR07476 Three to tour days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63 AgU 1 , available from ATCC, No CRL 1597 The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine. ammopterin. and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids
The hybridoma cells will be screened in an ELISA for reactivity against PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO5800, PRO6000. PRO6016, PRO6018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 is within the skill in the art
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the antι-PRO5800, antι-PRO6000, antι-PRO6016, antι-PRO6018, antι-PR06496. antι-PR07154, antι-PRO7170. antι-PR07422, antι-PR07431 or antι-PR07476 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 Altemativelv 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 U ni versity Blvd , Manassas VA 201 10-2209. USA (ATCC)
Material ATCC Deposit No Deposit Date
DNA 108912-2680 PTA- 124 May 25 , 1999
DNA 102880-2689 PTA-383 July 20, 1999
DNA96881 -2699 PTA-553 August 17. 1999 DNA98565-2701 PTA-481 August 3, 1999
DNA 1 19302-2737 PTA-520 August 10. 1999 DNA 108760-2740 PTA-548 August 17. 1999
DNA 108722-2743 PTA-552 August 17. 1999
DNA 1 19536-2752 PTA-551 August 17 1999
DNA 1 19542-2754 PTA-619 August 31. 1999 DNA 1 15253-2757 PTA-612 August 31. 1999
These deposits were made under the provisions ot the Budapest Treaty on the International Recognition ot the Deposit of Microorganisms tor the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) This assures the maintenance ot a viable culture ot the deposit for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms ot the Budapest Treaty, and subject to an agreement between Genentech, Ine , and the ATCC. which assures permanent and unrestricted availability ot the progeny of the culture ot the deposit to the public upon issuance ot 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 U S.C § 122 and the Commissioner's rules pursuant thereto (including 37 C F R § 1 14 with particular reference to 886 OG 638)
The assignee ot 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 ot 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 ot this invention The deposit ot material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice ot any aspect ot 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 ot 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 ithin the scope of the appended claims

Claims

WHAT IS CLAIMED IS
1 An isolated antibody that binds to a PRO5800 PRO6000 PRO6016 PRO6018, PR06496. PR07154. PRO7170 PR07422. PR07431 or PR07476 polypeptide
2 The antibody ot Claim 1 which specifically binds to said polypeptide
3 The antibody ot Claim 1 which induces the death of a cell that expresses said polypeptide
4 The antibody of Claim 3, wherein said cell is a cancer cell that overexpresses said polypeptide as compared to a normal cell of the same tissue type
5 The antibody of Claim 1 which is a monoclonal antibody
6 The antibody of Claim 5 which comprises a non-human complementarity determining region (CDR) or a human framework region (FR)
7 The antibody of Claim 1 which is labeled
8 The antibody ot Claim 1 which is an antibody fragment or a single-chain antibody
9 A composition of matter which comprises an antibody ot Claim 1 in admixture with a pharmaceutically acceptable carrier
10 The composition ot matter of Claim 9 which comprises a therapeutically effective amount of said antibody
1 1 The composition of matter of Claim 9 which further comprises a cytotoxic or a chemotherapeutic agent
12 An isolated nucleic acid molecule that encodes the antibody of Claim 1
13 A vector comprising the nucleic acid molecule ot Claim 12
14 A host cell comprising the vector ot Claim 13
15 A method tor producing an antibody that binds to a PRO5800. PRO6000. PRO6016. PRO6018, PR06496, PR07154. PR07170. PR07422. PR07431 or PR07476 polypeptide. said method comprising culturing the host cell of Claim 14 under conditions sufficient to allow expression of said antibody and recovering said antibody from the cell culture
16 An antagonist of a PRO5800 PRO6000. PRO6016. PRO6018. PR06496. PR07154. PR07170. PR07422. PR07431 or PR07476 polypeptide
17 The antagonist of Claim 16 herein said antagonist inhibits tumor cell growth
18 An isolated nucleic acid molecule that hybridizes to a nucleic acid sequence that encodes a PRO5800. PRO6000. PRO6016, PRO6018. PR06496. PR07154, PRO7170, PR07422. PR07431 or PR07476 polypeptide. or the complement thereof
19 The isolated nucleic acid molecule of Claim 18. wherein said hybridization is under stπngent hybridization and wash conditions
20 A method for determining the presence ot a PRO5800, PRO6000. PRO6016. PRO6018. PR06496, PR07154. PRO7170. PR07422, PR07431 or PR07476 polypeptide in a sample suspected ot containing said polypeptide, said method comprising exposing the sample to an antι-PRO5800. antι-PRO6000, antι-PRO6016, anti- PRO6018. antι-PR06496, antι-PR07154, antι-PRO7170. antι-PR07422, antι-PR07431 or antι-PR07476 antibody and determining binding ot said antibody to said polypeptide in said sample
21 The method of Claim 20. wherein said sample comprises a cell suspected of containing a PRO5800, PRO6000. PRO6016, PRO6018. PR06496. PR07154. PR07170, PR07422. PR07431 or PR07476 poiypeptide
22 The method ot Claim 21 , wherein said cell is a cancer cell
23 A method of diagnosing tumor in a mammal, said method comprising detecting the level ot expression of a gene encoding a PRO5800 PRO6000 PRO6016, PRO6018, PR06496. PR07154. PRO7170. PR07422. PR07431 or PR07476 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, as compared to the control sample, is indicative ot the presence of tumor in the mammal from which the test tissue cells were obtained
24 A method of diagnosing tumor in a mammal, said method comprising (a) contacting an anti- PRO5800. antι-PRO6000. antι-PRO6016. antι-PRO6018, antι-PR06496 antι-PR07154. antι-PRO7170. anti- PR07422. antι-PR07431 or antι-PR07476 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation ot a complex between said antibody and a PRO5800. PRO6000. PRO6016. PRO6018. PR06496. PR07154. PRO7170 PR07422. PR07431 or PR07476 polypeptide in the test sample, wherein the tormation ot a complex is indicative ot the presence of a tumor in said mammal
25 The method ot Claim 24 wherein said antibody is detectablv labeled
26 The method of Claim 24 wherein said test sample ot tissue cells is obtained from an individual suspected of having neoplastic cell growth or proliferation
27 A cancer diagnostic kit comprising an antι-PRO5800, antι-PRO6000. antι-PRO6016. antι-PRO6018, antι-PR06496. antι-PR07154 antι-PRO7170 antι-PR07422, antι-PR07431 or antι-PR07476 antibody and a carrier in suitable packaging
28 The kit of Claim 27 which further comprises instructions tor using said antibody to detect the presence ot a PRO5800, PRO6000. PRO6016 PRO6018, PR06496. PR07154, PR07170, PR07422, PR07431 or PR07476 pol peptide in a sample suspected of containing the same
29 A method for inhibiting the gro th of tumor cells, said method comprising exposing tumor cells that express a PRO5800. PRO6000. PRO6016. PRO6018, PR06496. PR07154, PRO7170, PR07422. PR07431 or PR07476 polypeptide to an effective amount of an agent that inhibits a biological activity of said polypeptide, wherein growth of said tumor cells is thereby inhibited
30 The method of Claim 29, wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type
31 The method of Claim 29, wherein said agent is an antι-PRO5800, antι-PRO6000, antι-PRO6016, antι-PRO6018. antι-PR06496. antι-PR07154. antι-PRO7170, antι-PR07422, antι-PR07431 or antι-PR07476 antibody
32 The method of Claim 31 w herein said antι-PRO5800 antι-PRO6000. antι-PRO6016. anti- PRO6018, antι-PR06496, antι-PR07154, antι-PR07170. antι-PR07422, antι-PR07431 or antι-PR07476 antibody induces cell death
33 The method of Claim 29, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent
34 A method for inhibiting the grow th of tumor cells, said method comprising exposing tumor cells that express a PRO5800. PRO6000. PRO6016 PRO6018, PR06496. PR07154. PRO7170, PR07422. PR07431 or PR07476 polypeptide to an effective amount ot an agent that inhibits the expression of said polypeptide wherein growth of said tumor cells is thereby inhibited
35 The method of Claim 34. wherein said tumor cells overexpress said polypeptide as compared to normal cells of the same tissue type
36 The method of Claim 34, wherein said agent is an antisense oligonucleotide that hybridizes to a nucleic acid which encodes the PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 polypeptide or the complement thereof
37 The method of Claim 36, wherein said tumor cells are further exposed to radiation treatment, a cytotoxic agent or a chemotherapeutic agent
38 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 and wherein the label on the container indicates that the composition is effective for treating conditions characterized by overexpression of a PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 polypeptide in said tumor cells as compared to in normal cells of the same tissue type
39 The article of manufacture of Claim 38, wherein said active agent inhibits a biological activity of and/or the expression of said PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 polypeptide
40 The article of manufacture of Claim 39, wherein said active agent is an antι-PRO5800, anti- PRO6000, antι-PRO6016, antι-PRO6018, antι-PR06496, antι-PR07154, antι-PRO7170, antι-PR07422, anti- PR07431 or antι-PR07476 antibody
41 The article of manufacture of Claim 39, wherein said active agent is an antisense oligonucleotide
42 A method of identifying a compound that inhibits a biological or immunological activity of a PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 polypeptide, said method comprising contacting a candidate compound with said polypeptide under conditions and for a time sufficient to allow the two components to interact and determining whether a biological or immunological activity of said polypeptide is inhibited
43 The method of Claim 42, wherein said candidate compound is an antι-PRO5800, antι-PRO6000, antι-PRO6016, antι-PRO6018, antι-PR06496, antι-PR07154, an tι-PR07170, antι-PR07422, antι-PR07431 or anti- PR07476 antibody
44. The method of Claim 42, wherein said candidate compound or said PRO5800. PRO6000, PRO6016. PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 polypeptide is immobilized on a solid support.
45. The method of Claim 44, wherein the non-immobilized component is detectably labeled.
46. A method of identifying a compound that inhibits an activity of a PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 polypeptide, said method comprising the steps of (a) contacting cells and a candidate compound to be screened in the presence of said polypeptide under conditions suitable for the induction of a cellular response normally induced by said polypeptide and (b) determining the induction of said cellular response to determine if the test compound is an effective antagonist, wherein the lack of induction of said cellular response is indicative of said compound being an effective antagonist.
47. A method for identifying a compound that inhibits the expression of a PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PR07170, PR07422, PR07431 or PR07476 polypeptide in cells that express said polypeptide, wherein said method comprises contacting said cells with a candidate compound and determining whether expression of said polypeptide is inhibited.
48. The method of Claim 47, wherein said candidate compound is an antisense oligonucleotide.
49. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), and Figure 20 (SEQ ID NO:20).
50. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ), Figure 3 (SEQ ID NO:3), Figure 5 (SEQ ID NO:5), Figure 7 (SEQ ID NO:7), Figure 9 (SEQ ID NO:9), Figure 1 1 (SEQ ID NO: 11 ), Figure 13 (SEQ ID NO: 13), Figure 15 (SEQ ID NO: 15), Figure 17 (SEQ ID NO: 17), and Figure 19 (SEQ ID NO: 19).
51. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the full-length coding sequence of the nucleotide sequence shown in Figure 1 (SEQ ID NO: l), Figure 3 (SEQ ID NO:3), Figure 5 (SEQ ID NO:5), Figure 7 (SEQ ID NO:7), Figure 9 (SEQ ID NO:9), Figure 1 1 (SEQ ID NO: l 1 ), Figure 13 (SEQ ID NO: 13), Figure 15 (SEQ ID NO:15), Figure 17 (SEQ ID NO: 17), and Figure 19 (SEQ ID NO: 19).
52. Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession number PTA- 124, PTA-383, PTA-553, PTA-481 ,PTA-520, PTA-548, PTA-552, PTA-551 , PTA-619 or PTA-612.
53. A vector comprising the nucleic acid of any one of Claims 49 to 52.
54. The vector of Claim 53 operably linked to control sequences recognized by a host cell transformed with the vector.
55. A host cell comprising the vector of Claim 53.
56. The host cell of Claim 55, wherein said cell is a CHO cell.
57. The host cell of Claim 55, wherein said cell is an E. coll.
58. The host cell of Claim 55, wherein said cell is a yeast cell.
59. The host cell of Claim 55, wherein said cell is a Baculovirus-infected insect cell.
60. A process for producing a PRO5800, PRO6000, PRO6016, PRO6018, PR06496, PR07154, PRO7170, PR07422, PR07431 or PR07476 polypeptide comprising culturing the host cell of Claim 55 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
61. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), and Figure 20 (SEQ ID NO:20).
62. An isolated polypeptide scoring at least 80% positives when compared to an amino acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), and Figure 20 (SEQ ID NO:20).
63. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence of the DNA deposited under ATCC accession number PTA- 124, PTA-383, PTA-553, PTA-48 l ,PTA-520, PTA-548, PTA-552, PTA-551, PTA-619 or PTA-612.
64. A chimeric molecule comprising a polypeptide according to any one of Claims 61 to 63 fused to a heterologous amino acid sequence.
65. The chimeric molecule of Claim 64, wherein said heterologous amino acid sequence is an epitope tag sequence.
66. The chimeric molecule of Claim 64, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.
67. An antibody which specifically binds to a polypeptide according to any one of Claims 61 to 63.
68. The antibody of Claim 67, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.
69. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO:10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), or Figure 20 (SEQ ID NO:20), lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO:l 8), or Figure 20 (SEQ ID NO:20), with its associated signal peptide; or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), or Figure 20 (SEQ ID NO:20), lacking its associated signal peptide.
70. An isolated polypeptide having at least 80% amino acid sequence identity to:
(a) the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), or Figure 20 (SEQ ID NO:20), lacking its associated signal peptide;
(b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18). or Figure 20 (SEQ ID NO:20), with its associated signal peptide; or
(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID N0:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID NO: 10), Figure 12 (SEQ ID NO: 12), Figure 14 (SEQ ID NO: 14), Figure 16 (SEQ ID NO: 16), Figure 18 (SEQ ID NO: 18), or Figure 20 (SEQ ID NO:20). lacking its associated signal peptide.
EP00930756A 1999-06-09 2000-05-15 Compositions and methods for the treatment of tumor Withdrawn EP1185642A2 (en)

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US14818899P 1999-08-10 1999-08-10
US148188P 1999-08-10
US14932799P 1999-08-17 1999-08-17
US14939699P 1999-08-17 1999-08-17
US14932099P 1999-08-17 1999-08-17
US149327P 1999-08-17
US149320P 1999-08-17
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US15011499P 1999-08-20 1999-08-20
US150114P 1999-08-20
US15173499P 1999-08-31 1999-08-31
US15170099P 1999-08-31 1999-08-31
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ATE440109T1 (en) 2009-09-15
JP4260778B2 (en) 2009-04-30
EP1683811A3 (en) 2006-10-11
WO2000075317A3 (en) 2001-02-08
DE60042799D1 (en) 2009-10-01
EP1683811A2 (en) 2006-07-26
JP4252501B2 (en) 2009-04-08
JP2006006330A (en) 2006-01-12
AU4851900A (en) 2000-12-28
ES2332916T3 (en) 2010-02-15
WO2000075317A2 (en) 2000-12-14
JP2006006331A (en) 2006-01-12
JP2003501087A (en) 2003-01-14
CA2371434A1 (en) 2000-12-14
ES2331657T3 (en) 2010-01-12
EP1657256A2 (en) 2006-05-17
EP1683811B1 (en) 2009-08-19
DE60043125D1 (en) 2009-11-19
JP2005027667A (en) 2005-02-03
ATE444971T1 (en) 2009-10-15

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