WO1999035170A9 - Compositions and methods for the treatment of tumor - Google Patents
Compositions and methods for the treatment of tumorInfo
- Publication number
- WO1999035170A9 WO1999035170A9 PCT/US1999/000106 US9900106W WO9935170A9 WO 1999035170 A9 WO1999035170 A9 WO 1999035170A9 US 9900106 W US9900106 W US 9900106W WO 9935170 A9 WO9935170 A9 WO 9935170A9
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- WO
- WIPO (PCT)
- Prior art keywords
- antι
- antibody
- pro
- pro509
- pro201
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Definitions
- cancers Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al . CA Cancel J Clin 43. 7 [ 1993])
- Cancer is characterized by the increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis)
- a cell proliferates under conditions in which normal cells would not grow Cancer manifests itself in a wide vapety 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 v ⁇ uld normally function to prevent malignant cell growth, and/or overexpression of certair dominant genes, such as oncogenes, that act to promote malignant growth
- tumor suppression genes which v ⁇ uld normally function to prevent malignant cell growth
- a well known mechanism of gene (e g oncogene) overexpression in cancer cells is gene amplification This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced The process involves unscheduled replication of the region of chromosome comp ⁇ sing the gene, followed by recombination of the replicated segments back into the chromosome (Alitalo et at , Adv Cancer Res 47, 235-281 [1986]) It is believed that the overexpression of the gene parallels gene amplification, I e is proportionate to the number of copies made
- Proto-oncogenesthat encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer
- the human ErbB2 gene e ⁇ />B2, also known as her2, or c-erbB-2
- c-erbB-2 which encodes a 185-kd transmembrane glycoprotem receptor (pl 85 HER2 , HER2) related to the epidermal growth factor receptor (EGFR)
- EGFR epidermal growth factor receptor
- gene amplification of a proto-oncogene is an event t> pically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome (Schwab et al Genes Chromosomes Cancer 1.
- 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 prevent ⁇ on)of certain cancers, and may act of predictors of the prognosis of tumor treatment
- the present invention concerns an isolated antibody which binds a polypeptide which is designated PRO201 (UNQ175), PR0292 (UNQ255), PR0327 (UNQ288), PR01265 (UNQ636), PR0344 (UNQ303), PR0343 (UNQ302), PR0347 (UNQ306), PR0357 (UNQ314).
- PR0715 (UNQ383), PRO 1017 (UNQ500), PRO 1 1 12 (UNQ555), PRO509 (UNQ329), PR0853 (UNQ419) or PR0882 (UNQ448), wherein "PRO” stands for “protein “ and "UNQ” stands for “unique” Throughout this specification, the “PRO” and “UNQ” numbers are used interchangeably to designate the respective proteins, wherein LTNQ designates the native human sequence only and PRO designates the native sequence and active variants thereof In one aspect, the antibody induces death of a cell overexpressmg a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12.
- the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues
- CDR complementarity 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 an anti-idiotypic antibody
- the invention concerns a composition
- a composition comprising an antibody which binds a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509 , PR0853 or PR0882 polypeptide in admixture with a pharmaceutically acceptable carrier
- the composition comprises a therapeutically effective amount of the antibody
- the composition comprises a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent
- the composition is sterile
- the invention concerns nucleic acid encoding an ant ⁇ -PRO201 , anti- PR0292. ant ⁇ -PR0327.ant ⁇ -PR01265,ant ⁇ -PR0344.ant ⁇ -PR0343,ant ⁇ -PR0347.ant ⁇ -PR0357,ant ⁇ -PR0715, ant ⁇ -PRO 1017, anti-PROl 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody, and vectors and recombinant host cells comprising such nucleic acid
- the invention concerns a method for producing an ant ⁇ -PRO201 , anti- PR0292, ant ⁇ -PR0327,ant ⁇ -PR01265,ant ⁇ -PR0344,ant ⁇ -PR0343,ant ⁇ -PR0347,ant ⁇ -PR0357 ant ⁇ -PR0715, ant ⁇ -PRO1017,ant ⁇ -PROl 1 12,ant ⁇ -PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody by cultu ⁇ ng a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the antibody from the cell culture
- the invention further concerns antagonists and agonists of a PRO201, PRO292, PRO327.
- the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides
- the nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions
- Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions
- sequences can be used as part of ribozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes
- the invention concerns a method for determining the presence of a PRO201,
- PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO 1 1 12, PRO509 , PR0853 or PR0882 polypeptide comprising exposing a cell suspected of containing the PRO201, PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide to an ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327, ant ⁇ -PR01265, ant ⁇ -PR0344, anti- PR0343, ant ⁇ -PR0347, ant ⁇ -PR0357, ant ⁇ -PR0715, ant ⁇ -PRO1017, ant ⁇ -PR01 1 12, ant ⁇ -PRO509, anti- PR0853 or ant ⁇ -PR0882 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 PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypept ⁇ de(a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample indicates the presence of tumor in the mammal from which the test tissue cells were obtained
- the present invention concerns a method of diagnosing tumor in a mammal, comprising (a) contacting an ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, ant ⁇ -PR01265, ant ⁇ -PR0344, anti- PR0343. ant ⁇ -PR0347, ant ⁇ -PR0357.
- ant ⁇ -PR0715 ant ⁇ -PRO1017, anti-PROl 1 12, ant ⁇ -PRO509, anti- PR0853 or ant ⁇ -PR0882 antibody with a test sample of tissue cells obtained from the mammal , and (b) detecting the formation of a complex between the ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265, ant ⁇ -PR0344,ant ⁇ -PR0343,ant ⁇ -PR0347, ant ⁇ -PR0357, ant ⁇ -PR0715, ant ⁇ -PRO1017, anti-PROl 1 12, anti- PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibody and the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide in the test sample
- the detection may be qualitative or quantitative, and may
- 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-
- the kit preferably contains instructions for using the antibody to detect the PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide
- the invention concerns a method for inhibiting the growth of tumor cells comprising exposing a cell which overexpressesa PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017 PROl 1 12, PRO509, PR0853 or PR0882 polypeptideto an effective amount of an agent inhibiting the expression and or activity of the PR 201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347 PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882 polypeptide
- the agent preferably is an ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327.
- the agent e g ant ⁇ -PRO201,ant ⁇ -PRO292,ant ⁇ -PRO327,ant ⁇ -PRO1265,ant ⁇ -PRO344,ant ⁇ -PRO343,ant ⁇ -PRO347,ant ⁇ - PRO357, ant ⁇ -PRO715,ant ⁇ -PRO1017,ant ⁇ -PROl 1 12,ant ⁇ -PRO509,ant ⁇ -PRO853 or ant ⁇ -PR0882 antibody induces cell death
- the tumor cells are further exposed to radiation treatment and/or
- the invention concerns an article of manufacture, comprising a container, a label on the container, and a composition comprising an active agent contained within the container, wherein the composition is effective for inhibiting the growth of tumor cells
- the label on the container indicates that the composition can be used for treating conditions characterized by overexpression of a PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343. PR0347, PR0357, PR0715, PRO1017, PROl 1 12. PRO509. PR0853 or PR0882 polypeptide
- the active agent in the composition is an agent inhibiting the expression and or activity of the PRO201.
- PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347. PR0357, PR0715 are examples of the composition.
- the active agent is an anti-PRO201 , anti-PR0292, anti-PR0327. anti-PRO 1265, anti-PR0344, anti-PR0343. anti-PR0347. anti- PRO357. anti-PRO715,anti-PRO1017,anti-PROl 1 12,anti-PRO509,anti-PRO853 or anti-PRO882 antibody.
- PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 polypeptide comprising contacting a candidate compound with a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 polypeptide under conditions and for a time sufficient to allow these two components to interact.
- PRO509. PR0853 or PR0882 polypeptide is immobilized on a solid support.
- Figure 1 is the nucleotide sequence of DNA30676 encoding a PRO201 (UNQ 175).
- Figure 2 is the amino acid sequence of the native human PRO201 (UNQ175) polypeptide.
- Figure 3 is the nucleotide sequence of DNA35617 encoding a PR0292.
- Figure 4 is the amino acid sequence of the native human PR0292 (UNQ266) polypeptide.
- Figure 5 is the nucleotide sequence of DNA381 13 encoding a PR0327.
- Figure 6 is the amino acid sequence of the native human PR0327 (UNQ288) polypeptide.
- Figure 7 is the nucleotide sequence of DNA60764 encoding a PR01265.
- Figure 8 is the amino acid sequence of the native human PRO 1265 (UNQ636) polypeptide.
- Figure 9 is the nucleotide sequence of DNA40592 encoding a PR0344 .
- Figure 10 is the amino acid sequence of the native human PR0344 (UNQ303) polypeptide.
- Figure 1 1 (SEQ ID NO: 1 1) is the nucleotide sequence of DNA43318 encoding a PR0343.
- Figure 12 is the amino acid sequence of the native human PR0343 (UNQ302) polypeptide.
- Figure 13 is the nucleotide sequence of DNA44176 encoding a PR0347.
- Figure 14 is the amino acid sequence of the native human PR0347 (UNQ306) polypeptide.
- Figure 15 is the nucleotide sequence of DNA44804 encoding a PR0357.
- Figure 16 is the amino acid sequence of the native human PR0357 (UNQ314) polypeptide.
- Figure 17 is the nucleotide sequence of DNA52722 encoding a PR0715
- Figure 18 is the amino acid sequence of the native human PR0715 (UNQ383) polypeptide
- Figure 19 is the nucleotide sequence of DNA561 12 encoding a PRO 1017
- Figure 20 is the amino acid sequence of the native human PRO 1017 (UNQ500) polypeptide
- Figure 21 is the nucleotide sequence of DNA57702 encoding a PROl 112
- Figure 22 is the ammo acid sequence of the native human PROl 1 12 (UNQ555)
- Figure 23 is the nucleotide sequence of DNA50148 encoding a PRO509
- Figure 24 is the am o acid sequence of the native human PRO509 polypeptide
- Figure 25 is the nucleotide sequence of DNA48227 encoding a PRO1350
- Figure 26 is the amino acid sequence of the native human PRO 1350 polypeptide (UNQ419)
- Figure 27 is the nucleotide sequence of DNA58125 encoding a PR0882
- Figure 28 is the ammo acid sequence of the native human PR0882 polypeptide (UNQ448)
- Figure 29 is the nucleotide sequence of DNA28710 used in the cloning of DNA 30676 encoding PRO201
- Figure 30 is the nucleotide sequence encoding EST 2452972
- Figure 31 is the nucleotide sequence encoding EST2099855
- F ⁇ gure32 is a map of chromosome 19 showing the mapping regions of DNA30676, DNA381 13 and DNA60764
- Figure 33 is a map of chromosome 1 1 showing the mapping region of DNA354617
- Figure 34 is a map of chromosome 16 showing the mapping region of DNA58125 and DNA43318
- Figure 35 is a map of chromosome 7 showing the mapping region of DNA561 12
- Figure 36A is map of chromosome ' 7 showing the mapping region of DNA52722
- Figure 36B is a map of chromosome 17 showing the mapping region of DNA48227
- Figures 37-39 are in situ hybridizations as described m
- the example section Figure 40 is a map of chromosome 16 showing the mapping region of DN A44804
- 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 "ampiicon "
- 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 and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized bv unregulated cell growth
- 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, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer
- Treatment refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented In
- the "pathology' of cancer includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc
- “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc Preferably, the mammal is human
- Carriers as used herein include pharmaceuticallyacceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being e> /osed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution
- physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyviny Ipyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccha ⁇ des,d ⁇ saccha ⁇ des,and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counte ⁇ ons such as sodium, and/or
- cytotoxic agent refers to a substance that inhibits or prevents the function of cells and orcauses destruction of cells
- the term is intended to include radioactive isotopes (e g I 131 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof
- a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer Examples of chemotherapeuticagents include ad ⁇ amyc ⁇ n,doxorub ⁇ c ⁇ n,ep ⁇ rub ⁇ c ⁇ n, 5-fluorourac ⁇ l, cytosine arabinoside ("Ara-C").
- cyclophosphamide thiotepa, busulfan, cytoxin, taxoids. e g paclitaxel (Taxol Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh ⁇ ne-Poulenc Rorer. Antony, Rnace). toxotere, methotrexate. cisplatin.melphalan, vinblastine.bleomycin.etoposide. ifosfamide.mitomycinC. mitoxantrone, vincnstine, vinorelbine, carboplatin, teniposide, daunomycin, carm omycin, aminopte ⁇ n.
- dactinomyc mitomycms, esperam ⁇ c ⁇ ns(see S Pat No 4,675,187), 5-FU, 6-th ⁇ oguamne,6-mercaptopu ⁇ ne actinomycin D, VP- 16.
- growth inhibitory agent when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressmg any of the genes identified herein, either in vitro or in vivo
- growth inhibitory agent is one which significantly reduces the percentage of cells overexpressmg 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 GI arrest and M-phase arrest
- Classical M-phase blockers include the vincas (vincnstine and vinblastme), taxol, and topo II inhibitors such as doxorubic , epirubicin, daunorubicin.etoposide and bleomycin
- doxorubic doxorubic
- epirubicin daunorubicin.etoposide and bleomycin
- Those agents that arrest G 1 also spill over into S-phase arrest, for example, DNA alkylating agents such as
- Doxorubicm is an athracycline antibiotic
- the full chemical name of doxorubicm is (8S-c ⁇ s)-10- [(3-am ⁇ no-2,3,6-t ⁇ deoxy- ⁇ -L-lyxo-hexapyranosy l)oxy]-7,8,9, 10-tetrahydro-6,8.1 1 -t ⁇ hydroxy-8- (hydroxyacetyl)- 1 -methoxy-5, 12-naphthacened ⁇ one
- cytokme is _ generic term for proteins released by one cell population which act on another cell as intercellular mediators
- lymphokines lymphokines. monokines. and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolact ⁇ n,placental lactogen, tumor necrosis factor- ⁇ and - ⁇ , mulle ⁇ an-inhibiting substance, mouse gonadotropm-associatedpeptide, ⁇ nh ⁇ b ⁇ n, act ⁇ v ⁇ n, vascular endothelial growth factor, ⁇ nteg ⁇ n,thrombopo ⁇ et ⁇ n (TPO), nerve growth factors such
- cytokme 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 See, e g Wilman, "Prodrugs in Cancer Chemotherapy ' , Biochemical Society Transactions, 14, pp 375-382, 615th Meeting, Harbor( 1986).
- the prodrugs of this invention include, but are not limited to, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containmg prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glysocylated prodrugs, ⁇ -lactam- containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytos ⁇ ne and other 5-fluorou ⁇ d ⁇ ne prodrugs which can be converted into the more active cytotoxic free drug
- Examples of cytotoxic drugs that can be de ⁇ vatized into a prodrugs form for use in this invention include, but are not limited
- PRO201 polypept ⁇ de encompass both native sequence and active variants thereof
- PR0292 polypept ⁇ de encompasses both native sequence and active variants thereof
- PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic means
- the term specifically encompasses naturally-occur ⁇ ngtruncatedor secreted forms (e g , an extracellular domain sequence), naturally-occurring
- PRO509, PR0853 or PR0882 polypeptide Fragments of the respective native polypeptides herein include, but are not limited, to polypeptide variants from which the native N-terminal signal sequence has been fully or partially deleted or replaced by another sequence, and extracellular domains of the respective native sequences, regardless whether such truncated (secreted) forms occur in nature Fragments are preferably sufficient in length for the production of an antibody specifically binding the corresponding native "PRO" polypeptide
- the terms “UNQ 175", “UNQ255”, “UNQ288”, “UNQ636”, “UNQ303”, “UNQ302”, “UNQ306”, “UNQ314", “UNQ383”, “UNQ500”, “UNQ555”, “UNQ329”, “UNQ419” and “UNQ448” are used to refer to the native sequence polypeptide corresponding to the PRO designated sequences above appearing as a full-length native presequence or a mature form of a PRO201 polypeptide shown in Figure 2 (SEQ ID NO 2), a PR0292 polypeptide shown in Figure 4 (SEQ ID NO 4), a PR0327 polypeptide shown in Figure 6 (SEQ ID NO 6), a PRO 1265 polypeptide shown in Figure 8 (SEQ ID NO 8), a PR0344 polypeptide shown in Figure 10 (SEQ ID NO 10), a PR0343 polypeptide shown in Figure 12 (SEQ ID NO 12), a PR0347 polypeptide shown in Figure 14 (SEQ ID NO
- a PR0715 polypeptide shown in Figure 18 (SEQ ID NO 18), a PROl 017 polypeptide shown in Figure 20 (SEQ ID NO 20), a PROl 1 12 poiypeptide shown in Figure 22 (SEQ ID NO 22).
- a PRO509 polypeptideshown m Figure 24 (SEQ ID NO 24), a PR0853 polypeptide shown in Figure 26 (SEQ IDNO 26), and a PR0882 polypeptideshown in Figure 28 (SEQ ID NO 28), respectively Said another way the "UNQ" designation refers to the specific native sequence, while the "PRO” designation refers to the native sequence and active variants thereof
- An "isolated" nucleic acid molecule encoding a PRO201 , PR0292, PR0327. PRO 1265. PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecuie with which it is ordinarily associated in the natural source of the PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-. PRO 1 1 12-, PRO509-.
- PR0853- or PR0882-encodmg nucleic acid An isolated PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-. PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucleic acid molecules therefore are distinguished from the PRO201-, PRO292-, PRO327-, PROl 265-, PR0344-, PR0343-.
- nucleic acid molecule encoding a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide includes nucleic acid molecules contained in cells that ordinarily express PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells
- control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism
- the control sequences that are suitable for prokaryot include a promoter, optionally an operator sequence, and a ribosome binding site
- Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
- Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence
- DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
- a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
- a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
- "operably linked” means that the DNA sequences being linked are contiguous, and.
- “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that ( 1 ) employ low ionic strength and high temperature for washing, for example 0 015 M sodium chlo ⁇ de/00015 M sodium c ⁇ trate/0 1% sodium dodecyl sulfate at 50°C , (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1% bovine serum album ⁇ n/0 1% F ⁇ coll/0 1% polyv ⁇ nylpyrrol ⁇ done/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 M 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 (
- dextran sulfate at 42°C with washes at 42°C in 0 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0 1 x SSC containing EDTA at 55°C
- Modely stringent conditions may be identified as described by Sambrook et al Molecular Cloning A Laboratory Manual, New York Cold Spring Harbor Press, 1989, and include the use of washing solutior and hyb ⁇ dization conditions (e g , temperature, ionic strength and % SDS) less st ⁇ ngen' than those described above
- epitope tagged when used herein refers to a chimeric polypeptide comprising a PRO201 ,
- tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused
- the tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes
- Suitable tag polypeptides generally have at least six 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” in the context of molecules identified based upon the PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptides (or their coding sequences) refers to polypeptides (e g antibodies) or organic or inorganic small molecules, peptides, ere which retain the biological and/or lmmunologicalactivities/properties of a native or naturally-occurring PRO201.
- Bioactivity in the context of an antibody or another molecule that can be identified by the screening assays disclosed herein is used to refer to the ability of such molecules to bind or complex with the polypeptides encoded by the amplified genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins
- a preferred biological activity is growth inhibition of a target tumor cell
- Another preferred biological activity is cytotoxic activity resulting in the death of the target tumor cell
- immunologicalproperty means immunological cross-reactivity with at least one epitope of a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343. PR0347, PR0357, PR0715, PRO1017, PROl 1 12.
- PRO509, PR0853 or PR0882 polypeptide "Immunological cross-react ⁇ v ⁇ ty"as used herein means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide having this activity with polyclonal antisera raised against the known active PRO201, PR0292, PR0327, PRO 1265, PR0344 PR0343, PR0347, PR0357.
- PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 poiypeptide 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 intraperitoneal or subcutaneous injection in incomplete Freunds
- the immunological cross-reactivity preferably is "specific", which means that the binding affinity of the immunologicallycross-reactive molecule (f g antibody) identified, to the corresponding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PRO357, PRO715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 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 8-t ⁇ mes higher) than the binding affinity of that molecule to any other known native polypeptide
- antagonist is used in the broadest sense, and includes any molecule that p -* ⁇ ally or fully blocks, inhibits, or neutralizes a biological activity of a native PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, or PRO509 oolypeptide disclosed herein
- agonist is used in the broadest sense and inch des any molecule that mimics a biological activity of a native PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide disclosed herein
- Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or am o acid sequence variants of native polypeptides, peptides, small organic molecules, etc
- a "small molecule ' is defined herein to have a molecular weight below about 500 Dalton
- Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characte ⁇ stics While antibodiesexhibit bindingspecificityto a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas
- antibody is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies polyclonal antibodies. mult ⁇ spec ⁇ ficant ⁇ bod ⁇ es(e 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 Dalton, composed of two identical light (L) chains and two identical heavy (H) chains Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes Each 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
- variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
- CDRs complementarity-determining regions
- hyperva ⁇ able regions both in the light-chain and the heavy-chain variable domains
- the more highly conserved portions of variable domains are called the framework (FR)
- the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configui anon, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -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-b dingsite of antibodies (see Kabat et al MH Publ No 91-3242, Vol I, pages 647-669 ( 1991))
- the constant domains are not involved
- hyperva ⁇ ableregion when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding
- the hyperva ⁇ able region comprises amino acid residues from a ' complementa ⁇ tydeterminingregion " to "CDR" (i e residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H I ), 50-65 (H2) and 95- 102 (H3) in the heavy chain variable domain, Kabat et al Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institute of Health, Bethesda, MD [ 1991 ]) and or those residues from a "hyperva ⁇ ableloop” (I e residues 26-32 (L 1 ), 50-52 (L2) and 91 -96 (L3) in the light chain variable domain and 26-32 (H 1 ), 53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain , Clothia and Les
- Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibodv
- antibody fragments include Fab, Fab', F(ab'),, and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng 8(10) 1057-1062 [ 1995]), single-chain antibodv molecules, and multispecific antibodies formed from antibody fragments
- Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab” fragments, each with a single antigen-bindingsite, and a residual "Fc” fragment whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab') : fragment that has two antigen-combining sites and is still capable of cross-linking antigen
- Fv is the minimum antibody fragment which contains a complete antigen-recognitionand -binding site This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association It is in this configuration
- the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) of the heavy chain Fab fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH I domain including one or more cystemes from the antibody hinge region Fab'-SH is the designation herein for Fab' in which the cysteine res ⁇ due(s) of the constant domains bear a free thiol group F(ab'), antibody fragments originally were produced as pairs of Fab' fragments which have hinge cystemes between them Other chemical couplings of antibody fragments are also known
- the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the ammo acid sequences of their constant domains
- immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e g , IgG 1 , IgG2, IgG3, IgG4. IgA, and IgA2
- the heavy-chain constant domains that corres;'_ nd to the different classes of immunoglobulins are called ⁇ , ⁇ , e, ⁇ , and ⁇ , respectively
- the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known
- the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ; e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore, in contrast to conventional (polyclonal)ant ⁇ body preparationswhich typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminatedby other immunoglobulins
- the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method
- the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first
- the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al Nature. 352 624-628 [ 19 1 ] and Marks e. ⁇ / J Mol Biol . 222 581-597 ( 1991 ). for example
- the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cha ⁇ n(s) is identical with or homologous to correspondingsequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U S Patent No 4,816.567 Morrison et al Proc Natl Acad Sci USA, 81 6851 -6855 [1984]) "Humanized" forms of non-human (e g , mu ⁇ ne) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv Fab.
- humanized antibodies are human ⁇ mmunoglobul ⁇ ns(rec ⁇ p ⁇ ent antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances.
- humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modificationsare made to further refine and maximize antibody performance
- the humanized antibody will comprise substantially all of at 'east 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 immunoglobulinconstant region (Fc), typically that of a human immunoglobulin
- Fc immunoglobulinconstant region
- the humanized antibody includes a PRIMATIZEDTM antibodv wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest
- Single-chain Fv or “sFv” antibody fragments comprise the V H and V L doma ⁇ nsof 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
- a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding
- diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H - V L )
- V H heavy-chain variable domain
- V L light-chain variable domain
- the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites
- Diabodies are described more fully in, for example, EP 404,097, WO 93/11 161 , and Hollinger et al , Proc Natl Acad S i US4. 90 6444-6448 ( 1993)
- an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceoussolutes
- 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 ammo 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
- 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), polysaccha ⁇ des (e g , agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones
- the solid phase can comprise the well of an assay plate, in others it is a purification column (e g , an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275,149
- a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as an PRO201 , PR0292, PR0327, PRO 1265. PR0344. PR0343, PR0347. PR0357, PR0715, PRO 1017. PROl 1 12. PRO509, P ⁇ 0853 or PR0882. or an antibody thereto and, optionally, a chemotherapeuticagent) to a mammal
- 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 (l e , is "heterologous"),and an immunoglobulinconstantdomain sequence
- the adhesin part of an lmmunoadhesin molecule typically is a contiguous am o 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,
- PRQ201 Preparation of the PRQ201. PRQ292. PRQ327. PRQ1265. PRQ344. PRQ343. PRQ347.
- PRQ357 PRQ715.
- PRO 1017 PRO 1 1 12.
- PRQ509. PRQ853 or PRQ882 polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO201 (UNQ 175), PR0292 (UNQ255). PR0327 (UNQ288).
- PRO 1265 (UNQ636).
- PR0344 (UNQ303), PR0343 (UNQ302), PR0347 (UNQ306), PR0357 (UNQ3 14), PRO715 (UNQ383), PRO 1017 (UNQ500), PRO l 1 12 (UNQ555), PRO509 (UNQ329), PR0853 (UNQ419) or PR0882 (UNQ448) In particular.
- the description below relates primarily to production of PRO201 , PR0292, PR0327, PRO 1265.
- PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides by cultur g cells transformed or transfected with a vector containing PRO201 -, PR0292-.
- PRO509, PR0853 or PR0882 polypeptides For instance, the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347 PR0357, PR0715.
- PROl 017, PROl 1 12, PRO509, PR0853 or PR08S" ⁇ polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e g , Stewan et al Solid-Phase Peptide Synthesis, W H Freeman Co , San Francisco, CA ( 1969), Mer ⁇ field, J Am Chem Soc , 85 2149- 2154 ( 1963)]
- In vitro protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions Various portions of the PRO201. PR0292, PR0327, PR01265.
- PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO201 , PR0292, PR0327, PRO 1265, PR0344. PR0343, PR0347. PR0357, PR0715. PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882
- PRO 1 1 12, PRO509, PR0853 or PR0882 may be obtained from a cDN A library prepared from tissue believed to possess the PRO201 PR0292, PR0327. PRO 1265, PR0344 PR0343, PR0347, PR0357. PR0715, PROl 017.
- PROl 1 12 PRO509 PR0853 or PR0882 mRNA and to express it at a detectable level Accordingly, human PRO201, PR0292, PR0327. PR01265, PR0344 PR0343. PR0347, PR0357, PR0715, PRO1017, PROl 1 12.
- PRO509, PR0853 or PR0882 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples PRO201-. PR0292-, PR0327-, PRO 1265-. PR0344-, PR0343-, PR0347-. PR0357-, PR0715-, PRO 1017-.
- PRO509-, PR0853- or PR0882-encod ⁇ ng gene may also be obtained from a genomic library or by oligonucleotide synthesis
- Probes such as antibodies to the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12.
- PRO509, PR0853 or PR0882 polypeptide, or oligonucleotides of at least about 20-80 bases designed to identify the gene of interest or the protein encoded by it Screening the cDN A 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)
- PR0853 or PR0882 is to use PCR methodology [Sambrook et al , supra. Dieffenbach et al PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995
- the oligonucleotide sequences selected as probes should be oi sufficient length and sufficiently unambiguous that false positives are minimized
- the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened
- Methods of labeling are well known in the art. and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
- Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases
- Sequence identity (at either the ammo acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as ALIGN, DNAstar. and INHERIT which employ various algorithms to measure homology
- Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and if necessary, using conventional primer extension procedures as described in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA b Selection and Transformation of Host Cells
- Host cells are transfected or transformed with expression or cloning vectors described herein for PRO201 , PR0292, PR0327, PRO 1265, PR0344 PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1112, PRO509, PR0853 or PR0882 production and cultured in conventionalnut ⁇ ent 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 culturescan be found in MammalianCell Biotechnology Practical Approach M Butler, ed (1RL Press. 1991 ) and Sambrook et al . supra
- transfection 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 or other cells that contain substantial cell-wall barriers
- Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al Gene.
- Suitable host cells for cloning or expressingthe DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells
- Suitable prokaryotes include but are not limited to eubacte ⁇ a, such as Gram- negative or Gram-positiveorganisms, for example, Enterobacte ⁇ aceaesuch as E coli
- Various /- coli strains are publicly available, such as £ coli Kl 2 strain MM294 (ATCC 31,446), E coli XI 776 (ATCC 31,537), E coli strain W31 10 (ATCC 27,325) and K5 772 (ATCC 53,635)
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO201 -, PR0292-, PR0327-, PRO 1265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO1017-.
- PROH 12-, PR 509-, PR0853- or PR0882-encod ⁇ ng vectors Saccharomvces cerevisiae is a commonly used lower eukaryotic host microorganism
- PR0853 or PR0882 are derived from multicellularorganisms
- invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells
- useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells More specific examples include monkey kidney CV l line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol , 36 59 (1977)), Chinese hamster ovary cellsADHFR (CHO, Urlaub and Chasm, Proc Natl Acad Set USA, TL 4216 ( 1980)), mouse sertoli cells (TM4, Mather, Bio
- PROl 1 12, PRO509, PR0853 or PR0882 may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression
- Various vectors are publicly available
- the vector may, for example, be in the form of a plasmid, cosmid.
- nucleic acid sequence may be inserted into the vector by a variety of 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 transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan
- PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide
- the signal sequence may be a component of the vector, or it may be a part of the PRO201 -, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-. PR0347-.
- 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 (mcludmgSaccharomvcesand Kluyveromvces ⁇ -factor leaders, the latter described in U S Patent No 5,010, 182), or acid phosphatase leader, the C ⁇ lbtc ⁇ ns glucoamylase leader (EP 362.179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990
- mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences
- Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected !• .st cells Such sequences are well known for a variety of bacteria, yeast, and viruses.
- the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for clonirg vectors in mammalian cells
- Selection genes will typically contain a selection gene, also termed a selectable marker
- Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e , ampicillin, neomycin, methotrexate, or tetracvcline, (b) complement auxotrophic deficiencies, or (c) supply c ⁇ tical nutrients not available from complex media, e , the gene encoding D-alanme racemase for Bacilli
- suitable selectable markers for mammalian cells are those that enable the identification of cells competentto take up the PRO201-.
- PRO853- or PR0882-encod ⁇ ng nucleic acid such as DHFR or thymidine kinase
- An appropriatehost cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaube/ ⁇ / , Proc Natl Acad Sci USA, 77.4216 (1980)
- a suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid YRp7 [Stmchcomb et al Nature, 282.39 ( 1979), Kingsman et al . Gene, 7 141 ( 1979), Tschemper et al .
- the /rpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example.
- ATCC No 44076 or PEP4-1 Japanese Patent Application Laid Generation
- Expression and cloning vectors usually contain a promoter operably linked to the PRO201 -, PR0292- . PR0327-, PRO 1265-, PR0344-. PR0343-, PR0347-, PR0357-, PR0715-, PRO 1017-.
- PRO 1 1 12-, PRO509- , PR0853- or PR0882-encod ⁇ ng nucleic acid sequence to direct mRNA synthesis Promoters recognized by a variety of potential host cells are well known Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al Nature, 275 615 ( 1978), Goeddel et al , Nature, 281 544 (1979)].
- 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 Enzvme Reg , 7 149 ( 1968), Holland, Biochemistry, 17 4900 ( 1978)], such as enolase. glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase.
- phosphofructokinase glucose-6-phosphate isomerase 3-phosphoglycerate mutase, pyruvate kinase, t ⁇ osephosphate isomerase, phosphoglucose isomerase, and glucokmase
- ye st promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2. isocytochromeC, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73,657
- PROl 1 12, PRO509, PR0853 or PR0882 transcription from vectors in mammalian host cells is controlled, for example, by oromoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 pub'ished 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarccma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems
- viruses such as polyoma virus, fowlpox virus (
- Enhancers are cis-actmg elements of DNA, usually about from 10 to 300 bp, that act on a promoterto increase its transcription
- Many enhancer sequences are now known from mammalian genes (globin, elastase. albumin, ⁇ -fetoprotein.
- an enhancer from a eukaryotic cell virus examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers
- the enhancer may be spliced into the vector at a position 5' or 3' to the PRO201 -, PR0292-. PR0327-, PRO 1265-, PR0344-, PR0343- PR0347-. PR0357-. PR0715- PRO 1017-. PRO1 1 12-. PRO509-. PRO853- or PRO882- coding sequence, but is preferably located at a site 5' from the promoter
- Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3'. untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylatedfragments in the untranslated portion of the mRNA encoding PRO201 , PR0292. PR0327. PR01265, PR0344, PR0343, PR0347, PR0357. PR0715. PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO201 ,
- PR0292 PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in recombinant vertebrate cell culture are described in Gething et al Nature, 293 620-625 ( 1981 , Mantel et al Nature, 28 . 40-46 ( 1979), EP 1 17,060, and EP 1 17,058 d Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting.
- Gene expression may be measured by immunological methods, such as immunohis. _chemicalstaining of cells or tissue sections and assay of cell culture or body fluids, to qi'- ntitate directly the expression of gene product
- Antibodiesuseful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal Conveniently, the antibodies may be prepared against a native sequence PRO201 , PR0292, PR0327, PRO ! 265, PR0344, PR0343, PR0347, PR0357, PR0715.
- PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PRO ! 1 12, PRO509, PR0853 or PR0882 polypeptides may be recovered from culture medium or from host cell lysates Ifmembrane-bound.it can be released from the membrane using a suitable detergent solution ( g T ⁇ ton-X 100) or by enzymatic cleavage Cells employed in expression of PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343 , PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysmg agents It may be desired to purify PRO201.
- PR0292, PR0327, PRO 1265. PR0344. PR0343, PR0347, PR0357. PR0715. PRO1017. PROl 1 12. PRO509. PR0853 or PR0882 from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC. chromatography on silica or on a cation-exchange resin such as DEAE.
- PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptides Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzvmology, 182 ( 1990), Scopes, Protein Purification Principles and Practice, Springer- Verlag, New York ( 1982) The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO201. PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357. PR0715, PRO1017.
- PROl 1 12, PRO509, PR0853 or PR0882 polypeptide produced 2 Amplification of Genes Encoding the PRO201. PRQ292. PRQ327. PRQ1265. PRQ344.
- the present invention is based on the ldentificationand characterization of genes which are amplified in certain cancer cells
- the genome of prokaryotic and eukaryotic organisms is subjected to two seemingly conflicting requirements One is the preservation and propagation of DNA as the genetic information in its ongmal form, to guarantee stable inheritance through multiple generations
- cells or organisms must be able to adapt to lasting environmental changes
- the adaptive mechanisms can include qualitative or
- genes involved in intracellular signaling and control of cell cycle progression can undergo amplification during malignant transformation This is illustrated by the amplification of the bcl-I and ras genes in various epithelial and lymphoid neoplasms
- CGH comparative genomic hybridization
- PCR polymerase chain reaction
- PCR-based assays are most suitable for the final identification of coding sequences, i e. genes in amplified regions
- such genes have been identified by quantitative PCR (S Gelmini et al , Clin Chem 43 752 [1997]), by comparing DNA from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus. testis, ovary, uterus, etc tumor, or tumor cell lines, with pooled DNA from healthy donors Quantitative PCR was performed using a TaqMan instrument (ABI) Gene-specific primers and fluorogenic probes were designed based upon the coding sequences of the DNAs
- Human lung carcinomacell lines include A549 (SRC768),Calu- 1 (SRC769). Calu-6 (SRC770), H 157 (SRC771 ), H441 (SRC772), H460 (SRC773), H522 (SRC832), H810 (SRC833), SKMES- 1 (SRC774) and SW900 (SRC775), all available from ATCC
- Primary human umg tumor cells usually derive from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas, and broncho alveolar carcinomas, and include, for example, SRC724 (squamous cell carcinoma abbreviated as "SqCCa”)(LT 1 ),SRC725 (non-small cell carcinoma, abbreviatedas "NSCCa”)(LT 1 a), SRC726 (adenocarcinoma, abbreviated as "AdenoCa”)(LT2), SRC727 (
- SRC728 (squamous cell carc ⁇ noma)(LT4), SRC729 (adenocarc ⁇ noma)(LT6), SRC730 (ader 'squamous cell carc ⁇ noma)(LT7), SRCC731 (adenocarc ⁇ noma)(LT9).
- SRC732 (squamous cell carc ⁇ noma)(LT10) SRC733 (adenocarc ⁇ noma)(LTl 1), SRC734 (adenocarc ⁇ noma)(LT12), SRC735 (broncho alveolar carcinoma, abbreviated as "BAC")(LT13), SRC736 (squamous cell carcmoma)(LT15), SRC737 (squamous cell carc ⁇ noma)(LT16),SRC738 (squamous cell carc ⁇ noma)(LT17), SRC739 (squamous cell carc ⁇ noma)(LTI 8), SRC740 (squamous cell carc ⁇ noma)(LT19), SRC741 (lung cell carcinoma, abbreviated as "LCCa”)(LT21), SRC81 1 (adenocarc ⁇ noma)(LT22)
- Colon cancer cell lines include, for example, ATCC cell lines S W480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRC777), Colo320 (carcinoma, SRCC778), Colo205 (carcinoma, SRC828), HCC2998 (carcinoma, SRC830), HT29 (adenocarcinoma, SRC779), HM7 (carcinoma. SRC780).
- KM 12 (carcinoma, SRC831 ), CaWiDr (adenocarc ⁇ noma,SRC781 ), HCT 15 (carcinoma, SRC829), HCTl 16 (carcinoma, SRC782), SKCOl (adenocarcinoma, SRC783), SW403 (adenocarcinoma, SRC784), LS174T (carcinoma, SRC785), and HM7 (a high mucin producing variant of ATCC colon adenocarcinomacell line LS 174T, obtained from Dr Robert Warren, UCSF)
- Primary colon tumors include colon adenoocarcinomas designated CT1 (SRC751 ), CT2 (SRC742), CT3 (SRC743).
- CT4 (SRC752), CT5 (SRC753), CT6 (SRC754), CT7 (SRC755), CT8 (SRC744), CT9 (SRC756), CT10 (SRC745), CT1 1 (SRC757), CT12 (SRC746), CT14 (SRC747).
- CT15 (SRC748).
- CT16 (SRC749).
- CT17 (SRC750).
- Human breast carcinomacell lines include, for example, HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761 ), MB468(SRCC762).MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3 (SRCC767)
- 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 appropriately labeled probe, based on the sequences provided herein
- antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes
- Gene expression in various tissues alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directlythe 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 RO201 ,
- General techniques for generating antibodies, and special protocols for Northern b' _tt ⁇ ng and in situ hybridization are provided hereinbelow
- the gene can be mapped to a particular chromosome, e g by radiation-hybrid analysis
- the amplification level is then determined at the location identified, and at neighboring genomic region Selective or preferential amplificationat the genomic region to which to gene has been mapped is consistent with the possibility that the gene amplification observed promotes tumor growth or survival
- Chromosome mapping includes both framework and epicenter mapping For further details see e g , Stewart et al . Genome Research 7, 422-433 ( 1997) 5 Antibodv Binding Studies
- the results of the gene amplification study can be further verified by antibody binding studies, in which the ability of ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, ant ⁇ -PR01265, ant ⁇ -PR0344. ant ⁇ -PR0343, ant ⁇ -PR0347,ant ⁇ -PR0357. ant ⁇ -PR0715. anti-PRO 1017, anti-PRO 1 1 12. ant ⁇ -PRO509, ant ⁇ -PR0853 or anti- PR0882 to inhibit the expression of the PRO201 , PR0292, PR0327. PR01265. PR0344, PR0343, PR0347, PR0357 PR0715. PRO 1017. PRO 1 1 12. PRO509. PR0853 or PR0882 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, Inc , 1987)
- Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected
- the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte.
- the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay)
- sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme For lmmunohistochemistry.
- the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example
- Cell-basedassays and animal models for tumors can be used to verify the findings of the gene amplification assay, and further understand th 'elationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth
- the role of gene products identified herein in the development and pathology of tumor or cancer can be tested by using primary tumor cells or cells lines that have been identified to amplify the genes herein Such cells include, for example, the breast, colon and lung cancer cells and cell lines listed above
- Suitable cells include, for example, stable tumor cells lines such as, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene)and r ⁇ _--transfectedNIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogemc growth
- stable tumor cells lines such as, the B104-1-1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene)and r ⁇ _--transfectedNIH-3T3 cells, which can be transfected with the desired gene, and monitored for tumorogemc growth
- Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit tumoroge c cell growth by exerting cytostatic or cytotoxic activity on the growth of the transformed cells, or by mediating antibody- dependentcellularcytotox ⁇ c ⁇ ty(ADCC) Cells transfected with the coding sequences of the genes identified herein can further
- 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
- Animal models oftumors and cancers include both non-recombmant and recombinant (transgenic) animals
- Non-recombinant animal models include, for example, rodent, e g , mu ⁇ ne models
- Such models can be generated by introducingfumor cells into syngeneic mice using standard techniques, e g subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule or orthopin implantation, e g colon cancer cells implanted in colonic tissue (See, e g PCT publication No WO 97/33551 , published September 18,
- the cells introduced into such anirr tls can be derived from known tumor/cancer cell lines, such as any of the above-listed tumor cell lines, and for example, the B104- 1 -1 cell line (stable NIH-3T3 cell line transfected with the neu protooncogene), Caco-2 (ATCC HTB-37) a moderately well- differentiatedgrade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or / fashion_» tumor, _» ⁇ / -_ «-,,_ Samples of tumor or cancer cells can be obtained from patients undergoing surgery, using standard conditions, involving freezing and storing in liquid nitrogen (Karmali et al Br J Cancer 48 689-696 [ 1983]) Tumor cells can be introduced into animals, such as nude mice, by a variety of procedures
- the subcutaneous (s c ) space in mice is very suitable for tumor implantation
- Tumors can be transplanted s c as solid blocks, as needle biopsies by use of a trochar, or as cell suspensions
- tumor tissue fragments of suitable size are introduced into the s c space
- Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected 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 neuroblastomacells (from which the neu oncogen was initially isolated), or neu -transformed NIH-3T3 cells into nude mice, essentially as described by Drebm 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 forexample bv Wang er ⁇ / Cancer Research 54 4726-
- Tumors that arise in animals can be removed and cultured in vitro Cells from the in vitro cultures can then be passaged to animals Such tumors can serve as targets for further testing or 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
- 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 study ⁇ ngthe ant ⁇ -tumoract ⁇ v ⁇ t ⁇ esofva ⁇ ous agents (PalIad ⁇ noe.
- 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 10x10" to 10xl0 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 the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL)
- SCCL small cell carcinoma of the lung
- Recombinant (transgen ⁇ c)an ⁇ mal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals
- 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 m ⁇ cro ⁇ nject ⁇ on(Hoppe and Wanger, U S Patent No 4,873,191 ), retrovirus-mediated gene transfer into germ lines (e g .
- transgenic animals include those that carrv the transgene only in part oftheir cells ("mosaic animals”).
- the transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction of a transgene into a particularcell type is also possible by following, for example, the technique of asko et al Proc Natl Acad Sci USA 89 6232-636 (1992)
- 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 immunocytochemistry The animals are further examined for signs of tumor or cancer development
- "knock out" animals can be constructed which have a defective or altered gene encoding a PRO201 , PR0292, PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal For example, cDNA encoding a particular PRO201 , PR0292, P10327. PRO 1265, PR0344, PR0343, PR0347, PR0357.
- PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques A portion of the genomic DNA encoding a particular PR0201.
- PR0292,PR0327, PR01265,PR0344,PR0343,PR0347, PR0357,PR0715,PR01017,PR01 1 12, PRO509, PR0853 or PR0882 polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
- another gene such as a gene encoding a selectable marker which can be used to monitor integration
- several kilobases of unaltered flanking DNA are included in the vector [see e g , Thomas and Capecchi, Cell.
- the vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombmed 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 harbo ⁇ ng the homologously recombmed DNA in their germ cells can be identified by standard techniques and used
- 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 mteractior 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- ⁇ mmunoglobul ⁇ n fusions, and. in particular, antibodies including, without lim nation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypicantibodies.
- the assays can be performed in a variety of formats, mcludingprotein-proteinbinding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art
- the polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, on a microtiter plate, by covalent or non-covalent attachments
- Non-covalentattachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying
- an immobilized antibody e g a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface
- the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e g the coated surface containing the anchored component When the reaction is complete, the non-reacted components are removed,
- the candidate compound interacts with but does not bind to a particular PRO201 , PR0292, PR0327, PR01265,PR0344,PR0343.PR0347.PR0357 PR0715, PROl 017, PROl 1 12 PRO509 PR0853 or PR0882 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-immunoprecipitation, and co-purification through gradients or chrom ⁇ tographiccolumns
- protein-proteminteractions can be monitored by using a yast-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
- GAL l-/ ⁇ rZ reporter gene under control of a GAL4-act ⁇ vated promoter depends on reconstitution of GAL4 activity via protein-protein interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ - galactosidase
- MATCHMAKERTM for identifying prote -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
- compositions useful in the treatment of tumors associated with the amplification of the genes identified herein include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides. antisense and ribozyme molecules, triple helix molecules, etc that inhibit the expression and/or activity of the target gene product
- antisense RNA and RNA molecule act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation
- oligodeoxy ⁇ bonucleotides derived from the translation initiation site e g between about -10 and +10 positions of the target gene nucleotide sequence
- Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA
- Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biolom 4 469-471 ( 1994), and PCT deviscation 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 pu ⁇ nes or py ⁇ midines on one strand of a duplex For further details see, e g PCT publication No WO 97 "3551, supra These molecules can be identified by any or any combination of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art
- 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 l Polyclonal Antibodies
- polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections
- the immunizing agent may include the PRO201, PR0292,
- PRO509, PR0853 or PR0882 polypeptideor a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized
- immunogenic proteins include but are not limited to keyhole limpet hemocyanin. serum albumin, bovine thvroglobulm.
- adjuvants examples include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate)
- MPL-TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate
- the immunization protocol may be selected by one skilled in the art without undue experimentation 11 Monoclonal Antibodies The ant ⁇ -PRO201 , ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265, ant ⁇ -PR0344, ant ⁇ -PR0343 anti-
- ant ⁇ -PRO1017, anti-PROl 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or anti- PR0882 antibodies may, alternatively, be monoclonal antibodies
- Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstem, 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 PRO201, PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof
- 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 [Godmg, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp 59-103]
- Immortalizedcell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, Dovme and human origin Usually, rat or mouse myeloma cell lines are employed
- Preferred immortalizedcell 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 immortalizedcell lines are mu ⁇ ne 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 , J_33 3001 ( 1984) Brodeur et al , Monoclonal Antibodv Production Techniques and Applications, Marcel Dekker, Inc , New York, ( 1987) pp 51-63]
- the culture medium in which the hybndoma ceils are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO20 I .
- the binding specificityof monoclonal antibodies produced by the hybridoma cells is determined by lmmunoprecipitation 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 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 lmmunoglobulinpu ⁇ fication 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 heavy and light chains of mu ⁇ ne antibodies)
- the hybridoma cells of the invention serve as a preferred source of such DNA
- the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myelom
- the antibodies may be monovalentantibodies
- Methods for preparing m -ovalentantibodiesarewell known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinkmg Alternatively, the relevant cysteine r e sidues are substituted with another amino acid residue or are deleted so as to prevent crosslinkmg
- ant ⁇ -PRO201 The ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265.
- ant ⁇ -PRO1017 The ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265.
- ant ⁇ -PRO1017 The ant ⁇ -PRO201, ant ⁇ -PR0292, ant ⁇ -PR0327, anti-PRO 1265.
- ant ⁇ -PRO1017 The ant ⁇ -PRO201, ant ⁇ -PR02
- anti-PROl 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or anti- PR0882 antibodies may further comprise humanized antibodies or human antibodies
- Humanized forms of non-human (e g , mu ⁇ ne) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin
- Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances.
- CDR complementary determining region
- Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences
- the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence
- the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al , Nature 321 522-525 ( 1986), Riechmann et al , Nature 332 323-329 ( 1988), and Presta, Curr Op Struct Biol , 2 593-596 (1992)]
- 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 a! . Nature.
- humanized antibodies are chimeric ant ⁇ bod ⁇ es(U S Patent No 4.816.567), wherein substantially le_s than an intact human variable domain has been substituted by the correspondmgsequencefrom a non-human species
- humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
- Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol B ol , 221 381 ( 19? 1 ), Marks et al , J Mol ⁇ /o/ , 222 581 (1991)]
- the techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner et al , J Immunol , 147( 1 ) 86-95 (1991 )]
- human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for
- the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activatmg enzyme which converts a prodrug (e g a peptidyl chemotherapeutic agent, see WO 81/01145) to an active anti-cancer drug See, for example. WO 88/07378 and U S Patent No 4,975,278
- a prodrug e g a peptidyl chemotherapeutic agent, see WO 81/01145
- the enzyme component of the immunoconjugate useful for ADEPT includes any enzvme capable of acting on a prodrug in such as way so as to convert it into its more active, cytotoxic form
- Enzymes that are useful in the method of this invention include, but are not limited to, glycosidase, glucose oxidase. human lysosyme, human glucuronidase, alkaline phosphatase usefulf or converting phosphate-contain ingprodrugs into free drugs, arylsulfataseuseful for converting sulfate-containing prodrugs into free drugs, cytosine deaminase useful for converting non-toxic 5-fluorocytos ⁇ ne into the anti-cancer drug 5-fluorourac ⁇ l, proteases, such as serratia protease, thermolysin, subtilism, carboxypeptidases (e g , carboxypeptidase G2 and carboxypeptidase A) and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs, D-alanylcarboxypeptidases.
- carbohydrate-cleavingenzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs
- ⁇ -lactamase useful for converting drugs de ⁇ vatized with ⁇ -lactams into free drugs
- penicillin amidases such as penicillin Vamidase or penicillin G amidase, useful for converting drugs de ⁇ vatized at their amine nitrogens with phenoxvacetyl or phenylacetyl groups, respectively, into free drugs
- antibodies with enzvmatic 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 , Massev, Nature 328 457-458 (1987))
- Antibody -abzymeconjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population
- the enzymes of this invention can be covalently bound to the ant ⁇ -PRO201 , ant ⁇ -PR0292, anti- PR0327, anti-PRO 1265, ant ⁇ -PR0344, ant ⁇ -PR03 ⁇ i3, ant ⁇ -PR0347, ant ⁇ -PR0357, ant ⁇ -PR0715, anti- PRO 1017, anti-PRO 1 1 12, ant ⁇ -PRO509, ant ⁇ -PR0853 or ant ⁇ -PR0882 antibodies by techniques well known in the art such as the use of the heterobifunctionalcross-linkingagents 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 ' 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
- PR0853 or PR0882, 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 often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, EMBO J., 10: 3655-3659 (1991).
- Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.
- the fusion preferably is with an immunoglobulinheavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH 1 ) containing the site necessary for light-chain binding present in at least one of the fusions.
- DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
- DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
- the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
- the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
- one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
- Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
- Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecfic antibodies can be prepared can be prepared using chemical linkeage. Brennan et al, Science 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
- TAB thionitrobenzoate
- One of the Fab'-TNB derivatives is then reconverted to the Fab -thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody
- the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes
- Fab' fragments may be directly recovered from E coli and chemically coupled to form bispecific antibodies Shalaby e. ⁇ / J Exp Med 175 217-225 ( 1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule Each Fab' fragment was separately secreted from E coli and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressmg the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol 148(5) 1547- 1553 (1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion The antibody homod
- ⁇ / 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 (V ⁇ ,) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-bindingsites
- sFv single-chain Fv
- t ⁇ specific antibodies can be prepared Tutt e/ ⁇ / J Immui ' 147 60 (1991)
- Exemplary bispecific antibodies may bind to two different epitopes on a given "Pro' protein herein
- an ant ⁇ -"PRO” protein arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecuoe (e g CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular "PRO" protein
- Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular "PRO" polypeptide
- a radionuc de chelator such as EOTUBE, DPTA, DOTA, or TETA
- Another bispecific antibody of interest binds the "PRO
- the antibody of the invention may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example
- cysteine res ⁇ due(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region
- the homodime ⁇ c antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al ,
- the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin), or a radioactive isotope (t 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 (t e , a radioconjugate)
- Enzymatically active protein toxins and fragments thereof which can be used include diphtheria A chain, nonbindmg active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonas aerugmos ⁇ ), ⁇ cin A chain, ab ⁇ n A chain, modeccin A chain, alpha-sarcin, Aleurttes fordu proteins, dianthm proteins, Phvtolaca amencana proteins (PAPI, PAPII, and PAP-S).
- Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succ ⁇ n ⁇ m ⁇ dyI-3-(2-py ⁇ dyld ⁇ th ⁇ ol) propionate (SPDP), lminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccmimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamme), bis-diazonium derivatives (such as b ⁇ s-(p-d ⁇ azon ⁇ umbenzoyl)- ethylened ⁇ amme),d ⁇ socyanates(such as tolyene 2,6-dnsocyanate), and bis-active fluorine compounds (such as 1 ,5-d ⁇ fluoro-2,4-d ⁇ n ⁇ tro
- the antibody may be conjugated to a "receptor” (such streptavidm) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e g avidin) which is conjugated to a cytotoxic agent (e g a radionucleotide) ix Immunoliposomes
- a "receptor” such streptavidm
- a "ligand” e g avidin
- cytotoxic agent e g a radionucleotide
- the antibodiesdisclosedherein may also be formulatedas immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al , Proc Natl Acad Sci USA, S2 3688 ( 1985), Hwang et al , Proc Natl Acad S t USA, T ⁇ 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
- Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-de ⁇ vatized phosphatidylethanolamme (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 Doxorubicm) is optionally contained with the liposome See Gabizon et al . J National Cancer inst 81(19.1484 (1989. 10 Pharmaceutical Compositions
- Antibodies specifically binding the product of an amplified gene identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of tumors, including cancers, in the form of pharmaceutical compositions If the protein encoded by the amplified gene is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred However, 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 uj on the variable region sequences of an antibody, peptide molecules can be designed whic ' retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e g Marascoe/ ⁇ / Proc Natl Acad Sci USA 90 7889-7893 [1993])
- Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed [1980]), in the form of lyophilized formulations or aqueous solutions
- Acceptable 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, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3- pentanol, and m-cresol),
- chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol.
- salt-formingcounter-ions such as sodium, metal complexes (e Zn- protein complexes), and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG)
- Non-antibody compounds identified by the screening assays of the present invention can be formulated in an analogous manner, using standard techniques well known in the art
- the formulation herein may also contain more than one active compound as necessary for the particularindication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively. or in addition, the composition may comprise a cytotoxic agent, cytokme or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the pu ⁇ ose intended
- the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsulesand poly-(mefhylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes. albumin microspheres. microemulsions. nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. ( 1980)
- the formulationsto be used for in V ⁇ administration must be sterile This is readily accomplished by filtration through sterile filtration membranes
- Sustained-releasepreparations may be prepared Suitable include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules
- sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vmylalcohol)), polylactides (U.S Pat No 3,773,9I 9),copolymersofL-glutam ⁇ cac ⁇ d and ⁇ ethyl-L-glutamate.
- non-degradable ethylene- vinyl acf rate degradable lactic acid-glyco c acid copolymers such as the LUPRON DEPOT TM .njectable microspheres composed of lactic acid-glycohc acid copolymer and leupro de acetate), and poly-D-(-)-3- hvdroxybuty ⁇ c acid
- polymers such as ethylene-vinyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days
- certain hydrogels release proteins for shorter time periods
- When encapsulated antibodies remain in the body for a long time they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity.
- Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions 11 Methods of Treatment
- the antibodies and other anti-tumor compounds of the present invention may be used to treat various conditions, including those characterized by overexpression and/or activation of the amplified genes identified herein Exemplary conditions or disorders to be treated with such antibodies and other compounds, including, but not limited to, small organic and inorganic molecules, peptides. antisense molecules. etc include benign or malignanttumors (e g renal, liver, kidnev, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, ling, vulval.
- benign or malignanttumors e g renal, liver, kidnev, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, ling, vulval.
- thyroid thyroid, hepatic carcinomas, sarcomas, glioblastomas.and various head and neck tumors
- leukemias and lymphoid malignancies other disorders such as neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoe c disorders, and inflammatory, angiogenic and immunologic disorders
- the anti-tumor agents of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, lntra-articular.intrasynovial. intrathecal, oral, topical, or inhalation routes Intravenous administration of the antibody is preferred
- chemotherapeutic agents may be administered to the patient Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' 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 ⁇ . nap ⁇ stone (see, EP 616812) in dosages known for such molecules
- the antibodies herein are co-administered with a growth inhibitory agent
- the growth inhibitory agent may be administered first, followed by an antibody of the present invention
- simultaneous administration or administration of the antibody of the present invention first is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the antibody herein
- an anti-tumor agent e g an antibody herein
- the appropriate dosage of an anti-tumor agent will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician
- the agent is suitably administered to the patient at one time or over a series of treatments
- ⁇ g/kg to 15 mg/kg (e g 0 1- 20mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations.or by continuous infusion
- a typical daily dosage might range from about 1 ⁇ g/kgto 100 mg/kg or more, depending on the factors mentioned above
- the treatment is sustained until a desired suppression of disease symptoms occurs
- other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays
- an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above comprises a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes
- the containers may be formed from a variety of materials such as glass or plastic
- the container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle)
- the active agent in the composition is usually an anti-tumor agent capable of interfering with the activity of a gene product identified herein, e g an antibody
- the label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice
- the article of manufacture may further comprise a second container comprising a 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 for use
- 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 for use
- antibodies directed against the proteins products of genes amplified in tumor cells can be used as tumor diagnostics or prognostics
- antibodies can be used to qu ⁇ . ⁇ tively or quantitatively detect the expression of proteins encoded by the amplified genes ("marker gene products")
- the antibody preferably is equipped with a detectable, e g fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluo ⁇ metry, or other techniques known ii the art These techniques are particularlysuitable, if the amplified gene encodes a cell surface protein, e g a growth factor
- binding assays are performed essentially as described in section 5 above
- tu detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy
- a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample This procedure also allows for determining the distribution of the marker gene product in the tissue examined It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in
- the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks Sambrook et al , Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press N Y , 1989, Ausubel et al , Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N Y , 1 89, Innis et al PCR Protocols A Guide to Methods and Applications, Academic Press, inc , N Y , 1990, Harlow et al , Antibodies A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor 1988, Gait, M J , Oligonucleotide Synthesis, IRL Press, Oxford, 1984, R I Freshney, Animal Cell Culture '987, Coligan et al , Current Protocols in Immunology, 1991
- PRQ347 PRQ357. PRQ715.
- PROl 017 PROl 1 12. PRO509. PRQ853 or PRQ882
- DNA381 13 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard , Manassas, V A d ' 10-2209, on December 10, 1997 and assigned ATCC Deposit No 209530
- EST Cluster No 86995 an EST cluster sequence from the LIFESEQ® database, designated EST Cluster No 86995 that was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homoiogies
- the homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , 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 w ith the program "phrap"
- One or more of the ESTs used in the assembly was derived from a cDNA iibrary prepared from RNA isolated from inflamed human adenoid tissue A consensus sequence was assembled, and
- the full length clone shown in Figure 7 contained a single open reading frame with an apparent translational initiation site at nucleotide positions 79-81 and ending at the stop codon found at nucleotide positions 1780- 1782 ( Figure 7, SEQ IDNO 7)
- SEQ 1D NO 8 is 567 amino acids long PRO 1265 has a calculated molecular weight of approximately 62,881 daltons and an estimatedpl of approximately 8 97 Additional features include a signal peptide sequence at about amino acids 1- 1 , potential N-glycosylation sites at about amino acids 54-57, 134-137. 220-223, and 559-562, and a region having amino acid sequence identity with D-ammo acid oxidase proteins at about ammo acids 61 -80
- ECD extracellulardoma ⁇ n sequences (includingthe secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases
- the EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ ® , Incyte Pharmaceuticals, Palo Alto.
- DNA40592 A consensus DNA sequence was assembled relative to other EST sequences using phrap The consensus DNA sequence was extended using repeated cycles of BLAST and phrap to extend the sequence as far as possible using the sources of EST sequences discussed above Using probes synthesized based upon the extended consensus sequence, a cDNA clone designated DNA40592 was isolated from a human fetal kidney tissue library
- Clone UNQ303 (DNA40592) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 227-229 and ending at the stop codon at nucleotide positions 956-958 ( Figure 9)
- the predicted polypeptide precursor is 243 amino acids long ( Figure 10)
- Important regions ofthe ammo acid sequence encoded by nucleotides 1 to 729 of PR0344 include the signal peptide, corresponding to amino acids 1-15, the start of the mature protein, corresponding to amino acids 16-67, and two potential N-myr ⁇ stoylat ⁇ ons ⁇ tes adj correspondingto amino acids 68-215 and 216- 243, respectively
- Clone DNA40592 (DNA40592-1242) was deposited with ATCC on November 21, 1997 and assigned ATCC deposit No 209492
- Clone UNQ302 (DNA43318) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 53-55 and ending at the stop codon at nucleotide positions 1004- 1006 ( Figure 1 1 ) The predicted polypeptide precursor is 317 amino acids long ( Figure 12) Various unique aspects of the PR0343 protein are shown in Figure 12 Clone UNQ302 (DNA43318-1217) was deposited with ATCC on November 21, 1997, and is assigned ATCC deposit no 209481
- a consensus sequence was determined Based on the consensus sequence, oligonucleotides were synthesized 1 ) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PR0347 RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (LIB228) The cDNA libraries used to isolate the cDN A clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA
- DNA44804 A consensus DNA sequence was assembled relative to other EST sequences using phrap Using probes synthesized based upon the consensus sequence, a cDNA clone designated DNA44804 was isolated from a human fetal liver library, and sequenced in its entirety The entire nucleotide sequence of DNA44804 is shown in Figure 15 (SEQ ID NO 15)
- DNA44804 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 137-139 and ending at the stop codon at nucleotide positions 1931-1933
- the predictedpolypeptideprecurso ⁇ s 598 amino acids long Figure 16.
- SEQ ID NO 16 Clone DNA44804-1248 was deposited with ATCC on December 10, 1997 and is assigned ATCC deposit No 209527
- Figure 18 has an estimated molecular weight of about 27,433 daltons and a pi of about 9 85
- PR0715 is a novel tumor necrosis factor protein
- a vector containing DNA52722 (DNA52722-1229) was deposited with ATCC on January 7, 1998 and was assigned ATCC deposit no 209570
- the extracellulardomain (ECD) sequences(mclud ⁇ ngthe secretion signal, if any) of from about 950 known secreted proteins horn the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases following the ECD homology search described above
- the EST databases included public EST databases (e g , GenBank, Merck/Wash U) and a proprietary EST DNA database (LIFESEQ ® ,
- a bacteriophage library of human retinal cDNA (commercially available from Clontech) was screened by hybridization with a synthetic oligonucleotide probe based on an EST sequence (GenBank locus AA021617).
- the cDNA inserts from three ofthe five bacte ⁇ ophageclones were excised from the vector arms by digestion with EcoRI, gel-pu ⁇ fied,and subcloned into pRK5 and sequenced on both strands
- the three clones contained an identical open reading frame (with the exception of an intron found in one ofthe clones)
- the pasicted amino acid sequence ofthe full length HVEM contains 283 amino acHs (See Fig 24, SEQ ID NO 24)
- a putative transmembrane region of the HVEM comprises amino acids 201- 225 of Fig 24 and a putative cytoplasmic region ofthe HVEM comprises amino acids 226-283 of Fig 24
- the sequence differs from the HVEM sequence reported in Montgomery et al , supra, in at least two amino acids as shown in Fig 26, codon 108 encodes a serine and codon 140 encodes an alanine
- An alignment (using the AlignTM imputer program) of a 58 amino acid long cytoplasmic region of HVEM with other known members of the human TNF receptor family showed some sequence similarity, in particular to CD40 ( 12 identities) and LT-beta receptor ( 1 1 identities)
- RNA for construction ofthe cDNA libraries was isolated from human fetal kidney tissue (LIB228)
- the cDNA libraries used to isolate the cDNA clones were contracted by standard methods using commercially available reagents such as those from Invitrogen. San Diego, CA
- PR0882 (UNQ448) is identical with cardiotrophin- 1
- the amino acid sequence of this 201 aminoa ⁇ d protein is present in the public Dayhoff database under Accession Nos P R83967. P W29238 and CTF1_HUMAN. among others, and is shown in Figure 28
- the nucleotide of the DNA encoding the PR0882 (UNQ448) is shown in Figure 29 (SEQ ID NO 29)
- Cardiotrophin- 1 has also been disclosed in WO9730146, published on 21 August 1997 and W09529237, published on 2 November 1995
- EXAMPLE 2 Gene Amplification This example shows that the PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-.
- PRO1017-, PROH 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng genes are amplified in the genome of certain human lung, colon and/or breast cancers and/or cell lines
- Amplification is associated with overexpression of the gene product, indicating that the binding specificities for at least two different rtigens In the present case, one ofthe binding specificities is for the PRO201.
- PRO509, PR0853 or PR0882 proteins are useful targets for therapeutic intervention in certain cancers such as colon, lung, breast and other cancers
- Therapeutic agent may take the form of antagomstsof binding specificifcs for at least two different antigens In the present case, one of the binding specificities is for the PRO201 -, PR0292-, PR0327- , PRO 1265-, PR0344-, PR0343-.
- PR0347-, PR0357-, PR0715-, PRO 1017-, PRO 1 1 12-, PRO509-, PR0853- or PR0882-encodmg genes for example, mu ⁇ ne-human chimeric, humanized or human antibodies against a binding specificities for at least two different antigens
- one ofthe binding specificities is for the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 polypeptide
- the starting material for the screen was genomic DNA isolated from a variety cancers
- the DNA is quantitated precisely, e g fluoromet ⁇ cally As a negative control.
- DNA was isolated from the cells often normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (not shown)
- the 5' nuclease assay for example, TaqManTM
- real-time quantitative PCR for example, ABI P ⁇ zm 7700 Sequence Detection SystemTM (Perkin Elmer.
- PRO509, PR0853 or PR0882 is over-represented in any ofthe 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 rumors ofthe type and stage as indicated in Table 1
- An explanation of the abbreviations used for the designation ofthe primary tumors sted in Table 1 and the primary tumors and cell lines referred to throughout this example has been given hereinbefore
- the results ofthe TaqmanTM are reported in delta ( ⁇ ) CT units
- One unit corresponds 1 PCR cycle or approximately a 2-fold amplification relative to normal, two units corresponds to 4-fold, 3 units to 8-fold amplification and so on Quantitation was obtained using pnmers and a TaqmanTM fluorescent prove derived from the binding specificities for at least two different antigens
- one of the binding specificities is for the PRO201-, PR0292-, PR0327-, PROl 265-, PR0344-, PR0343-, PR0347-, PR0357-, PR0715-, PRO10 I 7-, PROH 12-, PRO509-, PR0853- or PR0882-encod ⁇ ng gene Regions of binding specificities for at least two different antigens
- one ofthe binding specificities is for the PRO201 , PR0292, PR0327.
- PRO 1265 PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 1 12, PRO509, PR0853 or PR0882 which are most likely to contain unique nucleic acid seouences and which are least likely to have spliced out introns are preferred for the primer and probe derivation, e g 3-untranslated region
- the sequences for the primers and probes (forward, reverse and probe) used for the PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PRO 1 , ' 2, PRO509, PR0853 or PR0882 gene amplification were as follows PRO201 (DNA306761 30676 tm f
- PRQ292 (DNA35617) 35617 tm f 5'-GATCCTGGG CGACGTCTTC-3' (SEQ ID NO 35)
- PRQ327 (DNA381 13) 381 13 tm f 5'-CTCAAGAAGCACG CGTACTGC-3' (SEQ ID NO 38) 381 13.tm.p
- PRQ344 (DNA40592): 40592.tm.fl
- PRQ343 (DNA43318): 43318.tm.fl
- PRQ357 (DNA44804.: 44804.tm.fl 5'-CCTCGGTCTCCTCATCTGTGA-3' (SEQ ID NO: 53)
- PRO 1 1 12 (DNA57702): 57702.tm.fl
- PRO509 (DNA50148.: 50148.tm.fl 5'-GGAGGAGACAATACCCTCATTCA-3' (SEQ ID NO: 65)
- the 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor amplification in real time
- Two oligonucleotidep ⁇ mers are used to generate an ampiicon typical of a PCR reaction
- a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers
- the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe
- the TAQ DNA polymerase enzyme cleaves the probe in a template-dependent manner
- the resultant probe fragments disassociate in solution, and signal from 'he released reporter dye is free from the quenching effect of the second fluorophore
- the system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer
- the system amplifies samples in a 96-well format on a thermocycler During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD
- the system includes software for 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
- the ⁇ Ct values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer DNA results to normal human DNA results
- Table 1 describes the stage, T stage and N stage of various primary tumors which were used to screen the PRO201 , PR0292, PR0327. PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12.
- PRO509, PR0853 or PR0882 compounds ofthe invention Table 1 Primary Lung and Colon Tumor Profiles
- DNA was prepared from cultured cell lines, primary tumors, normal human blood The isolation was performed using purification kit, buffer set and protease and all from Quiagen. according to the manufacturer's instructions and the description below
- Buffer Cl (10 mL, 4°C) and ddH2O (40 mL, 4°C ) were then added to the 10 mL of cell suspension, mixed by inverting and incubated on ice for 10 minutes
- the cell nuclei were pelleted by cent ⁇ fuging in a Beckman swinging bucket rotor at 2500 ⁇ m at 4°C for 15 minutes The supernatant was discarded and the
- nuclei were suspended with a vortex into 2 mL Buffer C 1 (at 4°C) and 6 mL ddH 2 0, followed by a second 4°C cent ⁇ fugation at 2500 ⁇ m for 15 minutes
- the nuclei were then resuspended into the residual buffer using 200 ⁇ l per tip G2 buffer ( 10 ml) was added to the suspended nuclei while gentle vortexing was applied Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds Quiagen protease (200 ⁇ l, prepared as indicated above) was added and incubated at 50 D C for 60 minutes The incubation and
- DNA levels in each tube were quantified by standard A260, A280 spectrophotometry on a 1 20 dilution (5 ⁇ l DNA + 95 ⁇ l ddH 2 0) using the 0 1 ml quartz cuvetts in the Beckman DU640 spectrophotometer A260/A280 ratios were in the range of 1 8- 1 9
- Each DNA samples was then diluted further to approximately 200 ng/ml in TE (pH 8 5) If the original material was highly concentrated (about 700 ng/ ⁇ l), the material was placed at 50°C for several hours until resuspended
- Fluoromet ⁇ c D A quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidehnesas modified below This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes The Hoechst dye working solution (#H33258, 10 ⁇ l. prepared within 12 hours of use) was diluted into 100 ml 1 x TNE buffer A 2 ml cuvette was filled with the fluorometer solution, placed into the machine, and the machine was zeroed pGEM 3Zf(+) (2 ⁇ l.
- PRQ201 PRQ327 and PRO 1265
- PRO201 (UNQ 175) ( DNA10676).
- PR0327 (UNQ288)(DNA381 13) and PR01265 (UNQ636XDNA60764) were also reexamined along with selected tumors from the above initial screen with framework mapping Figure 32 and Table 3 indicate the chromosomal mapping of the framework markers that were used in the present example
- the tramework markers are located approximately every 20 megabases and were used to control aneuploidy
- PRO201 (UNQ175)(DNA30676), PR0327 (UNQ288)(DNA381 13) and PR01265 (UNQ636)(DNA60764) were also reexamined with epicenter mapping
- the markers indicated in Tables 4A, 4B and 4Care located in close proximity (in the genome) to DNA30676.
- the distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers.
- cR centirays
- SHGC-35441 is the marker found to be the closest to the location on chromosome 19 where DNA30676 maps, is closest to DNA381 13 and SHGC-33698 is closest to DNA60764
- Table 6 6 land 6 2 indicate the Ct values for results of epicenter mapping relative to DNA30676 DNA381 11 and DNA60764 respectivelv indicating the relative amplification in the region more immediate to the actual location of DN A30676 DN A381 11 and DNA670764 along chromosome 19
- Table 6 2 indicates the Ct ⁇ alues for the results of epicenter mapping relative to DNA60764, indicating relative amplification in the region more immediate to the actual location of DNA60764 along chromosome 19
- DNA34151, DNA40620 and DNA54002 are other independently identified molecules which have been observed to map to to the same region of chromosome 1 as DNA60764
- PR0292 (UNQ266) (DNA35617) was also examined with framework mapping Figure 33 and Table 7 indicate the chromosomal mapping of the framework markers that were used in this analysis
- the framework markers are located approximately every 20 megabases and were used to control aneuploidy
- PR0343 (UNQ302) (DNA433 I 8) and PR0882 (UNQ448) (DNA58125) were also examined with both framework and epicenter mapping Figure 34 and Table 9 indicate the chromosomal mapping of the framework markers that were used in this analysis
- the framework markers are located approximately every 20 megabases and were used to control aneuploidy
- Tables 9 1 and 9 2 in indicate the epicenter markers used tor the mapping of DNA43318 and DNA58125
- the markers shown in Tables 9 1 and 9 2 are located in close proximity (in the genome) to DNAs DNA43 18 and DNA58125 respectively, and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein the respective molecules map
- the distance between individual markers in measured in centiravs (cR) which is a radiation breakage unit roughly equal to a 17r chance of a breakage between iwo markers
- One cR is very roughly equn alent to 20 kilobases.
- Table 10.1 and 10.2 indicate the ⁇ Ct values from epicenter mapping relative to DNA43 18 and DNA58125, respectively, indicating the relative amplification in the region more immediate to the actual location of the respective molecules along chromosome 16.
- PRO 1017 (UNQ500) (DNA561 12) was also examined with framework mapping.
- Figure 35 and Table 1 1 indicate the chromosomal mapping of the framework markers that were used in this analysis.
- the framework markers are located approximately every 20 megabases and were used to control aneuploidy.
- PROl 017 (UNQ500)(DNA561 12) was also examined with epicenter mapping.
- Table 1 1.1 indicates the epicenter markers which are located in close proximity to DNA561 12 which were employed to assess the relative amplification in the immediate vicinity of chromosome 7 wherein DNA56112 is located. The distance between individual markers is measured in centirays (cR), which is a radiation breakage unit approximately equal to a 1% chance of a breakage between two markers. One cR is very roughly equivalent to 20 kilobases.
- the marker SHGC-22698 is the marker found to be the closest to the location on chromosome 7 where DNA561 12 maps.
- Table 1 1 Framework Marker used on Chromosome 7 for DNA 561 12
- Table 1 1 2 indicated the DCt values for the results of epicenter mapping relative to DNA561 12, indicating the relative amplification in the region more immediate to the actual location of DNA561 12 along chromosome 7
- PR0715 (UNQ383) (DNA52722) and PR0853 (UNQ419) (DNA48227) were also reexamined with both framework and epicenter mapping
- Figures 16A and 36B and Table 13 indicate the chromosomal localizations of the framework markers that were used for the procedure
- the framework markers are located approximately every 20 bases and were used to control aneuploidy
- Tables 14A and 14B indicate the epicenter mapping markers that were used in the procedure
- the epicenter markers were located in close proximity to DNA52722 and DNA48226, respectively, and are used to determine the relative DNA amplification in the immediate vicinity of DNA52722 and DNA48226
- the distance between individual markers is measured in centirays, which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between two markers
- One cR is very roughly equivalent to about 20 kilobases
- "BAC" means bacterial artificial chromosome The ends of a BAC clone which contained the gene of interest were sequenced
- Table 16 indicates the ⁇ Ct values o the above described framework markers along chromosome 17 relative to DNA52722 and DNA48227 for selected tumors. While not shown, the similar ⁇ Ct values for the framework markers in the analysis of DNA48227 were reported.
- Table 18 indicates the ⁇ Ct values for the indicated epicenter markers indicating the relative amplification along chromosome 17 in the immediate vicinity of DNA52722
- Tables 18A and 18B indicate the ⁇ Ct values for the indicated epicenter markers, indicating the relative amplification of selected lung and colon tumors, respectively, along chromosome 17 in the immediate vicinity of DNA48227.
- PR0357 (UNQ324, DNA44804) was reexamined with selected tumors from the above initial screen with framework mapping Figure 40 and Table 19 indicate the chromosomal mapping of the framework markers thai were used in the present example
- the framework markers are located approximately every 20 megabases and were used to control aneuploidv
- PR0357 (UNQ124 DNA44804) was also examined with epicenter mapping
- the markers indicated in Table 20 are located in close proximity (in the genome) to DNA44804 and are used to assess the relative amplification in the immediate vicinity of Chromosome 16 wherein DNA44804 is located
- the distance between individual markers is measured in centirays (cR). which is a radiation breakage unit approximately equal to a 1 % chance of a breakage between the two markers
- cR centirays
- SHGC-6154 is the marker tound to be the closest to the location on chromosome 16 where DNA44804 maps
- Table 22 indicates the ⁇ Ct values for the results of epicenter mapping relative to DNA44804. indicating the relative amplification in the region more immediate to the actual location of DNA44804 along chromosome 16.
- DNA30676 is the gene responsible for the amplification of the particular region on Chromosome 19. Because amplification of DNA30676 occurs in various lung and colon tumors and cell lines (especially colon), it is highly probable to play a significant role in tumor formation or growth As a result, antagonists (e g., antibodies) directed against the protein encoded by DNA30676 (PRO201 ) would be expected to have utility in cancer therapy.
- the ⁇ Ct and average ⁇ Ct values for these hits are- ( 1 ) primary lung tumors: 1 35, 1 93, 2 15, 2.33, 1.42, (2) primary colon tumors: 3.54, 1.52, 1.63, 1.58, (3) lung tumor cell lines: 1.13, 1.08; and (4) colon tumor cell lines.
- Amplification of DNA35617 was also confirmed by framework mapping Table 8 indicates that significant amplification of DNA35617 was confirmed in LT12, LT13, LT15, LT16 and CT2, CT8, CT10 and CT14.
- the reported ⁇ Ct values for the primary tumors were 1.61 , 1.95, 2 86 and 1 68, while for the primary colon tumors the values were 3.75, 1.76, 1.68 and 1 75 These represent a 3.05, 3.86, 7 26, 3.20- fold increase m gene copy for the lung tumors and a 13 45, 3 39, 3 20 and 3.36-fold increase in gene copy for the colon tumors
- the amplification of the closest known Iramework markers does not occur to a greater extent that that of DNA356 I 7 This strongly suggests that DNA35617 is the gene responsible for the amplification of the particular region on chromosome 17.
- DNA35617 Because amplification of DNA35617 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth. As a result, antagonist (e.g.. antibodies) directed against the protein encoded by DNA35617 would be expected to have utility in cancer therapy.
- antagonist e.g.. antibodies directed against the protein encoded by DNA35617 would be expected to have utility in cancer therapy.
- Table 2 indicates that significant amplification of DNA381 13 occurred in primary lung tumors: SRC734 (LT12), SRC735 (LT13), SRC736 (LT15), SRC737 (LT16), SRC738 (LT17), SRC740 (LT19); primary colon tumors: SRC742 (CT2), SRC743 (CT3), SRC744 (CT8), SRC745 (CT10), SRC746 (CT12), SRC747 (CT14), SRC748 (CT15), SRC749 (CT16), SRC750 (CT17), SRC751 (CTI ), SRC752 (CT4), SRC753 (CT5), SRC754 (CT6), SRC756 (CT9), SRC757 (CTI 1 ), SRC758
- CT18 lung tumor cell lines SRC771 (H157), SRC772 (H441 ), SRC773 (H460), SRC774 (SKMES-1 ), SRC832 (H522), SRC833 (H810); colon tumor cell lines: SRC777 (SW620), SRC778 (Colo320), SRC830 (HCC2998), SRC831 (KM 12).
- the ⁇ Ct values and average ⁇ Ct values for the primary lung tumor hits are: 2.57, 2.09, 3 44, 1.91 , 1.43, 3.58; primary colon tumors are: 3.64, 1.12, 1.64, 2.53, 1.88, 2.51 , 1.30, 1.47, 1.71 , 1.37, 2.22, 2.50, • 2.28, 1.58, 3.33 and 1.07; lung tumor cell lines 1.65, 2.23, 1.12, 1.18, 1.02 and 1.1 ; and colon tumor cell lines 2.24, 1.01 , 1.23 and 1.61.
- Amplification has been confirmed by framework mapping for DNA381 13 in primary lung tumors LT10 and in primary colon tumors CT2, CT3, CT8, CT10, CT12, CT14, CT16.
- the reported ⁇ Ct values for the primary lung tumor is 1.45; the primary colon tumors are 2 94, 1.23, 1 45, 1.72, 1.60, 2.03 and 1.07. Relative to normal tissue, this represents approximately a 2.73-fold increase for the lung tumor and a 7.67, 2.34, 2.73, 3.29, 3.03, 4.08 and 2.10-fold increase for the colon tumors.
- Epicenter mapping resulted in confirmation of significant amplification in: primary lung tumors LT12, LT13, LT15, LT16, LT17 and in primary colon tumors CTI , CT2, CT3, CT4, CT5, CT6, CT8, CT9, CT10, CTI 1 , CT12, CT14, CT16.
- the reported ⁇ Ct values for the primary lung tumors are: 1.57. 1.22, 2.47, 2.23, 1.02 and for the primary colon tumors are 1.38, 2.94, 1.23, 2.23, 2.51 , 2.29, 1.45. 1 59, 1.72, 3.34, 1.6, 2.03, 1.07 and 1.08. Relative to normal tissue, this represents a 2.97, 2.33, 5.54. 4 69.
- DNA381 13 is the gene which is driving the amplification of this particular region of Chromosome 19
- the amplification of marker S41 (which does not map closely to DNA381 13) could be an independent amplification event or even an error in the ordering of the markers
- antagonists e g , antibodies directed against the protein encoded by DNA381 13 (PR0327) would be expected to have utility in cancer therapy.
- PRO 1265 The ⁇ Ct values for DNA60764 (PR01265)(UNQ636) in a variety of lung tumors are reported in
- Table 2 A ⁇ Ct value of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
- Table 2 indicates that significant amplification of DNA60765 occurred in primary lung tumors LT3, LT12, LT13. LT15, LT16 and LT17 The ⁇ Ct values of these hits are 1.03, 2.17, 2.24, 3.51, 3.32 and 1.02 This represents an increase in gene copy of approximately 2.04, 4.50, 4.72, 11.39, 9.99 and 2.03
- Amplification has also been confirmed framework mapping for DNA60764 in LT16.
- the reported ⁇ Ct value was 1.37, which represents a 2.58 fold increase in gene copy relative to normal tissue.
- Epicenter mapping has also confirmed amplification of DNA60764 in LT12, LT13, LT15, LT16, CTI, CT4, CT5, CT7 and CTI 1.
- These tumors report ⁇ Ct values of 2.35, 2.37, 3.88, 3.32 in the lung tumors and 1.74, 1.86, 3.28, 1.29 and 2.32 in the colon tumors. Relative to normal tissue, this represents an increase in gene copy of approximately 5.10, 5.17, 14.72 and 9 98 in the lung tumors and 3.34, 3.63, 9.71 , 2.45 and 4.99 in the colon tumors.
- DNA60764 is ghe gene responsible for the amplification of the particular region in Chromosome 19 Because amplification of DNA60764 occurs various lung and colon tumors, it is highly probably to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA60764 would be expected to have utility in cancer therapy.
- PRQ344 The ⁇ Ct values for DNA40592 (PR0344, UNQ303) in a variety of lung and colon tumors are reported in Table 2. A ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA40592 occurred in primary lung tumors LT13 and LT19, in primary colon tumors CT2. CT14, CT15. CTI, CT4, CT5, CT11. The ⁇ Ct values and average ⁇ Ct of these hits are 1.34, 1.52 in the lung tumors, and 1.84, 1.16, 1 17, 1.24, 1.46, 1.51 and 1.35. This represents approximately a 2 53, 2.87 and 3.58, 2.23, 2.25, 2.36,
- the ⁇ Ct values for DNA43 18 (PRO343)(UNQ302) in a variety of lung and colon tumors are reported in Table 2 A ⁇ Ct of >1 was typically used as the threshold value tor amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA43318 occurred in primary lung tumor: LT13, LT15, LT19, CT2, CT8, CT10. CT12, CT14, CT15, CT16. CT17 and CT5.
- the ⁇ Ct and average ⁇ Ct values for the primary lung tumor hits are 1.67, 1 47 and 1.66. while the colon tumor hits are 2.72, 1.43, 1.68, 1.33, 2.02, 1.73, 1.16, 1.5, 1.34. This represents an increase in gene copy of approximately 3.18. 2.77 and 3.16 -fold for the lung tumors and 6.59, 2.69, 3.20, 2.51 , 4.06, 3.31 , 2.23, 2.83 and 2.53 -fold for the colon tumors.
- Relative to normal tissue this represents about a 2.23, 3.29, 6.63, 2.75 and 2.08-fold increase in gene copy for the lung tumors, and about a 1 1.63, 2.25, 6.19, 2.71 , 2.57, 3.20, 2.10 and 2.01-fold increase for the colon tumors.
- Epicenter mapping for DNA43318 confirmed significant amplification in LT12, LT13, LT15, LT16, CT4, CT5, CT6, CTI 1 and CT2.
- the reported ⁇ Ct values are 1.32, 1.94, 3.07, 1 83, 1.02, 2.40, 3.78, 1.51 and 2.48.
- Relative to normal tissue this represents a 2.50, 3.84, 8.40, 3.56-fold increase in the lung tumors and a 2.03, 5.28, 13.74, 2.85, 5 58-fold increase in gene copy for the colon tumors.
- DNA43318 is the gene responsible for the amplification of the particular region of Chromosome 16. Because amplification of DNA43318 occurs in various lung and colon tumors, it is highly probably to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA43318 would be expected to have utility in cancer therapy.
- the ⁇ Ct values for DNA44176 (PR0347, UNQ306) in a variety of lung and colon tumors are reported in Table 2.
- a ⁇ Ct of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
- Table 2 indicates that significant amplification of DNA44176 occurred in primary lung tumors LT15 and primary colon tumors CT2, CT8. CT14, CT15, CT16, CT5 and CTI 1.
- the ⁇ Ct values and average ⁇ Ct of these hits are 1.76 in the lung tumors, and 1.85, 1.12, 1.27, 1.07, 1.19, 1.80. 1.42 in the colon tumors This represents approximately a 3 .19. 3 61. 2 17. 2 41 , 2 10. 2.28. 1 48. 2 67-fold increase, respectively, in gene copy relative to normal tissue.
- DNA44176 Because amplification of DNA44176 occurs in various tumors, it is likely associated with tumor formation or growth. As a result, antagonists, (e g., antibodies) directed against the protein encoded by DNA44176 (PR0347) would be expected to be useful in cancer therapy
- ⁇ Ct values for DNA44804 (PR0357)(UNQ 14) in a variety of lung and colon tumor and cell lines are reported in Table 2
- a ⁇ Ct ot >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
- Table 2 indicates that significant amplification of DNA44804 occurred in primary lung tumors: LT9, LT10, LT1 1, LT12. LT13, LT15, LT16, LT17, LT19, LT21; in primary colon tumors CT2, CT8, CT10, CT14, CT15, CT16, CTI, CT4, CT5, CT6, CT7 and CTI 1.
- the ⁇ Ct and average ⁇ Ct values of the lung tumor hits are- 1.42, 1.63, 1 47, 1.42, 1.52, 2.18, 1.23, 1.71, 2.47. 1.66; and the colon tumor hits are 2.01 , 1.1, 1.42, 1.5, 1.25, 1.05, 1.04, 1.27, 1.69, 1.08, 1.73, 1.83.
- DNA44804 is the gene responsible for the amplification of the particular region on chromosome 16. Because amplification of DNA44804 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth . As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA44804 would be expected to have utility in cancer therapy.
- ⁇ Ct values for DNA52722 (PR0715)(UNQ383) in a variety of lung and colon tumor and cell lines are reported in Table 2.
- a ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
- Table 2 indicates that significant amplification of DNA52722 occurred in LT12, LT13.
- the ⁇ Ct and average ⁇ Ct values for the primary lung tumors are 1.29, 1.51 , 1.35, 1.22 and 1.55, while the primary colon tumors report values of 1.64, 1.12, 1 32. 1 97, 1 42. 1 53. 1 28. 1 31. 1 32. 1.59, 1.92. 1 41. 1 23. 2 29. 1.05 Relative to normal tissue, these represent 2 45, 2 85. 2 55. 2.33, 2 93-fold increase in gene copy tor the lung tumors and 3 12, 2 17. 2.50. 3 92, 2 68. 2 89. 2.42, 2 48, 2.50, 3 01 , 3 78. 2 69. 2 34.
- the reported ⁇ Ct values for the primary lung tumors were 1.07, 1.29, 1.33, 1.1 1 , 1.13, 1.21 , while the colon tumors indicate 1.22, 1.19, 1.26, 1.79, 1.71 , 1.19, 1.86, 2.29, 1.05. 1.27, 1.03 and 1.06. Relative to normal tissue, this represents a 2.10, 2 45, 2.51 , 2.16, 2.19, 2.31 -fold increase in the lung tumors and a 2.33, 2.28, 2.39, 3.46, 3.27, 2.28, 3.63, 4.89, 2.07, 2.41 , 2.04 and 2.08-fold increase in the colon tumors.
- DNA52722 is the gene responsible for the amplification of the particular region on Chromosome 17. Because amplification of DNA52722 occurs in various lung and colon tumors and cell lines, it is highly probably to play a significant role in tumor formation or growth. As a result, antagonists (e.g., antibodies) directed against the protein encoded by DNA52722 (PR0715) would be expected to have utility in cancer therapy.
- PRO1017 The ⁇ Ct values for DNA561 12 (PRO1017. UNQ500) in a variety of lung and colon tumors are reported in Table 2. A ⁇ Ct value of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy. Table 2 indicates that significant amplification of DNA561 12 occurred in: primary lung tumors.
- ⁇ Ct and average ⁇ Ct values for ( 1 ) the primary lung tumor hits are: 1.78, 2.37, 2.79, 1.1 1 , 2.73, 3.51 , 1.96, 2.20, 1.90. 2.24, 1.78, 1.76, 1.24 and 2.24; and (2) primary colon tumor hits are.
- LT4, LT7, LT9 LT10 LTI I LT12 LTI I LT15 LT16 LT18 and LT22 The reported ⁇ Ct values are 1 21 , 1 59 1 40, 1 07 1 98 1 15. 2 11 , 1 81, 2 79 2 22, 1 06 and 1 18 These values represent a 2 11, 3 01 , 2 64, 2 10, 1 95 2 22 4 96 3 56 6 92. 4 66 2 08 and 2 27-fold increase respectively, in gene copy for the indicated lung tumors
- CT16 and CT17 The ⁇ Ct values for the primary lung tumors are 1 47, 1 51 , 2 27, 1 62 and 1 03, and the primary colon tumors are 1 29, 1 46, 2 28, 1 84. 2 56. 1 22 and 1 52 These values represent a 2 77, 2 85, 4 82, 3 07, 2 04-fold increase, respectively, in the lung tumors and a 2 45. 2 75, 4 86, 3 58, 5 90, 2 33 and 2 87-fold increase, respectively, in the colon tumors
- DNA561 12 is the gene responsible for the amplification of the particular region on chromosome 17 Because amplification of DNA561 12 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth As a result, antagonist (e g , antibodies) directed against the protein encoded by DNA56112 would be expected to have utility in cancer therapy
- the ⁇ Ct values and average ⁇ Ct of these hits are 1 13, 1 52, 1 19, 1 63, 1 77 1 45 and 1 25 for the lung tumor and 2 26, 1 06, 1 57, 1 31 , 1 89, 1 46, 1 25, 1 24, 1 53, 1 97, 1 57 and 2 28 for the colon tumors This represents approximately a 2 19, 2 87, 2 28, 3 09, 3 41, 2 73, 2 38-fold amplification, respectively, for the lung tumor, and
- DNA57702 (PROl 1 12) would be expected to be useful in cancer therapy
- the ⁇ Ct values for DNA50148 (PRO509, UNQ329) in a variety of lung and colon tumors are reported in Table 2 A ⁇ Ct of >1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA50148 occurred in primary colon tumors CT15. CT17, CT6, CTI 1 and CT18 The ⁇ Ct values and average ⁇ Ct ot these hits are I 16. 1 02. 1 01. 1.34. 1.35. which represents a 2 23. 2 03. 2.01. 2.53, 2.55-fold increase, respectively, in gene copy relative to normal tissue.
- DNA50148 Because amplification of DNA50148 occurs in various tumors, it is likely associated with tumor formation or growth As a result, antagonists, (e.g., antibodies) directed against the protein encoded by DNA50148 (PRO509 would be expected to be useful in cancer therapy
- the ⁇ Ct values for DNA48227 (PR0853)(UNQ419) in a variety of lung and colon tumors and cell lines are reported in Table 2 A ⁇ Ct of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy.
- Table 2 indicates that significant amplification of DNA48227 occurred in primary lung tumors LT12 and lung cell line H522, as well as in primary colon tumors CT2, CT3, CT8.
- the ⁇ Ct and average ⁇ Ct values for these hits are 1.03, 1.02, 2.4, 1.52, 1.55, 1.97, 1.36, 1.75, 1.75, 1.1 1 , 1.1 1 ,
- DNA48227 is the gene responsible for the amplification of the particular region on chromosome 17. Because amplification of DNA47227 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA48227 would be expected to have utility in cancer therapy.
- PRQ882 The ⁇ Ct value for DNA58125 (DNA882, UNQ448) in a variety of lung and colon tumors and cell lines is reported in Table 2. A ⁇ Ct value of > 1 was typically used as the threshold value for amplification scoring, as this represents a doubling of gene copy Table 2 indicates that significant amplification of DNA58125 occurred in primary lung tumors: LTl a. LT3, LT6, LT9. LT10, LT1 1 , LT12, LT13, LT15, LT17, LT 19; primary colon tumors: CT2, CT3.
- lung tumor cell lines H441
- colon tumor cell lines SW620, Colo320, HT29, SKCOl , SW403, LS 147T, Colo205, HCT15, HCC2998 and KM 12.
- the ⁇ Ct and average ⁇ Ct values for the primary lung tumor hits are: 1.18, 1.26, 1.10, 1.81 , 1.95, 1 29, 1 07, 1.36, 1.58, 1.45, 1.87, the colon tumor hits report 2.18, 1 64, 1.10. 1 64. 1 05. 1 62. 1 25. 1.29. 1.17. 1.1. 2.03, 2 13. lung tumor cell lines-
- Amplification has been confirmed by framework mapping for DNA58125 in primary lung tumors LT3, LT13, LT15, LT17, LT18, CTI , CT2, CT3, CT4, CT5, CT6. CT8, CT10, CT12, CT14, CT15, CTI 8.
- the reported ⁇ Ct values for the primary lung tumors are 1.02, 1.33, 1.83, 1.03 and 1 08, while the primary colon tumors values are 1.08, 2.27, 1.34, 1.13, 2.17, 1.41 , 1.23, 1.74, 1.13, 1.74, 1.30 and 1.04. Relative to normal tissue, this represents approximately a 2.03, 2.51, 3.56, 2.04. 2.1 1 -fold increase, respectively, for the lung tumors, and a 2.1 1. 4.82, 2.53, 2.19, 4.50, 2.66, 2.34, 3.34, 2.19, 3.34, 2.46 and 2.06-fold increase, respectively, for the colon tumors.
- Epicenter mapping further confirmed significant amplification in primary colon tumors LT12,
- LT13, LT15, LT16, LT17 and in primary colon tumors CTI, CT4, CT6, CT7, CT9, CTI 1, CT2, CT8, CT10 and CTI 6.
- the reported ⁇ Ct values in the primary lung tumors are 1.02, 1.52, 2.04, 1.09 and 1.32, while the colon tumor values are 2.29, 1.49, 1.83, 1.20, 1.67, 1.02, 1.07, 2.27, 1.50 and 2.24 Relative to normal tissue, this represents approximately a 2.03, 2.87, 4.1 1, 2.13, 2.50-fold increase, respectively, in gene copy for the lung tumors and a 4.89, 2 81 , 3.56, 2.30, 3.18, 2.03, 2.10, 4.82, 2.83, 4.72-fold increase, respectively, for the colon tumors.
- DNA58125 is the gene responsible for the amplification of the particular region on chromosome 16. Because amplification ot DNA58125 occurs in various tumor tissues, especially colon tumors, it is highly probably to play a significant role in tumor formation or growth, in particular, colon tumor formation and growth.. As a result, antagonist (e.g., antibodies) directed against the protein encoded by DNA58125 would be expected to have utility in cancer therapy.
- In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences withm cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping In situ hybridization was performed following an optimized version of the protocol by
- the tubes were incubated at 37°C for one hour 1 0 ⁇ l RQ1 DNase were added, followed by incubation at 37 °C for 15 minutes 90 ⁇ l TE ( 10 mM T ⁇ s pH 7 6/l mM EDTA pH 8 0) were added, and the mixture was pipetted onto DE81 paper
- the remaining solution was loaded in a Microcon- 50 ultrafiltration unit, and spun using program 10 (6 minutes)
- the filtration unit was inverted over a second tube and spun using program 2 (3 minutes)
- 100 ⁇ l TE were added 1 ⁇ l of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II
- the probe was run on a TBE/urea gel 1-3 ⁇ l of the probe or 5 ⁇ l of RNA Mrk III were added to 3 ⁇ l of loading buffer After heating on a 95°C heat block for three minutes
- Hsbridization 1 0 10' cpm probe and 1 0 ⁇ l tRNA (50 mg/ml stock) per slide were heated at 95 °C for 1 minutes The slides were cooled on ice, and 48 ⁇ l hybridization buffer were added per slide After vortexing, 50 ⁇ l ''P mix were added to 50 ⁇ l prehybridization on slide The slides were incubated overnight at 55 C C
- Washes Washing was done 2x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0 25M EDTA V 4L), followed by RNaseA treatment at 37°C for 30 minutes (500 ⁇ l of 10 mg/ml in 250 ml Rnase buffer - 20 ⁇ g/ml), The slides were washed 2x10 minutes with 2 x SSC, EDTA at room temperature
- the stringency wash conditions were as follows 2 hours at 55 °C, 0 1 x SSC,
- DNA381 1 (Prolactin Rceptor Homlog)
- DNA52722 (TNF Homolog) dna52722p1 GGA TTC TAA TAC GAC TCA CTA TAG GGC CGC CCC GCC ACC TCC T dna52722p2 CTA TGA AAT TAA CCC TCA CTA AAG GGA CTC GAG ACA CCA CCT GAC CCA
- TCT 52722 p4 CTA TGA AAT TAA CCC TCA CTA AAG GGA CTA GGG GGT GGG AAT GAA
- AAG High expression levels were observed in many tissues, including placenta, osteoblasts. injured renal tubules, injured liver colorectal liver matastasis and gall bladder Tested sample had acetominophen induced liver injury and hepatic cirrhosis
- DNA48227 (Reductase Homolog) 607 p 1 GGA TTC TAA TAC GAC TCA CTA TAG GGC CCA ACA GCG GCA TCG G AA AGA 607.p2 CTA TGA AAT TAA CCC TCA CTA AAG GGA GGA GCA CCA GCC AAG CCA ATG
- the following method describes use of a nucleotide sequence encoding a PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 112, PRO509, PR0853 or PR0882 polypeptide as a hybridization probe
- DNA comprising the coding sequence of full-length or mature PRO201 (Fig.1, SEQ ID NO. 1), PR0292 (Fig. 3, SEQ ID NO 3), PR0327 (Fig 5, SEQ ID NO: 5), PR01265 (Fig 7, SEQ ID NO: 7), PR0344 (Fig. 9, SEQ ID NO 9), PR0343 (Fig 1 1, SEQ ID NO. 1 1), PR0347 (Fig. 13, SEQ ID NO: 13), PR0357 (Fig. 15, SEQ ID NO 15), PR0715 (Fig. 17, SEQ ID NO 17), PRO1017 (Fig. 19, SEQ ID NO: 19), PROl 1 12 (Fig. 21 , SEQ ID NO. 21), PRO509 (Fig. 23, SEQ ID NO. 23), PR0853 (Fig.
- SEQ ID NO: 25 is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347. PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in human tissue cDNA libraries or human tissue genomic libraries
- Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions
- Hybridization of radiolabeled PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882-de ⁇ ved probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0 1 % SDS, 0 1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42"C for 20 hours
- Washing of the filters is performed in an aqueous solution of 0 1 x SSC and 0 l% SDS at 42°C DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347,
- EXAMPLE 5 Expression of PRO201. PRQ292. PRQ327. PRQ1265, PRQ344, PRQ343, PRQ347. PRQ357, PRQ715, PRO1017. PROl 1 12. PRO509. PRQ853 or PRQ882 in E colt This example illustrates preparation ol an unglycosylated form of PRO201. PR0292 PR0327. PR01265. PR0344 PR0343, PR0347. PR0157. PR0715, PRO1017. PROl 1 12, PRO509. PR0851 or PR0882 by recombinant expression in E coli
- a variety of expression vectors mav be employed An example of a suitable vector is pBR322 (derived from £ coli, see Bolivar et al , Gene.
- the vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357. PR0715. PRO1017, PROl 112, PRO509, PR0853 or PR0882 coding region, lambda transcriptional terminator, and an argU gene
- the ligation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on
- the cells can be harvested by centrifugation
- the cell pellet obtained by the centrifugation can be solubi zed using various agents known m the art. and the solubihzed PRO201 , PR0292. PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein
- PRO201 Expression of PRO201. PRQ292. PRQ327, PRQ1265, PRQ344 PRQ343. PRQ347. PRQ357. PRQ715.
- the vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector
- PR0144, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 112, PRO509, PR0853 or PR0882 DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA using ligation methods such as described in Sambrook et al .
- the selected host cells may be 293 cells Human 293 cells (ATCC
- CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics.
- medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics.
- pRK5-PR0357, pRK5-PR0715, pRK-1017, pRK5-PR01 1 12., pRK5-PRO509, pRK5-PR0853, pRK5- PR0882 DNA is mixed with about 1 ⁇ g DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 ⁇ l of 1 mM T ⁇ s-HCl, 0.1 mM EDTA, 0.227 M CaCl, To this mixture is added, dropwise, 500 ⁇ l of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP0 4 , and a precipitate is allowed to form for 10 minutes at 25"C.
- the precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C.
- the culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds.
- the 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
- the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ⁇ Ci/ml 3, S-cyste ⁇ ne and 200 ⁇ Ci/ml "S-methionine.
- the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel.
- the processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO201, PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12.
- PRO509, PR0853 or PR0882 , or EBAF-2 polypeptide may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
- PR0344, PR0343
- PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 DNA may be introduced into 293 cells transiently using the dextran sullate method described by Somparyrac et al , Proc Natl. Acad. Sci..12:7575 (1981).
- 293 cells are grown to maximal density in a spinner flask and 700 ⁇ g pRK5-PRO201, pRK5-PR0292, pRK5-PR0327, pRK5-PR01265, pRK5-PR0344, pRK5-PR0343, pRK5-PR0347, pRK5-PR0357, pRK5-PR0715, pRK-1017, pRK5-PR01 1 12., pRK5-PRO509, pRK5- PR0853, pRK5-PR0882 DNA is added The cells are first concentrated from the spinner flask by centrifugation and washed with PBS.
- the DNA-dextran precipitate is incubated on the cell pellet for tour hours.
- the cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re- mtroduced into the spinner flask containing tissue culture medium, 5 ⁇ g/ml bovine insulin and 0.1 ⁇ g/ml bovine transfemn. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris.
- the sample containing expressed PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PRO l 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography
- PRO509. PR0853 or PR0882 can be expressed in CHO cells The pRK5-PRO201 , pRK5-PR0292, pRK5-PR0327. pRK5-PR01265. pRK5-PR0344. pRK5-PR0141. pRK5-PR0147. pRK5-PR0157. pRK5-PR07 15. pRK- 1017.
- pRK5-PR01 1 12 pRK5- PRO509, pRK5-PR0853.
- pRK5-PR0882 vector can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone ) or medium containing a radiolabel such as "S-methionine. After determining the presence of PRO201. PR0292. PR0327, PRO 1265. PR0344, PR0343. PR0347, PR0357, PR0715, PRO 1017. PRO l 1 12, PRO509, PR0853 or PR0882 polypeptide.
- the culture medium may be replaced with serum tree medium
- the cultures are incubated for about 6 days, and then the conditioned medium is harvested.
- PR0715, PRO1017, PRO l 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method.
- Epitope-tagged PRO201 , PR0292, PR0327, PROl 265, PR0344, PR0343, PR0347, PR0357, PR0715, PROl 017, PROl 1 12, PRO509, PR0853 or PR0882 may also be expressed in host CHO cells.
- PRO509, PR0853 or PR0882 may be subcloned out of the pRK5 vector.
- the subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-His tag into a Baculovirus expression vector.
- the poly-His tagged PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones.
- the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression.
- PROl 1 12, PRO509, PR0853 or PR0882 can then be concentrated and purified by any selected method, such as by Nr + -chelate affinity chromatography.
- PR0292 (UNQ266) and PRO509 (UNQ329) were expressed in CHO cells by a transient expression procedure
- PRO1017 (UNQ500) and PRO509 (UNQ329) were stably expressed in CHO cells.
- the proteins were expressed as an IgG construct (lmmunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins were iused to an IgG l constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
- IgG construct immunoadhesin
- CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's
- the vector used expression in CHO cells is as described in Lucas et al.. Nucl. Acids Res. 24: 9 ( 1774-1779 ( 1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR).
- DHFR expression permits selection for stable maintenance of the plasmid following transfection Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Supertect ® (Quiagen) Dosper J or Fugene 5 ' (Boeh ⁇ nger Mannheim) The cells were grown and described in Lucas et al , supra Approximately 1 x 10 7 cells are frozen in an ampule for further growth and production as described below
- the ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing The contents were pipetted into a centrifuge tube containing 10 mLs of media and cent ⁇ tuged at 1000 ⁇ m for 5 minutes The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0 2 ⁇ m filtered PS20 with 5% 0 2 ⁇ m diafiltered fetal bovine serum) The cells were then aliquoted into a 100 mL spinner containing 90 mL of selective media After 1 -2 days, the cells were transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37°C After another 2-3 days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3 x 10 5 cells/mL The cell media was exchanged with fresh media by centrifugation and resuspension in production medium Although any suitable CHO media may be employed
- the proteins were purified using a Ni-NTA column (Qiagen) Before purification, imidazole was added to the conditioned media to a concentration of 5 mM The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min at 4°C After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0 14 M NaCl and 4% mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C lmmunoadhesin (Fc containing) construct
- EXAMPLE 7 Expression of PRQ201. PRQ292. PRQ327 PRQ 1265. PRQ344. PRQ141 PRQ147 PRQ357. PRQ715, PRQ1017. PRO l 1 12. PRO509. PRQ851 or PRQ882 in Yeast
- the following method describes recombinant expression of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 in yeast.
- yeast expression vectors are constructed for intracellular production or secretion of
- PRO509, PR0853 or PR0882 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROH 12, PRO509, PR0853 or PR0882.
- DNA encoding PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715.
- PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 Yeast cells, such as yeast strain AB I 10, can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
- the transformed yeast supernatants can be analyzed by precipitation with 10% t ⁇ chloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
- Recombinant PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
- the concentrate containing PRO201 , PR0292, PR0327, PRO 1265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROl 1 12, PRO509, PR0853 or PR0882 may further be purified using selected column chromatography resins.
- PRQ201 Expression of PRQ201 .
- PR0292. PRQ127.
- PRQ1017 PROl 1 PRO509. PRQ851 or PRQ882 in Baculovirus-Infected Insect Cells
- the following method describes recombinant expression in Baculovirus-infected insect cells.
- PRO201 The sequence coding for PRO201. PR0292. PR0127, PROl 265, PR0344. PRO.343. PR0347, PR0357. PR0715, PRO1017, PROl 1 12, PRO509. PR0853 or PR0882 is fused upstream of an epitope tag contained within a baculovirus expression vector
- epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG).
- plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen).
- Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9") cells (ATCC CRL 171 1 ) using l ⁇ ofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28 ' C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors- A Laboratory Manual, Oxford: Oxford University Press ( 1994). Expressed poly-His tagged PRO201. PR0292, PR0327, PR01265. PR0344. PR0343,
- PR0853 or PR0882 can then be purified, for example, by Nr + -chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature. 362.175-179 ( 1993). Briefly, Sf9 cells are washed, resuspended in somcation buffer (25 mL Hepes, pH 7.9, 12.5 mM MgCl 2 ; 0 1 mM EDTA, 10% glycerol; 0.1 % NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice.
- somcation buffer 25 mL Hepes, pH 7.9, 12.5 mM MgCl 2 ; 0 1 mM EDTA, 10% glycerol; 0.1 % NP-40; 0.4 M KCl
- the sonicates are cleared by centrifugation. and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 ⁇ m filter.
- loading buffer 50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8
- a N ⁇ 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
- the filtered cell extract is loaded onto the column at 0.5 mL per minute.
- the column is washed to baseline A 28(1 with loading buffer, at which point fraction collection is started.
- PR0327 (UNQ288 PR0344 (UNQ103), PRO509 (UNQ129) were expressed in baculovirus infected Sf9 insect cells While the expression was actually performed in a 0 5 2 L scale, it can be readily scaled up for larger (e g 8 L) preparations
- the proteins were expressed as an IgG construct (lmmunoadhesin), in which the protein extracellular region was fused to an IgG l 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 Pharmingen
- Sf9' Spodoptera frugiperda
- Lipofectm Gibingen
- pb PH IgG and pb PH His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences
- the cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone) Cells were incubated for 5 days at 28°C The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells
- the first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0 1 Cells were incubated for 3 days at 28°C The supernatant was harvested and filtered Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed
- the conditioned medium from the transfected cells (0 5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0 22 micron filters
- the protein construct were purified using a Ni-NTA column (Qiagen) Before purification, imidazole was added to the conditioned media to a concentration of 5 mM The conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCl and 5 mM imidazole at a flow rate of 4-5
- 11- lmmunoadhesin ( Fc containing) constructs of proteins were purified from the conditioned media as follows The conditioned media were pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid.
- the DNA encoding the desired sequence was amplified with suitable systems, such as Pfu (Stratagene), or fused upstream (5'-of) of an epitope tag contained with a baculovirus expression vector
- 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 of recombinant proteins from the baculovirus le 1 promoter in stably-transformed insect cells ( 1 )
- the plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences known to be important for lel -mediated gene expression in umnfected insect cells as well as the hr5 enhancer element pIEl-1 and pIEl-2 include the te translation initiation
- H ⁇ 5 cells are grown to a confluency of 50% under the conditions of, 27°C, no C0 2 , NO pen/strep
- 30 ug of pIE based vector containing the sequence was mixed with 1 ml Ex-Cell medium (Media Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401 -78P (note this media is light sensitive)), and in a separate tube, 100 ul of CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 ml of Ex-Cell medium
- the two solutions were combined and allowed to incubate at room temperature for 15 minutes 8 ml of Ex-Cell media was added to the 2ml of DNA/CellFECTIN mix and this is layered on H ⁇ 5 cells that have been washed once with Ex-Cell media The plate is then incubated in darkness for 1 hour at room temperature The DNA/CellFECTIN mix is
- Immunogens that may be employed include purified PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, fusion proteins containing PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882, and cells expressing recombinant PRO201 , PR0292, PR0327, PR01265, PR0344. PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 on the cell surface Selection of the immunogen can be made by the skilled artisan without undue experimentation
- mice such as Balb/c are immunized with the PRO201 , PR0292.
- the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads
- the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant Thereafter, lor several weeks, the mice mav also be boosted w ith additional immunization injections Serum samples mav be periodically obtained Irom the mice by retro-orbital bleeding for testing in ELISA assays to detect ami PRO201 PR0292.
- PR0344 PR0143, PR0347, PR0357, PR0715, PRO 1017, PRO l 1 12, PRO509, PR0851 or PR0882 antibodies After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PRO201 , PR0292.
- PR0327 PR01265, PR0344, PR0343, PR0347, PR0157, PR0715 can be injected with a final intravenous injection of PRO201 , PR0292.
- mice Three to four days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35% polyethylene glycol) to a selected mu ⁇ ne myeloma cell line such as P3X63AgU 1 , available from ATCC, No CRL 1597 The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopte ⁇ n, and thymidine) medium to inhibit proliferation of non-fused cells, mveloma hybrids, and spleen cell hybrids
- HAT hyperxanthine, aminopte ⁇ n, and thymidine
- hybridoma cells will be screened in an ELISA for reactivity against PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO1017, PROl 1 12, PRO509, PR0853 or PR0882 is within the skill in the art
- the positive hybridoma cells can be injected lntrape ⁇ toneally into syngeneic Balb/c mice to produce ascites containing the anti- PRO201 , PR0292, PR0327, PR01265, PR0344, PR0343, PR0347, PR0357, PR0715, PRO 1017, PROH 12, PRO509, PR0853 or PR0882 monoclonal antibodies
- the hybridoma cells can be grown in tissue culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
Abstract
Description
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Priority Applications (343)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU22122/99A AU2212299A (en) | 1998-01-05 | 1999-01-05 | Compositions and methods for the treatment of tumor |
US09/918,585 US20030060406A1 (en) | 1997-10-17 | 2001-07-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/941,992 US20030082546A1 (en) | 1996-11-06 | 2001-08-28 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/946,374 US20030073129A1 (en) | 1998-09-01 | 2001-09-04 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,192 US20020177553A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,189 US6972325B2 (en) | 1997-10-17 | 2001-10-15 | PRO273 polypeptides |
US09/978,295 US20020156006A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,298 US20030134785A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,193 US20030073624A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,191 US20030050239A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,188 US20030139328A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,194 US20030195333A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,299 US20030199435A1 (en) | 1997-10-17 | 2001-10-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,375 US7196165B2 (en) | 1997-10-17 | 2001-10-16 | PRO363 polypeptides |
US09/978,643 US20030104998A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,608 US20030045462A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/981,915 US20030054986A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,697 US20020169284A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,824 US20050124789A9 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,403 US20030050240A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,544 US20030199436A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,665 US7294700B2 (en) | 1997-10-17 | 2001-10-16 | Anti-PRO846 antibodies |
US09/978,681 US20030195148A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/981,915 US7285623B2 (en) | 1997-10-17 | 2001-10-16 | PRO337 polypeptides |
US09/978,757 US20030083248A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,423 US20030069178A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,585 US20030049633A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,802 US20030199674A1 (en) | 1997-10-17 | 2001-10-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/978,564 US7195760B2 (en) | 1997-10-17 | 2001-10-16 | Anti-pro363 antibodies |
US09/978,824 US20030055216A1 (en) | 1997-10-17 | 2001-10-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/152,388 US20040223964A1 (en) | 1998-03-17 | 2001-10-18 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/210,028 US20030203446A1 (en) | 1998-10-07 | 2001-10-18 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/164,829 US20030194780A1 (en) | 1998-04-29 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/145,017 US20030186365A1 (en) | 1998-03-26 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/164,749 US20040029218A1 (en) | 1998-10-07 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/165,036 US20050227342A1 (en) | 1998-10-07 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/145,089 US7208575B2 (en) | 1998-10-07 | 2001-10-19 | PRO531 polypeptides |
US10/164,728 US20030186368A1 (en) | 1998-05-13 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/166,709 US20030104536A1 (en) | 1998-10-07 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/165,067 US7279553B2 (en) | 1998-05-13 | 2001-10-19 | PRO1083 polypeptides |
US10/143,029 US7105640B2 (en) | 1997-10-17 | 2001-10-19 | Anti-pro792 antibodies |
US10/164,929 US20030194781A1 (en) | 1998-03-30 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/160,502 US7220835B2 (en) | 1998-07-30 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/162,521 US7067628B2 (en) | 1998-03-17 | 2001-10-19 | PRO788 polypeptides |
US10/165,247 US7112657B2 (en) | 1998-10-07 | 2001-10-19 | PRO697 polypeptides |
US10/145,124 US20030190701A1 (en) | 1998-04-30 | 2001-10-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,922 US20030195345A1 (en) | 1997-10-17 | 2001-10-21 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,083 US20030148376A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,081 US20030049684A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,829 US20030195344A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/020,445 US20030198994A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,084 US20030203402A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,832 US20020192706A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,830 US20030077700A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,834 US20030064407A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,833 US6916648B2 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/999,832 US7132283B2 (en) | 1997-10-17 | 2001-10-24 | PRO273 polypeptides |
US10/017,085 US6974696B2 (en) | 1997-10-17 | 2001-10-24 | PRO853 nucleic acids |
US10/002,967 US20030148373A1 (en) | 1997-10-17 | 2001-10-24 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,086 US7122375B2 (en) | 1997-10-17 | 2001-10-24 | PRO274 nucleic acids |
US10/013,927 US7189529B2 (en) | 1997-10-17 | 2001-10-25 | PRO792 nucleic acids |
US10/013,925 US7037710B2 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/016,177 US20030073131A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,918 US20030211091A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,921 US20030068648A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,917 US7029874B2 (en) | 1998-03-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,929 US7019124B2 (en) | 1997-10-17 | 2001-10-25 | PRO788 nucleic acids |
US10/013,920 US20040006219A1 (en) | 1997-10-17 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,923 US7169912B2 (en) | 1997-10-17 | 2001-10-25 | PRO1017 nucleic acids |
US10/013,926 US7074593B2 (en) | 1998-04-01 | 2001-10-25 | PRO 703 nucleic acids |
US10/013,928 US20030215905A1 (en) | 1998-10-07 | 2001-10-25 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,711 US20030032023A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,436 US20020198148A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/993,583 US7074897B2 (en) | 1997-06-16 | 2001-11-14 | Pro943 polypeptides |
US09/990,726 US20030054359A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/991,163 US20020132253A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,438 US20030027754A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,562 US20030027985A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/991,073 US20020127576A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,440 US20030060407A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,443 US20030054987A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/993,469 US20030068623A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,427 US20030073809A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/992,521 US20030083461A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/992,598 US6956108B2 (en) | 1997-06-16 | 2001-11-14 | PRO1184 antibodies |
US09/993,748 US20030069403A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,442 US20020132252A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,444 US6930170B2 (en) | 1997-06-16 | 2001-11-14 | PRO1184 polypeptides |
US09/991,854 US20030059780A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/993,687 US20020198149A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/993,667 US20030022187A1 (en) | 1997-06-16 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,456 US20020137890A1 (en) | 1997-03-31 | 2001-11-14 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,529 US7309761B2 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,601 US20030054404A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,349 US7034106B2 (en) | 1997-06-16 | 2001-11-15 | Pro1159 polypeptides |
US09/997,440 US20030059833A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/998,041 US20030119001A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,559 US20030054403A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,614 US20030124531A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,585 US7166282B2 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,641 US7112656B2 (en) | 1997-06-16 | 2001-11-15 | PRO1312 polypeptides |
US09/998,041 US7309775B2 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,666 US20030027163A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/998,156 US20030044806A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,542 US20030068647A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,384 US20030087305A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,666 US7244816B2 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,641 US20030224358A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,573 US20030049682A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,585 US20030119055A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,514 US7019116B2 (en) | 1997-06-16 | 2001-11-15 | PRO 1387 polypeptides |
US09/997,333 US6953836B2 (en) | 1997-06-16 | 2001-11-15 | PRO844 polypeptides |
US09/997,601 US7189814B2 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,384 US7119177B2 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,529 US20030134284A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,683 US20030059783A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,428 US20030027162A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,628 US20030059782A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,857 US20030064375A1 (en) | 1997-06-16 | 2001-11-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/997,653 US7034122B2 (en) | 1997-06-16 | 2001-11-15 | Antibodies to PRO1159 polypeptides |
US09/991,157 US7101687B2 (en) | 1997-06-16 | 2001-11-16 | Nucleic acids encoding PRO943 |
US09/991,181 US6913919B2 (en) | 1997-06-16 | 2001-11-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/991,150 US20030194760A1 (en) | 1997-06-16 | 2001-11-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,437 US20030045463A1 (en) | 1997-06-16 | 2001-11-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/991,172 US20030050457A1 (en) | 1997-06-16 | 2001-11-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/990,441 US7041804B2 (en) | 1997-06-16 | 2001-11-16 | Antibodies to PRO1387 polypeptides |
US09/989,862 US20030130182A1 (en) | 1997-11-05 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,722 US20020072067A1 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,735 US6972185B2 (en) | 1997-06-16 | 2001-11-19 | Nucleic acids encoding PRO844 polypeptides |
US09/989,721 US20020142961A1 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,734 US7491529B2 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,328 US7056736B2 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,732 US7037679B2 (en) | 1997-06-16 | 2001-11-19 | Nucleic acids encoding PRO1184 polypeptides |
US09/989,279 US7083978B2 (en) | 1997-06-16 | 2001-11-19 | Nucleic acid encoding PRO1111 polypeptides |
US09/989,727 US20020072497A1 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,726 US7018811B2 (en) | 1997-06-16 | 2001-11-19 | Nucleic acids encoding PRO189 polypeptides |
US09/989,723 US20020072092A1 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,729 US20030059831A1 (en) | 1997-06-16 | 2001-11-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,293 US7034136B2 (en) | 1997-06-16 | 2001-11-20 | Nucleic acids encoding PRO1159 polypeptides |
US09/989,730 US7157247B2 (en) | 1997-06-16 | 2001-11-20 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,725 US20030139329A1 (en) | 1997-06-16 | 2001-11-20 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US09/989,728 US7029873B2 (en) | 1997-06-16 | 2001-11-20 | Nucleic acids to PRO1387 polypeptides |
US09/989,731 US20020103125A1 (en) | 1997-06-16 | 2001-11-20 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/001,054 US20020192209A1 (en) | 1997-09-17 | 2001-11-30 | Methods and compositions for inhibiting neoplastic cell growth |
US10/006,116 US20030082626A1 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,063 US20030114652A1 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,041 US6951921B2 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/007,194 US7041805B2 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,485 US7026448B2 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,818 US20030054406A1 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,856 US7538086B2 (en) | 1998-09-01 | 2001-12-06 | PRO1303 polypeptides |
US10/007,236 US7034123B2 (en) | 1998-09-01 | 2001-12-06 | Anti-PRO1347 antibodies |
US10/006,117 US7071304B2 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,768 US6936697B2 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,130 US7098312B2 (en) | 1998-09-01 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,746 US7026449B2 (en) | 1999-01-05 | 2001-12-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/006,172 US7081514B2 (en) | 1998-09-01 | 2001-12-06 | PRO1347 polypeptides |
US10/011,833 US6951920B2 (en) | 1998-09-01 | 2001-12-07 | PRO1340 polypeptides |
US10/012,137 US20030187189A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/011,671 US20030096954A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/011,795 US7012131B2 (en) | 1998-09-01 | 2001-12-07 | Pro1410 polypeptides |
US10/012,121 US7022817B2 (en) | 1998-09-01 | 2001-12-07 | PRO1325 polypeptides |
US10/012,755 US20030096955A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/012,101 US20030187239A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/012,149 US7038019B2 (en) | 1998-09-01 | 2001-12-07 | Antibodies to PRO1410 |
US10/012,237 US20030191281A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/011,692 US20030109672A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/012,754 US20030187191A1 (en) | 1998-09-01 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/012,064 US6953841B2 (en) | 1999-01-05 | 2001-12-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/012,752 US7026455B2 (en) | 1998-09-01 | 2001-12-07 | Anti-pro 1343 antibodies |
US10/012,754 US7375184B2 (en) | 1998-09-01 | 2001-12-07 | PRO1382 polypeptides |
US10/012,231 US6924355B2 (en) | 1998-09-01 | 2001-12-07 | PRO1343 polypeptides |
US10/012,753 US7488796B2 (en) | 1998-09-01 | 2001-12-07 | PRO1269 polypeptides |
US10/015,822 US20030130491A1 (en) | 1998-09-01 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,915 US20030204053A1 (en) | 1998-09-01 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,910 US7057018B2 (en) | 1999-01-05 | 2001-12-10 | Pro 1474 polypeptides |
US10/013,911 US20030187193A1 (en) | 1998-09-01 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,909 US20030186318A1 (en) | 1999-01-05 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,913 US20030083462A1 (en) | 1999-01-05 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,907 US20030064925A1 (en) | 1998-09-01 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,430 US20030092883A1 (en) | 1998-09-01 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/013,906 US20030191282A1 (en) | 1998-09-01 | 2001-12-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,869 US20030073130A1 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,671 US6946263B2 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,653 US20030187195A1 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,389 US6936436B2 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,499 US20030065142A1 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,393 US6951737B2 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,480 US7074912B2 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,519 US7033785B2 (en) | 1998-09-01 | 2001-12-11 | PRO1347 nucleic acids |
US10/015,869 US7189530B2 (en) | 1998-09-01 | 2001-12-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,388 US20030191299A1 (en) | 1998-09-01 | 2001-12-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,715 US7033786B2 (en) | 1998-09-01 | 2001-12-12 | Pro1340 nucleic acids |
US10/015,390 US20030216562A1 (en) | 1998-09-01 | 2001-12-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,391 US20030120053A1 (en) | 1998-09-01 | 2001-12-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,385 US20030195347A1 (en) | 1998-09-01 | 2001-12-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,387 US20030135034A1 (en) | 1998-09-01 | 2001-12-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/015,386 US7022498B2 (en) | 1998-09-01 | 2001-12-12 | Pro 1325 nucleic acids |
US10/017,610 US20030113795A1 (en) | 1998-09-01 | 2001-12-13 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,407 US20030125535A1 (en) | 1998-09-01 | 2001-12-13 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/020,063 US20030119097A1 (en) | 1999-01-05 | 2001-12-13 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,867 US20030180792A1 (en) | 1998-09-01 | 2001-12-13 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,527 US20030082628A1 (en) | 1998-09-01 | 2001-12-13 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/017,306 US20030170718A1 (en) | 1998-09-01 | 2001-12-13 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/028,072 US20030004311A1 (en) | 1997-06-18 | 2001-12-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/053,107 US20020192752A1 (en) | 1998-09-09 | 2002-01-17 | Compositions and methods for the treatment of immune related diseases |
US09/978,187 US20030096744A1 (en) | 1997-10-17 | 2002-01-28 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/062,923 US20030191283A1 (en) | 1998-04-23 | 2002-01-31 | Nsp molecules |
US10/081,056 US20040043927A1 (en) | 1997-09-19 | 2002-02-20 | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
US10/119,480 US20040087769A1 (en) | 1998-09-10 | 2002-04-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,051 US20030092147A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,045 US20030073210A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,047 US20030077778A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,040 US20030082759A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,046 US20030194791A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,042 US20030096386A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,044 US20030190717A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,059 US20030190721A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,041 US20030077776A1 (en) | 1997-03-31 | 2002-04-11 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,043 US7220831B2 (en) | 1997-03-31 | 2002-04-12 | PRO235 polypeptides |
US10/121,050 US20030054516A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,056 US20030082760A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,054 US20030199054A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,057 US20030190719A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,049 US20030022239A1 (en) | 1997-06-18 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,061 US20030082761A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,048 US20030199051A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,055 US20030190718A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,063 US20030199055A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,058 US20030190720A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,053 US20030199053A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,052 US20030199052A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,062 US20030077779A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/121,060 US20030190722A1 (en) | 1997-03-31 | 2002-04-12 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,261 US20030068796A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,215 US7291329B2 (en) | 1997-03-31 | 2002-04-15 | Antibodies against PRO4406 |
US10/123,214 US7343721B2 (en) | 1997-03-31 | 2002-04-15 | PRO4406 polypeptide |
US10/123,154 US20030190724A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,212 US7276577B2 (en) | 1997-03-31 | 2002-04-15 | PRO1866 polypeptides |
US10/123,108 US7635478B2 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,156 US20030194792A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,292 US20030073211A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,322 US20030199059A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,155 US20030068794A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,236 US20030068795A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,262 US20030049816A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,213 US20030199057A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,157 US20030190725A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,291 US20030199058A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,213 US7193048B2 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,235 US20030082762A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,109 US20030190723A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,771 US20030199060A1 (en) | 1997-03-31 | 2002-04-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,906 US20030190726A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,911 US7408032B2 (en) | 1997-03-31 | 2002-04-16 | PRO1188 polypeptides |
US10/123,912 US20030100087A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,913 US20030203462A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,905 US7285625B2 (en) | 1997-06-18 | 2002-04-16 | PRO536 polypeptides |
US10/123,904 US20030022328A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,903 US20030073212A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,909 US7193049B2 (en) | 1997-03-31 | 2002-04-16 | PRO862 polypeptides |
US10/123,902 US20030077781A1 (en) | 1997-03-31 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,907 US7084258B2 (en) | 1997-03-31 | 2002-04-16 | Antibodies against the PRO862 polypeptides |
US10/123,908 US7335728B2 (en) | 1997-03-31 | 2002-04-16 | PRO1310 polypeptides |
US10/123,905 US20030087344A1 (en) | 1997-06-18 | 2002-04-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/123,910 US7329404B2 (en) | 1997-03-31 | 2002-04-16 | Antibodies against PRO1310 |
US10/124,814 US7105335B2 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,704 US7357926B2 (en) | 1997-03-31 | 2002-04-17 | Antibodies against PRO1879 and the use thereof |
US10/124,813 US7312307B2 (en) | 1997-03-31 | 2002-04-17 | PRO1056 polypeptides |
US10/125,795 US7304131B2 (en) | 1997-03-31 | 2002-04-17 | PRO1483 polypeptides |
US10/124,820 US20030190729A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,821 US20030199023A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,824 US20030077659A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,817 US20030077786A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,816 US20030190728A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,819 US7285626B2 (en) | 1997-03-31 | 2002-04-17 | PRO1076 polypeptides |
US10/124,822 US7109305B2 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,805 US20030194794A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,823 US20030199062A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/124,818 US20030082763A1 (en) | 1997-03-31 | 2002-04-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,927 US20030190731A1 (en) | 1997-03-31 | 2002-04-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,924 US7342097B2 (en) | 1997-03-31 | 2002-04-19 | PRO1309 polypeptides |
US10/125,922 US7309762B2 (en) | 1997-03-31 | 2002-04-19 | PRO1360 polypeptides |
US10/125,931 US20030199063A1 (en) | 1997-03-31 | 2002-04-19 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/125,932 US7317079B2 (en) | 1997-03-31 | 2002-04-19 | PRO812 polypeptides |
US10/127,831 US20030082689A1 (en) | 1997-03-31 | 2002-04-22 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/128,689 US20030087365A1 (en) | 1997-03-31 | 2002-04-23 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/131,823 US7304132B2 (en) | 1997-03-31 | 2002-04-24 | PRO1693 polypeptides |
US10/131,817 US7291701B2 (en) | 1997-03-31 | 2002-04-24 | PRO1777 polypeptides |
US10/131,825 US7282566B2 (en) | 1997-03-31 | 2002-04-24 | PRO1779 polypeptide |
US10/137,867 US20030207349A1 (en) | 1997-03-31 | 2002-05-03 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/137,868 US20030082764A1 (en) | 1997-03-31 | 2002-05-03 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/137,865 US20030032155A1 (en) | 1997-03-31 | 2002-05-03 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,024 US20040058424A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,474 US20030032156A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/139,980 US7247710B2 (en) | 1997-03-31 | 2002-05-06 | PRO4395 antibodies |
US10/140,023 US20030207416A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,020 US20030207415A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,470 US20030022331A1 (en) | 1997-03-31 | 2002-05-06 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/139,963 US7288625B2 (en) | 1997-03-31 | 2002-05-06 | PRO4395 polypeptides |
US10/140,809 US20030207418A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,864 US20030207419A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,921 US7317080B2 (en) | 1997-03-31 | 2002-05-07 | PRO4303 polypeptides |
US10/140,865 US20030207420A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,805 US20030207417A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,925 US20030073215A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,860 US7307151B2 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/140,808 US7425621B2 (en) | 1997-03-31 | 2002-05-07 | Antibodies against the PRO4401 polypeptide |
US10/140,928 US20030068798A1 (en) | 1997-03-31 | 2002-05-07 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/141,760 US7342104B2 (en) | 1997-03-31 | 2002-05-08 | Antibodies against the PRO4320 polypeptide |
US10/141,755 US7297764B2 (en) | 1997-03-31 | 2002-05-08 | PRO4318 polypeptides |
US10/141,756 US7488586B2 (en) | 1997-03-31 | 2002-05-08 | PRO4409 polypeptides |
US10/141,701 US20030207421A1 (en) | 1997-03-31 | 2002-05-08 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/141,754 US7361732B2 (en) | 1997-03-31 | 2002-05-08 | PRO4400 polypeptides |
US10/142,425 US20030207424A1 (en) | 1997-03-31 | 2002-05-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/142,430 US7309766B2 (en) | 1997-03-31 | 2002-05-09 | PRO5774 polypeptides |
US10/143,114 US20030036180A1 (en) | 1997-03-31 | 2002-05-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/142,417 US7304133B2 (en) | 1997-03-31 | 2002-05-09 | PRO4389 polypeptides |
US10/143,113 US7329730B2 (en) | 1997-03-31 | 2002-05-09 | PRO4348 polypeptides |
US10/142,419 US7153941B2 (en) | 1997-03-31 | 2002-05-10 | Antibodies that bind PRO4994 polypeptides |
US10/142,423 US20030049817A1 (en) | 1997-03-31 | 2002-05-10 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/143,032 US7408033B2 (en) | 1997-03-31 | 2002-05-10 | PRO5995 polypeptides |
US10/142,431 US7285629B2 (en) | 1997-03-31 | 2002-05-10 | Pro5005 polypeptides |
US10/146,792 US20030207428A1 (en) | 1997-03-31 | 2002-05-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/146,730 US20030207427A1 (en) | 1997-03-31 | 2002-05-15 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,528 US20030219885A1 (en) | 1997-03-31 | 2002-05-16 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,536 US20040077064A1 (en) | 1997-03-31 | 2002-05-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,519 US20030077791A1 (en) | 1997-03-31 | 2002-05-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/147,492 US20030082765A1 (en) | 1997-03-31 | 2002-05-17 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/152,395 US7189534B2 (en) | 1997-03-31 | 2002-05-21 | PRO4320 polynucleotide |
US10/153,934 US20030129695A1 (en) | 1997-03-31 | 2002-05-22 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/156,843 US20030207805A1 (en) | 1997-06-18 | 2002-05-28 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/157,782 US20030077792A1 (en) | 1997-03-31 | 2002-05-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/157,786 US20030208055A1 (en) | 1997-03-31 | 2002-05-29 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/158,782 US20030082766A1 (en) | 1997-03-31 | 2002-05-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/158,791 US20030207429A1 (en) | 1997-03-31 | 2002-05-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/160,498 US20030073216A1 (en) | 1997-03-31 | 2002-05-30 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/176,913 US20030022298A1 (en) | 1997-09-15 | 2002-06-20 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US10/213,199 US7381809B2 (en) | 1998-09-09 | 2002-08-05 | Compositions and methods for the treatment of immune related diseases |
US11/025,607 US20050181478A1 (en) | 1998-09-01 | 2004-12-28 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US11/100,159 US7425613B2 (en) | 1997-11-05 | 2005-04-05 | PRO1375 polypeptides |
US11/341,175 US7468427B2 (en) | 1997-03-31 | 2006-01-27 | Antibodies to PRO1275 polypeptide |
US11/796,725 US20090197301A1 (en) | 1998-09-01 | 2007-04-27 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
Applications Claiming Priority (12)
Application Number | Priority Date | Filing Date | Title |
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US7044098P | 1998-01-05 | 1998-01-05 | |
US60/070,440 | 1998-01-05 | ||
US8350098P | 1998-04-29 | 1998-04-29 | |
US60/083,500 | 1998-04-29 | ||
US8641498P | 1998-05-22 | 1998-05-22 | |
US60/086,414 | 1998-05-22 | ||
US8874298P | 1998-06-10 | 1998-06-10 | |
US60/088,742 | 1998-06-10 | ||
US10778398P | 1998-11-10 | 1998-11-10 | |
US60/107,783 | 1998-11-10 | ||
US10930498P | 1998-11-20 | 1998-11-20 | |
US60/109,304 | 1998-11-20 |
Related Parent Applications (2)
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US21851798A Continuation-In-Part | 1996-11-06 | 1998-12-22 | |
PCT/US1999/005028 Continuation-In-Part WO1999046281A2 (en) | 1996-11-06 | 1999-03-08 | Novel polypeptides and nucleic acids encoding the same |
Related Child Applications (6)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1999/005028 Continuation-In-Part WO1999046281A2 (en) | 1996-11-06 | 1999-03-08 | Novel polypeptides and nucleic acids encoding the same |
US28429199A A-371-Of-International | 1996-11-06 | 1999-04-12 | |
US38013899A Continuation-In-Part | 1996-11-06 | 1999-08-25 | |
PCT/US1999/020111 Continuation-In-Part WO2000012708A2 (en) | 1997-03-31 | 1999-09-01 | Further pro polypeptides and sequences thereof |
US10/081,056 Continuation US20040043927A1 (en) | 1997-09-19 | 2002-02-20 | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
US10/119,480 Continuation US20040087769A1 (en) | 1998-09-10 | 2002-04-09 | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
Publications (3)
Publication Number | Publication Date |
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WO1999035170A2 WO1999035170A2 (en) | 1999-07-15 |
WO1999035170A8 WO1999035170A8 (en) | 1999-10-21 |
WO1999035170A9 true WO1999035170A9 (en) | 2000-03-09 |
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Family Applications (1)
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PCT/US1999/000106 WO1999035170A2 (en) | 1996-11-06 | 1999-01-05 | Compositions and methods for the treatment of tumor |
Country Status (2)
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AU (1) | AU2212299A (en) |
WO (1) | WO1999035170A2 (en) |
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US8212004B2 (en) | 1999-03-02 | 2012-07-03 | Human Genome Sciences, Inc. | Neutrokine-alpha fusion proteins |
US6812327B1 (en) | 1996-10-25 | 2004-11-02 | Human Genome Sciences, Inc. | Neutrokine-alpha polypeptides |
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US6774106B2 (en) | 2000-05-12 | 2004-08-10 | Amgen Inc. | Methods and compositions of matter concerning APRIL/G70, BCMA, BLYS/AGP-3 and TACI |
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-
1999
- 1999-01-05 AU AU22122/99A patent/AU2212299A/en not_active Abandoned
- 1999-01-05 WO PCT/US1999/000106 patent/WO1999035170A2/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
WO1999035170A8 (en) | 1999-10-21 |
WO1999035170A2 (en) | 1999-07-15 |
AU2212299A (en) | 1999-07-26 |
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