DE60035077T2 - In tumors amplified gene sequences and their diagnostic uses - Google Patents

Description

Field of the invention

The The present invention relates to compositions and methods for tumor diagnosis and tumor treatment.

Background of the invention

Malignant Tumors (cancers) are the second most common after heart disease Cause of death in the United States (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 normal tissue and which multiply to form a tumor mass by invasion adjacent tissue through these neoplastic tumor cells and through the formation of malicious Cells that are ultimately over the blood or the lymphatic system on local lymph nodes and spread more distant sites (metastasis). In a cancerous State, a cell proliferates under conditions in which normal cells would not multiply. Cancer manifests in many forms, characterized by varying degrees of invasiveness and aggressiveness are.

The change Gene expression is closely related to the uncontrolled one Growth and dedifferentiation, which is a common feature all cancers are. It has been shown that the genomes of certain well-studied tumors decreased expression recessive Have genes that are usually are referred to as tumor suppressor genes which are normally present in their function the malignant Cell growth and / or overexpression certain dominant genes, such as B. oncogenes, their effect malicious Promotes growth, prevent. Each of these genetic changes seems to introduce some To be responsible for the overall characteristics of the complete neoplastic phenotype (Hunter, Cell 64, 1129 (1991); Bishop, Cell 64, 235-248 (1991)).

One well-known mechanism of gene (eg, oncogene) overexpression in cancer cells is the gene amplification. This is a process at in the chromosome of the stem cell multiple copies of a particular gene to be produced. The process includes non-scheduled replication the chromosome region comprising the gene, followed by recombination the replicated segments back into the chromosome (Alitalo et al., Adv. Cancer Res. 47, 235-281 (1986)). It is believed that overexpression the gene is associated with gene amplification, d. H. proportional to the number of copies made.

It has been found that proto-oncogenes encoding growth factors and growth factor receptors play important roles in the pathogenesis of various human malignancies, including breast carcinomas. For example, it has been found that the human ErbB2 gene (erbB2, also known as her2 or c-erbB-2) encoding a 185 kD transmembrane glycoprotein receptor (p185 ^HER2 ; HER2) is associated with the epidermis Growth Factor Receptor (EGFR) is overexpressed in approximately 25% to 30% of human breast carcinomas (Slamon et al., Science 235, 177-182 (1987); Slamon et al., Science 244, 707-712 (1987); 1989)).

It has been reported that gene amplification of a proto-oncogene is an event typically involved in more virulent cancers and could serve as a predictor of clinical outcome (Schwab et al., Genes Chromosome Cancer 1, 181-193 (1990); Alitalo et al., supra). Thus, erbB2 overexpression is commonly considered to be a predictor of poor prognosis, particularly in patients with a primary disease involving axillary lymph nodes (Slamon et al., (1987) and (1989), Ravdin and Chamness, Gene 159, 19-27 (1995); and Hynes and Stern, Biochim., Biophys, Acta 1198, 165-184 (1994)), and is associated with susceptibility and / or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and Fluorouracil) and anthracyclines (Baselga et al., Oncology 11 (3 Suppl. 1), 43-48 (1997)). However, despite the association of erbB2 overexpression with poor prognosis, the differences in HER2-positive patients who respond clinically to treatment with taxanes were three times higher than those of HER2-negative patients (ibid). A recombinant humanized monoclonal anti-ErbB2 (anti-HER2) antibody (a humanized version of the murine anti-ErbBG2 antibody 4D5, referred to as rhuMAb HER2 or Herceptin ^®) was clinically active in patients with ErbB2-overexpressing metastatic breast cancer who have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol 14, 737-744 (1996)).

in the Lichte of course, of course Interest in the identification of new processes and compositions, which are useful for the diagnosis and treatment of tumors, which are related to gene amplification.

The PRO4980 protein and the one for it encoding nucleic acids are known as KIAA1094 from EMBL database entry A029017. This document contains however, no indications or suggestions as to possible uses or applications of these sequences as disclosed herein.

Summary of the invention

A. Embodiments

The The present invention relates to compositions and methods for Diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including People. The present invention is based on identification of genes that are amplified in the genome of tumor cells. From Such gene amplification is expected to interfere with overexpression of the gene product and contributes to tumorigenesis. Accordingly, from the proteins encoded by the amplified genes, assumed that they have appropriate targets for diagnosis and / or Treatment (including prevention) of certain types of cancer and as forecasting means of prognosis could serve the tumor treatment.

In an embodiment The present invention relates to an isolated antibody which to a polypeptide referred to herein as PRO4980 polypeptide is used for a diagnosis. In one aspect, the binds isolated antibodies specific to a PRO4980 polypeptide. Often a cell is that Polypeptide expressed, a tumor cell that compared the polypeptide to overexpress a normal cell of the same tissue type. In another Aspect is the antibody a monoclonal antibody, preferably residues of a non-human complementarity determining Region (CDR) and remains of a human framework region (FR). Of the antibody may be labeled and immobilized on a solid support. In In another aspect, the antibody is an antibody fragment, a single chain antibody or a humanized antibody, which preferably binds specifically to a PRO4980 polypeptide.

Here in Nucleic acid molecules are disclosed code for anti-PRO4980 antibody, and vectors and recombinant host cells comprising such nucleic acid molecules.

In addition, will discloses herein a process for producing an anti-PRO4980 antibody, the method comprising culturing a host cell with a for the antibody transformed nucleic acid molecule is under conditions sufficient to cause expression of the antibody permitting and recovering the antibody from the cell culture.

Furthermore, becomes an isolated nucleic acid molecule, the one for a PRO4980 polypeptide encoding nucleic acid molecule hybridizes, or the complement thereof disclosed. The isolated nucleic acid molecule is preferably DNA, and the hybridization preferably takes place under stringent Hybridization and washing conditions instead. Such nucleic acid molecules can be described as Antisense molecules serve the amplified genes identified herein, which in turn in the modulation of transcription and / or translation of the respective amplified Gene or as an antisense primer in amplification reactions application can find. Furthermore you can such sequences are used as part of a ribozyme and / or a triple helix sequence which in turn are used in the regulation of amplified genes can be.

In The invention may include a method for determining the presence of a PRO4980 polypeptide in a sample believed to be a PRO4980 polypeptide contains use, wherein the method of exposing the sample to a PRO4980 antibodies and determining the binding of the antibody to a PRO4980 polypeptide in the sample.

The The invention may include a method for determining the presence of a PRO4980 polypeptide in a cell, wherein the method involves exposing the cell across from an anti-PRO4980 antibody and determining the binding of an antibody to the cell.

In another embodiment, the present invention relates to a method of diagnosing a tumor in a mammal, comprising detecting the expression level of a PRO4980 polypeptide-encoding gene (a) in a sample of mammalian tissue cells and (b) a control sample of be knew normal tissue cells of the same cell type, wherein a higher expression level in the sample compared to the control indicates the presence of a tumor in the mammal from which the test tissue cells were obtained.

In a further embodiment The present invention relates to a method for diagnosing a tumor in a mammal, comprising (a) contacting an anti-PRO4980 antibody with a sample of mammalian tissue cells; and (b) detecting the formation of a complex of the anti-PRO4980 antibody and a PRO4980 polypeptide in the sample, wherein the formation of a complex the presence of a tumor in the mammal displays. The detection can be qualitative or quantitative and can be compared with the monitoring complex formation in a control of known normal Tissue cells of the same cell type take place. A larger amount Complexes that form in the sample show the presence a tumor in the mammal from which the test tissue cells were obtained. The antibody preferably bears a detectable marker. The complex formation can be, for example by light microscopy, flow cytometry, fluorimetry or monitors other methods known in the art become.

The Sample usually comes of an individual suspected of having neoplastic (s) Cell growth or proliferation (eg, cancer cells).

In a further embodiment The present invention relates to a cancer diagnostic kit comprising a Anti-PRO4980 antibodies and a carrier (eg a buffer) in suitable packaging. The set includes preferably instructions for using the antibody to detect the presence a PRO4980 polypeptide in a sample suspected of having this contains.

B. Additional embodiments

Here in discloses an isolated nucleic acid molecule having a nucleotide sequence that covers for a PRO4980 polypeptide and hereinafter referred to as "PRO".

In In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, still more preferably at least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, still more preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, still more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 91% sequence identity, still more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, still more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, still more preferably at least about 98% sequence identity, and more preferably at least about 99% sequence identity, with (a) a DNA molecule, that for a polypeptide having a full-length amino acid sequence, as disclosed herein, an amino acid sequence encoding the signal peptide lacks, as disclosed herein, an extracellular domain of a transmembrane protein, with or without signal peptide as disclosed herein or another specifically defined fragment of the full length amino acid sequence as disclosed herein or (b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% sequence identity, more preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably at least about 90% sequence identity, more preferably at least about 91 % Sequence identity, more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity t, more preferably at least about 97% sequence identity, more preferably at least about 98% sequence identity, and even more preferably at least about 99% sequence identity, with (a) a DNA molecule encoding the coding sequence of a full-length PRO polypeptide cDNA as herein discloses the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane bran PRO polypeptide, with or without signal peptide as disclosed herein, or the coding sequence of another specific defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule according to (a) ,

Also discloses an isolated nucleic acid molecule having a nucleotide sequence with at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, still more preferably at least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, still more preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, still more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 91% sequence identity, still more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, still more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, still more preferably at least about 98% sequence identity, and more preferably at least about 99% sequence identity, with (a) a DNA molecule, that for encodes the same mature polypeptide as deposited with the ATCC human protein cDNA as disclosed herein, or (b) the complement of the DNA molecule according to (a).

Also discloses an isolated nucleic acid molecule having a nucleotide sequence includes that for encodes a PRO polypeptide that either deletes transmembrane domains or transmembrane domains inactivated or complementary to such a coding nucleotide sequence, wherein the transmembrane domain (s) such polypeptide is / are disclosed herein. That's why come soluble extracellular domains of PRO polypeptides described herein are also contemplated.

Also disclosed are fragments of a coding for a PRO polypeptide Sequence or its complement, for example, as hybridization probes for coding for Fragments of a PRO polypeptide can be used, the if necessary for encode a polypeptide having a binding site for an anti-PRO antibody can, or as antisense oligonucleotide probes. Such nucleic acid fragments are common about 20 nucleotides long, preferably at least about 30 nucleotides, even more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 60 Nucleotides, more preferably at least about 70 nucleotides, still more preferably at least about 80 nucleotides, more preferably at least about 90 nucleotides, more preferably at least about 100 nucleotides, even more preferably at least about 110 nucleotides, more preferably about 120 nucleotides, more preferably at least about 130 nucleotides, even more preferably at least about 140 nucleotides, more preferably at least about 150 nucleotides, more preferably at least about 160 Nucleotides, more preferably at least about 170 nucleotides, still more preferably at least about 180 nucleotides, more preferably at least about 190 nucleotides, more preferably at least about 200 nucleotides, even more preferably at least about 250 nucleotides, more preferably at least about 300 nucleotides, more preferably at least about 350 Nucleotides, more preferably at least about 400 nucleotides, still more preferably at least about 450 nucleotides, more preferably at least about 500 nucleotides, more preferably at least about 600 nucleotides, even more preferably at least about 700 nucleotides, more preferably at least about 800 nucleotides, more preferably at least about 900 Nucleotides, and more preferably at least about 1000 nucleotides long, wherein in this context the term "about" refers to said nucleotide sequence length plus or minus 10% of said length refers. It should be noted that new fragments of one for a PRO polypeptide encoding nucleotide sequence in a routine manner by a comparative Arrangement of for a PRO polypeptide-encoding nucleotide sequence with other known ones Nucleotide sequences, well-known sequence ordering programs can be used and determining which one (s) for a PRO polypeptide new coding nucleotide sequence fragment (s) is / are new can be. All such for a PRO polypeptide-encoding nucleotide sequences are included herein in question. Also suitable are the PRO polypeptide fragments, for which these nucleotide molecule fragments encode, preferably those PRO polypeptide fragments, the one binding site for an anti-PRO antibody include.

Also disclosed herein is an isolated PRO polypeptide for which a any of the above identified isolated nucleic acid sequences coded.

Also disclosed is an isolated PRO polypeptide having an amino acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, even before at least about 86% sequence identity, more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity, more preferably at least about 98 % Sequence identity, and more preferably at least about 99% sequence identity, with a PRO polypeptide having a full length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, m it or without signal peptide as disclosed herein or another specifically defined fragment of the full-length amino acid sequence as disclosed herein.

Also discloses an isolated PRO polypeptide having an amino acid sequence with at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, still more preferably at least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, still more preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, still more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 91% sequence identity, still more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, still more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, still more preferably at least about 98% sequence identity, and more preferably at least about 99% sequence identity, with an amino acid sequence, for the any of the human protein cDNAs deposited with the ATCC encoded as disclosed herein.

Also discloses an isolated PRO polypeptide having an amino acid sequence their comparison with the amino acid sequence of a PRO polypeptide with a full-length amino acid sequence, as disclosed herein, an amino acid sequence encoding the signal peptide lacks, as disclosed herein, an extracellular domain of a transmembrane protein, with or without signal peptide as disclosed herein or another specifically defined fragment of the full length amino acid sequence as herein is at least about 80% positive, preferably at least about 81% positive, more preferably at least about 82% positive, even more preferably at least about 83% positive, more preferably at least about 84% positive, more preferably at least about 85% positive, more preferably at least about 86% positive, still more preferably at least about 87% positive, more preferably at least about 88% positive, more preferably at least about 89% positive, even more preferably at least about 90% positive, more preferably at least about 91% positive, more preferably at least about 92% positive, more preferably at least about 93% positive, still more preferably at least about 94% positive, more preferably at least about 95% positive, more preferably at least about 96% positive, even more preferably at least about 97% positive, more preferably at least about 98% positive, and more preferably at least about 99% positive, fails.

Also disclosed an isolated PRO polypeptide without N-terminal signal sequence and / or initiating methionine for which a nucleotide sequence encoded for an amino acid sequence encoded as described above. Process for the preparation the same are also described herein, wherein these methods culturing a host cell containing a vector with the appropriate comprising nucleic acid molecule encoding under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.

Also discloses an isolated PRO polypeptide that either deletes transmembrane domains or transmembrane domains inactivated is. Methods of making the same are also described herein. wherein said methods comprise cultivating a host cell containing a Vector comprising the appropriate coding nucleic acid molecule, under expression the PRO polypeptide suitable conditions and winning the PRO polypeptide from cell culture.

Also disclosed are vectors comprising DNA encoding one of the polypeptides described herein. Host cells comprising any such vector are also provided. The host cells may be, for example, CHO cells, E. coli cells, yeast cells or baculovirus-infected insect cells. A method of producing any of the polypeptides described herein is also described and involves culturing the host cells under conditions conducive to the expression of the ge desired polypeptide, and recovering the desired polypeptide from the cell culture.

Also discloses hybrid molecules, include any of the molecules described herein heterologous polypeptide or an amino acid sequence. An example for such hybrid molecules includes any of the polypeptides described herein fused to an epitope tagging sequence or an Fc region of an immunoglobulin.

The The invention optionally uses an antibody specific to a any of the polypeptides described above or below binds. The antibody is optionally a monoclonal antibody, a humanized antibody Antibody fragment or a single chain antibody.

Also discloses oligonucleotide probes responsible for the isolation of genomic and cDNA nucleotide sequences or as antisense probes wherein these probes are of any of the above or below described nucleotide sequences can be derived.

Brief description of the drawings

1 Figure 4 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA containing a nucleotide sequence encoding the native sequence PRO4980, wherein the nucleotide sequence (SEQ ID NO: 1) is a clone, referred to herein as DNA97003 2649 is designated. In addition, the positions of the respective start and stop codons are printed in bold and underlined.

2 Figure 4 shows the amino acid sequence (SEQ ID NO: 2) of a native sequence PRO4980 polypeptide derived from that described in U.S. Pat 1 derived coding sequence of Seq.-ID No. 1 is derived.

Detailed description of the invention

I. Definitions

The Expressions "gene amplification" and "gene duplication" become interchangeable used and refer to a process by which multiple copies a gene or gene fragment in a particular cell or cell line be formed. The duplicated region (a section amplified DNA) becomes common referred to as "amplicon." Usually increases the amount of messenger RNA (mRNA) produced, i. H. the Extent of Gene expression, also in proportion to the number of copies made of each expressed Gene.

"Tumor" refers As used herein, refers to any neoplastic cell growth and multiplication, whether evil or benign, and on all precancerous and cancerous Cells and tissues.

The The terms "cancer" and "cancerous" refer to or describe the physiological state in mammals, typically characterized by uncontrolled cell growth is. Examples of cancer include, but are not limited to, carcinomas, lymphomas, Blastomas, sarcomas and leukemia. More specific examples for such cancers include breast carcinomas, prostate cancers, Colon carcinomas, squamous cell carcinomas, small cell lung carcinomas, non-small cell lung carcinomas, gastrointestinal carcinomas, pancreatic carcinomas, Glioblastomas, cervical carcinomas, ovarian cancers, liver carcinomas, Bladder carcinoma, hepatoma, colorectal carcinoma, endometrial carcinoma, Salivary gland carcinoma, Renal carcinomas, liver carcinomas, vulvar carcinomas, thyroid carcinomas and different types of head and neck cancer.

"Treatment" is an intervention who performed with the intention is to prevent the development of the pathology of a disorder or this to change. Accordingly, relates itself "treatment" on both therapeutic Treatment, as well as prophylactic or preventive measures. Those who require treatment include those who the disorder already have, as well as those in which the disorder is to be prevented. at The tumor (eg, cancer) treatment may be a therapeutic agent can directly diminish the pathology of tumor cells or can tumor cells more receptive for one Treatment with other therapeutic agents, eg. B. irradiation and / or chemotherapy.

The "pathology" of cancer includes all phenomena that affect the patient's health including, without limitation, abnormal or uncontrollable growth, metastasis, disruption of the normal function of neighboring cells, release of cytokines or other secretory products in abnormal proportions, suppression or exacerbation of inflammatory or immunological reactions, etc.

"Mammal" for the purpose treatment refers to any animal classified as a mammal, including humans, Domestic and farm animals and zoo, sports or pets, such. Dogs, horses, Cats, cattle, pigs, sheep, etc. Preferably, the mammal the human being.

"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal exposed thereto at the dosages and concentrations employed, and often the physiologically acceptable carrier is an aqueous, pH buffered solution acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as e.g. B. polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides or other carbohydrates, including glucose, mannose or dextrins; chelating agents, such as EDTA, sugar alcohols, e.g. Mannitol or sorbitol; salt-forming counterions, such as sodium, and / or nonionic surfactants, such as sodium; TWEEN ^™ , polyethylene glycol (PEG) and PLURONICS ^™ .

Administration "in combination with "one or several other therapeutic agents, the simultaneous (simultaneous) and sequential administration in any order.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or inhibits the function of cells and / or causes the destruction of cells.The term is intended to include radioactive isotopes (eg, I ¹³¹ , I ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ ), chemotherapeutic agents and toxins, such as enzymatically active toxins derived from bacteria, fungi, plants or animals, or fragments thereof.

A "chemotherapeutic agent" is a compound useful in the treatment of cancer Examples of chemotherapeutic agents include adriamycin, do xorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thioetepa, busulfan, cytoxin, taxoids, e.g. , Paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosamide, mitotoxin C, Mitoxantrone, vincistrine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Patent No. 4,675,187 ), 5-FU, 6-thioguanine, 6-mercatopurine, actinomycin D, VP-16, chlorambucil, melphalan and other related nitrogen vacancies. Also included in this definition are hormonal agents whose action regulates or inhibits the action of hormones on tumors, such as, e.g. Tamoxifen and onapristone.

A "growth-inhibiting Means " when used herein refers to a compound or composition, which inhibits the growth of a cell, in particular a cancer cell, any of the genes identified herein either in vitro or overexpressed in vivo. Consequently, the growth-inhibiting agent is one that is the percent Proportion of cells overexpressing such genes in the S-phase of the cell cycle, significantly reduced. Examples of growth inhibiting agents include Means that the progression of the cell cycle (in one place, the not the S phase is) blocked, such as. As means, the G1-locking and M-phase locking trigger. Classical M-phase blockers include vincas (vincristine and vinblastine), taxol and topo II inhibitors, such as. Doxorubicin, epirubicin, daunorubicin, Etoposide and bleomycin. Those agents that arrest the G1 swell also in the S-phase locking over, for example DNA-alkylating agents, such as. Tamoxifen, prednisone, dacarbazine, Mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel (ed.), Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplasmic drugs "of Murakami et al., WB Saunders, Philadelphia (1995), in particular P. 13.

"Doxorubicin" is an anthracycline antibiotic. The complete chemical name of doxorubicin is (8S-cis) -10 - [(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl) oxy] -7,8,9,10-tetrahydro-6, 8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5,12-naphthacenedione.

The term "cytokine" is a general term for proteins released from a population of cells that act on a cell other than intracellular mediators.Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines included are wax tumor hormone, such as. Human growth hormone, human N-methionyl 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; prolactin; placental lactogen; Tumor necrosis factor-α and -β; Müllerian inhibitor; Mouse gonadotropin-associated peptide; inhibin; activin; vascular; integrin; Thrombopoietin (TPO); Nerve growth factors, such. NGF-β; Platelet-derived growth factor; transforming growth factors (TGFs) such. TGF-α and TGF-β; insulin-like growth factor-I and -II; Erythropoietin (EPO); bone-forming factors; Interferons, such as B. interferon-α, -β and -γ; colony stimulating factors (CSFs), such as Macrophage CSF (M-CSF); Granulocyte macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); Interleukins (ILs), such as. IL-1, IL-1α; IL-2; IL-3; IL-4; IL-4; IL-6; IL-7; IL-8; IL-9; IL-11; IL-12; a tumor necrosis factor, such as B. TNF-α or TNF-β; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active counterparts of the native sequence cytokines.

The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the related agent and is capable of being enzymatically activated or converted to the more active related form. see, eg, Wilman, "prodrugs in Cancer chemotherapy" Biochemical Society Transactions 14, 375-382, ^615th meeting Belfast (1986) and Stella et al, "prodrugs: A Chemical Approach to Targeted Drug Delivery."., Directed Drug Delivery, Borchardt et al., Eds., Humana Press, 247-267 (1985) The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfated prodrugs, peptide-containing prodrugs, D-amino acid derivatives. modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenylacetamide-containing prodrugs and optionally substituted pheny lacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized to a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents previously described.

An "effective Amount "of one here disclosed polypeptide or an agonist thereof with respect to Inhibition of neoplastic cell growth, tumor growth or Cancer cell growth is an amount that is capable of growth to inhibit target cells to some extent. The name comprises an amount capable of inhibiting growth, cytostatic and / or cytotoxic action and / or apoptosis of the To cause target cells. An "effective amount" of a PRO polypeptide or an agonist of it for the purposes of inhibiting neoplastic cell growth, tumor growth or cancer cell growth can be determined empirically and routinely become.

A "therapeutic effective amount "in Terms of treating a tumor refers to a lot which is capable of producing one or more of the following effects: (1) Inhibition of tumor growth to some extent, including slowing and complete Arrest of tumor growth; (2) reduction in the number of tumor cells; (3) reduction of tumor size; (4) Inhibition (i.e., reduction, slowing, or complete arrest) tumor cell infiltration into peripheral organs; (5) inhibition (i.e. H. Reduction, slowing or complete arrest) of metastasis; (6) Promotion the anti-tumor immune response, those for regression or repulsion lead the tumor can, but does not have to; and / or (7) relief in a certain sense Extent of one or more symptoms associated with the disease are. A "therapeutic effective amount of "one PRO polypeptide or an agonist thereof may be for the purpose tumor treatment can be determined empirically and routinely.

A "growth-inhibiting Amount "of a PRO antagonist is an amount that is capable of growing a cell, in particular a tumor, z. A cancer cell, either in vitro or in vivo, to inhibit. A "growth-inhibiting Amount "of a PRO antagonist can be used empirically and to inhibit neoplastic cell growth determined routinely become.

A "cytotoxic Amount "of a PRO antagonist of it is a lot that is capable of destroying one Cell, in particular a tumor, z. A cancer cell, either in vitro or in vivo. A "cytotoxic amount" of a PRO antagonist can be used empirically and to inhibit neoplastic cell growth determined routinely become.

The terms "PRO polypeptide" and "PRO" as used herein, and immediately followed by a number designation, refer to various polypeptides, wherein the full text of the claims drawing (ie, PRO / Number) to specific polypeptide sequences as described herein. The terms "PRO / number polypeptide" and "PRO / number", wherein the term "number" is provided herein as an actual numerical designation, encompass native sequence polypeptides and polypeptide variants (to be further defined herein) The PRO polypeptides described herein can be isolated from many different sources, such as human tissue types or from another source, or can be prepared by recombinant or synthetic methods.

A "native sequence PRO polypeptide" comprises a polypeptide with the same amino acid sequence like the corresponding naturally derived PRO polypeptide.

Such Native sequence PRO polypeptides can from natural Sources can be isolated or can by means of Recombination or synthesis methods are produced. The term "native sequence PRO polypeptide" includes in particular Naturally occurring, truncated or secreted forms of the specific PRO polypeptide (e.g., an extracellular domain sequence), naturally occurring Variants (eg alternatively spliced forms) and naturally occurring ones Allelic variants of the polypeptide. In various embodiments of the invention are the native sequence PRO polypeptides disclosed herein mature or full length native sequence polypeptides which include the full length amino acid sequences shown in the accompanying figures. Begin- and stop codons are bolded and underlined in the figures. While, however the PRO polypeptides disclosed in the accompanying figures, as starting with methionine residues, referred to herein as amino acid position 1 in the figures, it is also conceivable and possible that other methionine residues either upstream or downstream of the amino acid position 1 is used in the Figures as the starting amino acid residues for the PRO polypeptides become.

The "extracellular domain" of a PRO polypeptide or "ECD" refers to a form of the PRO polypeptide substantially free of the transmembrane and cytoplasmic domains. Usually points a PRO polypeptide ECD less than about 1% of such transmembrane and / or cytoplasmic domains and preferably less than 0.5% of such domains. It is understood that that every transmembrane domain, the for the PRO polypeptides of the present invention are identified according to U.S. Pat Field of the invention routinely used Identified criteria for identifying this type of hydrophobic domains become. The exact boundaries of a transmembrane domain can vary, however most likely not more than about 5 amino acids every end of the domain, as defined initially herein. If necessary, therefore, a extracellular domain of a PRO polypeptide has about 5 or fewer amino acids on each side of the border between transmembrane domain and extracellular domain as in included in the examples or description, and Such polypeptides, with or without the associated signal peptide, and nucleic acids, the for they encode are considered in the present invention.

Of the approximate Place of the "signal peptides" of the different PRO polypeptides disclosed herein is in the present description and / or the accompanying figures shown. It should be noted, however, that the C-terminal limit of a signal peptide, but very likely to vary not more than about 5 amino acids on either side of the C-terminal signal peptide border as initially herein wherein the C-terminal border of the signal peptide according to US Pat Field of the Invention for Identifying This Type of Amino Acid Sequence Element used routinely Criteria can be identified (eg Nielsen et al., Prot. Closely. 10, 1-6 (1997), and Heinje et al., Nucl. Acids. Res. 14, 4683-4690 (1986)). About that In addition, it is also recognized that in some cases, cleavage of a signal sequence from a secreted polypeptide does not take place quite uniformly, which leads to more than one secreted species. These polypeptides in which the signal peptide within not more than about 5 amino acids either side of the C-terminal border of the signal peptide as herein is cleaved and the polynucleotides encoding them are treated by the present invention.

"PRO polypeptide variant" refers to an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein extracellular domain of a PRO polypeptide, with or without signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Such PRO polypeptide variants include, for example, PRO polypeptides wherein one or more amino acid residues are present at the N- or C- Typically, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, even more preferably at least about 83% amino acid sequence identity t, more preferably at least about 84% Amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, even more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, even more preferably at least about 89% amino acid sequence identity, even more preferably at least about 90% amino acid sequence identity , even more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, even more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, even more preferably at least about 95% amino acid sequence identity, even more preferably at least about 96% amino acid sequence identity more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, and most preferably at least about 99% Am inosäuresequence identity, with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide as disclosed herein or with any other A specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Typically, PRO polypeptide variants are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about 50 in length Amino acids, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 in length More preferably, amino acids, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more.

As As shown below, Table 1 provides the complete source code for the ALIGN-2 computer program ready for sequence comparison. This source code can be routinely used for To be compiled to a UNIX operating system To provide ALIGN-2 sequence comparison computer program.

Tabelle 2A PRO XXXXXXXXXXXXXXX (Länge = 15 Aminosäuren) Vergleichsprotein XXXXXYYYYYYY (Länge = 12 Aminosäuren)
% Aminosäuresequenzidentität =
(Anzahl an identisch übereinstimmenden Aminosäureresten zwischen den zwei Polypeptidsequenzen, wie durch ALIGN-2 bestimmt) dividiert durch (Gesamtzahl an Aminosäureresten des PRO-Polypeptids) =
5 dividiert durch 15 = 33,3% Tabelle 2B PRO XXXXXXXXXX (Länge = 10 Aminosäuren) Vergleichsprotein XXXXXYYYYYYZZYZ (Länge = 15 Aminosäuren)
% Aminosäuresequenzidentität =
(Anzahl an identisch übereinstimmenden Aminosäureresten zwischen den zwei Polypeptidsequenzen, wie durch ALIGN-2 bestimmt) dividiert durch (Gesamtzahl an Aminosäureresten des PRO-Polypeptids) =
5 dividiert durch 10 = 50% Tabelle 2C PRO-DNA NNNNNNNNNNNNNN (Länge = 14 Nucleotide) Vergleichs-DNA NNNNNNLLLLLLLLLL (Länge = 16 Nucleotide)
% Nucleinsäuresequenzidentität =
(Anzahl an identisch übereinstimmenden Nucleotiden zwischen den zwei Nucleinsäuresequenzen, wie durch ALIGN-2 bestimmt) dividiert durch (Gesamtzahl an Nucleotiden der PRO-DNA-Nucleinsäuresequenz) =
6 dividiert durch 14 = 42,9% Tabelle 2D PRO-DNA NNNNNNNNNNNN (Länge = 12 Nucleotide) Vergleichs-DNA NNNNLLLVV (Länge = 9 Nucleotide)
% Nucleinsäuresequenzidentität =
(Anzahl an identisch übereinstimmenden Nucleotiden zwischen den zwei Nucleinsäuresequenzen, wie durch ALIGN-2 bestimmt) dividiert durch (Gesamtzahl an Nucleotiden der PRO-DNA-Nucleinsäuresequenz) =
4 dividiert durch 12 = 33,3%In addition, Tables 2A-2D show hypothetical examples of the use of the percent amino acid sequence identity determination method (Tables 2A-2B) and percent nucleic acid sequence identity (Tables 2C-2D) described below using the ALIGN-2 sequence comparison computer program in which "PRO" stands for the amino acid sequence of a hypothetical PRO polypeptide of interest, "control protein" means the amino acid sequence of a polypeptide to which the "PRO" polypeptide of interest is compared, "PRO DNA" for a hypothetical PRO-encoding nucleic acid sequence of interest, "reference DNA" means the nucleotide sequence of a nucleic acid molecule to which the "PRO DNA" nucleic acid molecule of interest is compared, "X", "Y" and "Z" each represent different hypothetical amino acid residues, and " N "," L "and" V "each represent different hypothetical nucleotides hen. Table 1

Table 2A PER XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparative protein XXXXXYYYYYYY (Length = 12 amino acids)
% Amino acid sequence identity
(Number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (total number of amino acid residues of the PRO polypeptide) =
5 divided by 15 = 33.3% Table 2B PER XXXXXXXXXX (Length = 10 amino acids) Comparative protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% Amino acid sequence identity
(Number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (total number of amino acid residues of the PRO polypeptide) =
5 divided by 10 = 50% Table 2C PRO-DNA nnnnnnnnnnnnnn (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% Nucleic acid sequence identity
(Number of identically matched nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (total number of nucleotides of the PRO DNA nucleic acid sequence)
6 divided by 14 = 42.9% Table 2D PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% Nucleic acid sequence identity
(Number of identically matched nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (total number of nucleotides of the PRO DNA nucleic acid sequence)
4 divided by 12 = 33.3%

"Percent (%) Amino acid sequence identity "with respect to PRO polypeptide sequences identified herein are expressed as a percentage at amino acid residues defined in a candidate sequence with the amino acid residues in a PRO sequence after alignment of the sequences and introduction of Gaps, if necessary to achieve maximum percent sequence identity, and without consideration of any conservative substitutions as part of the sequence identity are. Matching for the purpose of determining the percent amino acid sequence identity can be various types are achieved in the field of the invention are known, for example, using commonly available Computer programs such. B. BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign software (DNASTAR). Professionals can determine appropriate parameters for measuring alignment, including all Algorithms that are needed to get maximum balance over the full length to achieve the sequences to be compared. For the present purposes, however become% amino acid sequence identity values using the computer program for sequence comparison ALIGN-2 as described below, wherein the complete source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 computer program for sequence comparison was designed by Genentech, Inc., and the Source code shown in Table 1 below was user documentation in U.S. Pat. Copyright Office, Washington D.C., 20559, filed where he is under the U.S. copyright registration number TXU510087 is registered. The ALIGN-2 program is through Genentech, Inc., South San Francisco, California, publicly available or may be from the source code provided in Table 1 below be compiled. The ALIGN-2 program should be available for use a UNIX operating system, preferably digital UNIX V4.0D compiled become. All sequence comparison parameters are through the ALIGN-2 program fixed and do not vary.

For the present purposes, the% amino acid sequence identity of a particular amino acid sequence A to, with or against a particular amino acid sequence B (which may alternatively be described as a particular amino acid sequence A having or having a particular% amino acid sequence identity to or against a particular amino acid sequence B) includes) calculated as follows: 100 times the break X / Y where X is the number of amino acid residues recorded by the sequence alignment program ALIGN-2 in this alignment of the program of A and B as identical matches, and Y is the total number of amino acid residues in B. It is understood that, unless the length of amino acid sequence A is in agreement with the length of amino acid sequence B, the% amino acid sequence identity from A to B is not equal to the% amino acid sequence identity of B to A. As examples of% amino acid sequence identity calculations, Table 2A and 2B show how the% amino acid sequence identity of the amino acid sequence referred to as "control protein" is calculated to the amino acid sequence referred to as "PRO".

Except specific otherwise noted, all% amino acid sequence identity values used herein are as described above using the ALIGN-2 computer program receive. % Amino acid sequence identity values can but also using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25, 3389-3402 (1997)). The NCBI BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nig.gov become. NCBI-BLAST2 uses several search parameters, where all these search parameters are set to default values, including, for example unmask = yes, beach = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.

In situations where NCBI-BLAST2 is used for amino acid sequence comparisons, the% amino acid sequence identity of a particular amino acid sequence A is to, with or against a particular amino acid sequence B (which may alternatively be described as a particular amino acid sequence A that has a particular% amino acid sequence identity, with or against a particular amino acid sequence B) is calculated as follows: 100 times the break X / Y where X is the number of amino acid residues recorded as identical matches of the sequence matching program NCBI-BLAST2 in the program match of A and B, and Y is the total number of amino acid residues in B. It will be understood that, unless the length of amino acid sequence A corresponds to the length of amino acid sequence B, the% amino acid sequence identity from A to B is not equal to the% amino acid sequence identity of B to A.

Furthermore % amino acid sequence identity may also be below Using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266, 460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to default values. Those who are not at default values are set, d. H. the changeable Parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11 and scoring matrix = BLOSUM62. For the present purposes is a% amino acid sequence identity value Divide (a) the number of matches identical amino acid residues between the amino acid sequence the PRO polypeptide of interest having a sequence selected from the derived native PRO polypeptide, and the comparative amino acid sequence of interest (i.e., the sequence with which the PRO polypeptide of Interest, which may be a PRO polypeptide variant can) as determined by WU-BLAST-2 by (b) the total number amino acid residues of the PRO polypeptide of interest. For example, in the statement "a Polypeptide containing the amino acid sequence A comprising at least 80% amino acid sequence identity with the amino acid sequence B has "the amino acid sequence A is the comparative amino acid sequence of interest, and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.

"PRO Polynucleotide Variant" or "PRO Nucleic Acid Sequence Variant" refers to a nucleic acid molecule that encodes an active PRO polypeptide as defined below and that has at least about 80% nucleic acid sequence identity with a nucleic acid sequence that is for a full-length native sequence PRO polypeptide sequence as disclosed herein a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without Signal peptide as disclosed herein or with any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein. Typically, a PRO polynucleotide variant will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, even more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85%. Nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, even more preferably at least about 90% nucleic acid sequence identity, even more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, even more preferred r at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity, and even more preferably at least about 99% nucleic acid sequence identity, with the nucleic acid sequence which discloses a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein , or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Variants do not include the native nucleotide sequence.

Usually PRO polynucleotide variants are at least about 30 nucleotides in length, often at least one length of at least about 60 nucleotides, more often a length of at least about 90 nucleotides, more often a length of at least about 120 nucleotides, more often a length of at least about 150 nucleotides, more often a length of at least about 180 nucleotides, more often a length of at least about 210 nucleotides, more often a length of at least about 240 nucleotides, more often a length of at least about 270 nucleotides, more often a length of at least about 300 nucleotides, more often a length of at least about 450 nucleotides, more often a length of at least about 600 nucleotides, more often a length of at least about 900 nucleotides, or more.

"Percent (%) Nucleic acid sequence identity "with respect to herein identified PRO polypeptide coding nucleic acid sequences is the percentage of nucleotides in a candidate sequence defined with the nucleotides in the PRO polypeptide-encoding nucleic acid sequence of interest, after aligning the sequences and introducing Gaps, if required to achieve maximum percent sequence identity, are identical. Matching for the purpose of determining percent nucleic acid sequence identity may occur different ways can be achieved in the area of the area of the invention, for example, using public available Computer software such as BLAST, BLAST-2, ALIGN or Megalign software (DNASTAR). Professionals can determine appropriate parameters for measuring alignment, including all Algorithms that are needed to get maximum balance over the full length to achieve the sequences to be compared. For the purposes herein, however % nucleic acid sequence identity values as described below using the sequence comparison computer program ALIGN-2 determines where the full source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. and listed below in Table 1 was supplied with user documents in U.S. Pat. Copyright Office, Washington D.C., 20559, where it is filed under U.S. Copyright Registration Number TXU510087 is registered. The ALIGN-2 program is over Genentech, Inc., South San Francisco, California, Public available or can be prepared from that provided in Table 1 below Source code to be compiled. The ALIGN-2 program should be for use on a UNIX operating system, preferably digital UNIX V4.0D, be compiled. All sequence comparison parameters are determined by the ALIGN-2 program set and do not vary.

For the purposes herein, the% nucleic acid sequence identity of a particular nucleic acid sequence C to, with or against a particular nucleic acid sequence D (which may alternatively be described as a particular nucleic acid sequence C having or having a particular% nucleic acid sequence identity, with or against a particular nucleic acid sequence D) includes) calculated as follows: 100 times the break W / Z where W is the number of nucleotides that match as identical matches by the sequence ALIGN-2 are listed in the program match of C and D, and where Z is the total number of nucleotides in D. It is understood that, unless the length of nucleic acid sequence C corresponds to the length of nucleic acid sequence D, the% nucleic acid sequence identity of C to D does not equal the% nucleic acid sequence identity of D to C. As examples of% nucleic acid sequence identity calculations, Tables 2C-2D show how the% nucleic acid sequence identity of the nucleic acid sequence, referred to as the "control DNA", is calculated to the nucleic acid sequence designated as "PRO DNA".

Except specific otherwise noted, all% nucleic acid sequence identity values used herein are as described above using the ALIGN-2 computer program obtained.% nucleic acid sequence identity values can but also using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25, 3389-3402 (1997)). The NCBI BLAST2 sequence comparison program can from the address http: //www.ncbi.nlm.nih. gov be downloaded. NCBI-BLAST2 uses several search parameters, in which all these search parameters are set to default values, including unmasked, for example = yes, beach = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.

In situations where NCBI-BLAST2 is used for sequence comparisons, the% nucleic acid sequence identity of a particular nucleic acid sequence C to, with or against a particular nucleic acid sequence D (which may alternatively be described as a particular nucleic acid sequence C, which assigns a particular% nucleic acid sequence identity, with or against a particular nucleic acid sequence D or comprises) calculated as follows: 100 times the break W / Z where W is the number of nucleotides recorded as identical matches by the sequence alignment program NCBI-BLAST2 in the program alignment of C and D, and Z is the total number of nucleotides in D. It is understood that, unless the length of nucleic acid sequence C is equal to the length of nucleic acid sequence D, the% nucleic acid sequence identity of C to D does not correspond to the% nucleic acid sequence identity of D to C.

In addition,% nucleic acid sequence identity values also using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266, 460-480 (1996)). Most of the WU-BLAST-2 search parameters are at default values set. Those that are not set to default, d. H. the changeable Parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11 and scoring matrix = BLOSUM62. For the For purposes herein, a% nucleic acid sequence identity value is used Divide (a) the number of matches identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding Nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparative nucleic acid molecule of interest (i.e., the sequence with which the PRO polypeptide-encoding nucleic acid molecule of interest which may be a PRO polynucleotide variant) such as by WU-BLAST-2 determined by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest. For example, the statement "an isolated nucleic acid molecule that has a nucleic acid sequence A comprising at least 80% nucleic acid sequence identity with the nucleic acid sequence B has "the nucleic acid sequence A is the comparison nucleic acid molecule of interest, and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.

In other embodiments, PRO polynucleotide variants are nucleic acid molecules that encode an active PRO polypeptide and that are capable of hybridizing, preferably under stringent hybridization and washing conditions, to nucleotide sequences that are unique to that described in U.S. Pat 2 full-length PRO polypeptide shown (SEQ ID NO: 2). PRO polypeptide variants may be those encoded by a PRO polynucleotide variant.

The term "positives" in the context of the amino acid sequence identity comparisons performed as previously described includes not only amino acid residues in the compared sequences that are identical but also those having similar properties, amino acid residues that have a positive value to an amino acid residue of interest are those which are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 3 below) niert) of the amino acid residue of interest.

For the present purposes, the% value of positives of a particular amino acid sequence A to, with or against a particular amino acid sequence B (which may alternatively be described as a particular amino acid sequence A, which is a certain% positive, with or against a particular amino acid sequence B has or includes) calculated as follows: 100 times the break X / Y where X is the number of amino acid residues that have a positive value as previously defined by the sequence alignment program ALIGN-2 or NCBI-BLAST2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that, unless the length of amino acid sequence A is the length of amino acid sequence B, the% positives from A to B are not equal to the% positives from B to A.

"Isolated", if used, to describe the various polypeptides disclosed herein, denotes a polypeptide that is identified and separated and / or was obtained from a component of its natural environment. Preferably, the isolated polypeptide is free of compounds with all components, with which it is associated in nature. Contaminating components his natural Environment are materials that are typically diagnostic or therapeutic uses for disturb the polypeptide would and can Enzymes, hormones and other proteinaceous or non-proteinaceous solutes integrated. In preferred embodiments For example, the polypeptide (1) is used to a sufficient degree by use a centrifuge tube sequencer purified to at least 15 residues of N-terminal or internal amino acid sequence or (2) by SDS-PAGE under non-reducing or reducing Conditions by Coomassie blue or, preferably, silver staining until cleaned to homogeneity. Isolated polypeptide closes Polypeptide in situ within recombinant cells, since at least one Component of the natural Environment of PRO does not exist. Usually becomes isolated Polypeptide produced by at least one purification step.

An "isolated" for a PRO polypeptide coding nucleic acid molecule or a solubilized "coding for an anti-PRO antibody Nucleic acid molecule is a Nucleic acid molecule that identifies and separated from at least one contaminating nucleic acid molecule with which it is normally coded in the natural source of PRO nucleic acid or for Anti-PRO encoding nucleic acid is associated. Preferably, the isolated nucleic acid is free of connections with all Components with which it is associated in nature. An isolated, for PRO coding nucleic acid molecule or a insulated, for Anti-PRO coding nucleic acid molecule is located in a different form or condition than it does in nature can be found. Isolated nucleic acid molecules are therefore used by PRO encoding nucleic acid molecule or from for the Anti-PRO-encoding nucleic acid molecule, as is in natural Cells exist, differentiated. One coding for a PRO polypeptide isolated nucleic acid molecule or a for one Anti-PRO antibodies but encoding isolated nucleic acid molecule ever closes PRO nucleic acid molecules or Anti-PRO nucleic acid molecules, contained in cells, which are usually PRO polypeptides or Anti-PRO antibodies expressing, for example, the nucleic acid molecule on a other chromosomal site than in natural cells.

The Designation "Control Sequences" refers to DNA sequences used to express an operably linked coding sequence in a particular host organism are required. The control sequences, for example for Prokaryotes are suitable, include a promoter, optionally an operator sequence, and a ribosome binding site. eukaryotic Cells are known for Promoters, polyadenylation signals and enhancers.

For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a preprotein involved in the secretion of the polypeptide a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to support translation. in that the DNA sequences that are linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Binding is by ligation at appropriate restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are processed according to conventional practices applies.

The The term "antibody" is used broadly Sinn uses and covers in particular, for example, single monoclonal Anti-PRO antibodies (including Antagonist and neutralizing antibodies), anti-PRO antibody compositions with polyepitope specificity, single-chain anti-PRO antibodies and fragments of anti-PRO antibodies (see below). The term "monoclonal antibody" as used herein refers to an antibody, obtained from a population of substantially homogeneous antibodies was, d. H. that the individual antibodies that make up the population exists, are identical, with the exception of possible, naturally occurring Mutations that may be present in small amounts.

"Stringency" of hybridization reactions can Professionals easily determine and are generally based on an empirical Calculation, by probe length, Washing temperature and salt concentration depends. Generally require longer Probes higher Temperatures for correct annealing, while shorter probes lower temperatures require. Hybridization in general depends on the ability denatured DNA from reanimating when complementary strands in an environment below their melting temperature are present. The higher the degree of desired homogeneity between the probe and the hybridizable sequence is the higher also the relative temperature that can be used. As result follows that higher relative temperatures would tend to be the reaction conditions more stringent while lower temperatures would require this less. For additional details and explanations to the Stringency of hybridization reactions see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995).

"Stringent Conditions "or" Conditions high stringency "like defined herein are identified as those conditions which: (1) low ionic strength and high temperatures for use the washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C; (2) during hybridization use denaturing agent such as formamide, e.g. B. 50% by volume of formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% Polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 at 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardts Solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a high stringency wash step consisting of 0.1 × SSC, contains the EDTA, at 55 ° C, use.

"Moderately stringent Conditions "can be like by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press (1989) and close the use of washing solution and hybridization conditions (e.g., temperature, ionic strength, and % SDS) that are less stringent than those previously described were. An example for moderately stringent Conditions is overnight incubation at 37 ° C in a solution, comprising: 20% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7,6), 5 × Denhardts Solution, 10% dextran sulfate and 20 mg / ml denatured sheared salmon sperm DNA; followed by washing the filters in 1 x SSC at about 35-50 ° C. professionals will detect how to adjust temperature, ionic strength, etc. as needed are factors such as probe length and the like.

Of the The term "epitope tagged" refers to Use herein of a hybrid polypeptide comprising a PRO polypeptide fused to a "marker polypeptide" includes. The marker polypeptide has enough residues to provide an epitope, against that an antibody can be prepared, however, it is short enough that it minimizes the activity of the polypeptide, it is merged does not bother. The marker polypeptide is also preferably pretty unique, so the antibody is essentially not cross-reacted with other epitopes. Suitable marker polypeptides generally have at least six amino acid residues, and usually between about 8 and 50 amino acid residues on (preferably between about 10 and 20 amino acid residues).

"Active" or "Activity" for the purpose herein refers to the form (s) of PRO polypeptides having an immunological Activity / property a native or natural occurring PRO polypeptide, wherein "immunological" activity on the ability refers to the production of an antibody to an antigenic epitope induce that from a native or naturally occurring PRO polypeptide is shown.

The Designation "immunological Activity "refers to immunological cross-reactivity with at least one epitope of a PRO polypeptide.

"Immunological Cross-reactivity "means when used herein that the candidate polypeptide is capable of producing the qualitative biological activity a PRO polypeptide having this activity with polyclonal, against the known active PRO polypeptide competitively inhibited antisera. Such antisera be on conventional By subcutaneous injection of, for example, goats or rabbits with the known active analog in complete Freund's adjuvant, followed by an intraperitoneal or subcutaneous booster injection in incomplete Freund's adjuvant. The immunological Cross-reactivity is preferably "specific," meaning that the binding affinity of the identified, immunologically cross-reactive molecule (eg. Antibody) across from the corresponding PRO polypeptide significantly higher (preferably at least about twice, more preferably at least about four times, more preferably at least about six times, most preferably at least about eight times, higher) as the binding affinity of this molecule to any other known native polypeptide.

A small Molecule "is herein with a Molecular weight defined as less than about 500 daltons.

"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins, which have the same structural properties. While antibodies a binding specificity have a specific antigen, immunoglobulins include both Antibody, as well as antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, in low dimensions produced by the lymphatic system and in increased degrees of myeloma. The term "antibody becomes used in the broadest sense and includes intact without limitation monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies), which are formed from at least two intact antibodies, and Antibody fragments, as long as you want biological activity exhibit.

"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain through a covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has interchain disulfide bridges at regular intervals. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has at one end a variable domain (V _L ) and a constant domain at its other end; the light chain constant domain is aligned with the first heavy chain constant domain and the variable domain of the light chain is aligned with the heavy chain variable domain. Certain amino acid residues are believed to be an interface between the light and heavy chain variable domains.

Of the Expression "variable" refers to the fact that certain sections of the variable domain are up under antibodies differ greatly in their sequence and in binding and in the specificity of each single antibody to be respective antigen can be used. However, the variability is not uniform over the variable domains of antibodies distributed. It is condensed into three segments, which determine complementarity Regions (CDRs) or hypervariable regions, and though in the variable domains both the light chain and the heavy chain. The more conserved Sections of variable domains become scaffolding regions Called (FR). The variable domains native heavy and light chains each comprise four FR regions, the mostly a β-sheet configuration occupied by three CDRs forming loops, which is the β-sheet structure connect and in some cases form part of it. The CDRs in each chain are replaced by the FR regions are kept close to each other and contribute with the CDRs another chain for the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publication No. 91-3242, Vol. 1, pages 647-669 (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, However, show different effector functions, such. Eg participation of the antibody on antibody-dependent cell toxicity.

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody responsible for antigen binding The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (ie residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable heavy chain domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and / or "hypervariable loop" residues (ie residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable heavy chain domain; Chothia & Lesk, J. Mol. Biol. 196, 901-917 (1987)). "Framework" or "FR" residues are those variable domain residues that are not the hypervariable region residues as defined herein.

"Antibody fragments" include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8 (10), 1057-1062 (1995)); single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, both having a single antigen binding site, and a remaining "Fc" fragment, the name of which reflects its ability to readily crystallize. Pepsin treatment yields an F (ab ') ₂ fragment which has two antigen-combining sites and is still capable of cross-linking antigen.

"Fv" is the minimal antibody fragment that contains a complete antigen recognition and antigen binding site, which consists of a dimer of a variable heavy chain and a light chain variable domain in a tight non-covalent link. It is this configuration in which the three CDRs of each variable Together, the six CDRs confer antigen-binding specificity on the antibody to the antibody, however, even a single domain (or half of one Fv, which has only three for one) interacts to define an antigen-binding site on the surface of the V _H -V _L dimer Antigen-specific CDRs) includes the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

The Fab fragment also contains the light chain constant domain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab 'fragments by the addition of a few residues at the carboxy-terminus of the heavy chain CH1 domain, including two cysteines from the antibody hinge region. Fab'-SH herein is the designation for Fab 'in which the cysteine residue (s) of the constant domains carry a free thiol group. F (ab ') ₂ antibody fragments were originally produced as pairs of Fab' fragments having hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be one of two clearly distinguishable Types are assigned based on the amino acid sequences their constant domains Kappa (κ) and lambda (λ) to be named.

In dependence from the amino acid sequence the constant domain their heavy chains can Immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can further divided into subclasses (isotypes), e.g. Eg IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The constant domains of Heavy chains that correspond to the different immunoglobulin classes, are called α, δ, ε, γ and μ. The subunit structures and three-dimensional configurations various immunoglobulin classes are well known.

The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations which are minor Monoclonal antibodies are highly specific and are directed against a single antigenic site, and in contrast to conventional (polyclonal) antibody preparations, which typically comprise several antibodies directed against different antigens (epitopes), each monoclonal antibody is directed against one In addition to their specificity, the monoclonal antibodies are advantageous because they are not synthesized by other immunoglobulins contaminated by the hybridoma culture.The modifier "monoclonal" characterizes the character of the antibody in that it is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975) or may be prepared by recombinant DNA technology (see, e.g. U.S. Patent No. 4,816,567 ). For example, the "monoclonal antibodies" can also be obtained from phage antibody libraries using the methods described in Clackson et al., Nature 352, 624-628 (1991) and US Pat Marks et al., J. Mol. Biol. 222, 581-597 (1991).

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class of antibody or subclass while the remainder of the chain (s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to a different class of antibody or subclass, as well as fragments of such antibodies, as long as they have 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 (eg, mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F (ab') _2, or other antigen-binding subsequences of antibodies) For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a CDR of the recipient are expressed by residues from a CDR of a non-human species (donor antibody), such as human donor antibodies For example, mouse, rat, or rabbit with the desired specificity, affinity, and capacity are replaced, In some cases, Fv-FR residues of the human immunoglobulin are replaced with corresponding non-human residues, and humanized antibodies may include residues that are not found in the mouse Recipient antibodies, still found in the imported CDR or framework sequences ability of the antibody to further refine and maximize. In general, the humanized antibody will comprise substantially all of at least one and typically two variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human Immunoglobulin sequence. The humanized antibody will also optionally comprise at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al., Nature 321, 522-525 (1986); Reichmann et al., Nature 332, 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992). The humanized antibody comprises a PRIMATIZED ^™ antibody, wherein the antigen-binding region of the antibody is derived from an antibody prepared by immunizing macaque monkeys with the antigen of interest.

"Single chain Fv" or "sFv" antibody fragments include the V _H and V _L antibody domains wherein these domains are in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V _H. and V _L domains, allowing the sFv to form the desired structure for antigen binding. For a review, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small antibody fragments having two antigen binding sites, the fragments comprising a variable heavy chain domain (V _H ) linked to a variable light chain domain (V _L ) in the same polypeptide chain (V _H -V _L ) For example, if a linker is too short to allow pairing between the two domains on the same chains, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites EP 404,097 ; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448 (1993).

An "isolated" antibody is one that identifies and consists of a component of its natural Environment has been separated and / or recovered. contaminating Components of his natural Surroundings are materials that are diagnostic and therapeutic Uses for the antibody to disturb would and can Enzymes, hormones and other proteinaceous or non-proteinaceous solutes include. In preferred embodiments becomes the antibody purified, namely (1) to more than 95% by weight of antibodies, such as determined by Lowry method, and particularly preferred more than 99% by weight, (2) to an extent sufficient to at least 15 residues of the N-terminal or internal amino acid sequence by use a centrifuge tube sequencer or (3) to homogeneity by SDS-PAGE under reducing and non-reducing conditions using Coomassie blue or preferably silver staining. An isolated antibody includes the antibody in situ in recombinant cells, since at least one component of the natural Environment of the antibody will not be available. For usually however, becomes an isolated antibody produced by at least one cleaning step.

The term "marker" as used herein refers to a detectable compound or to composition which is directly or indirectly conjugated to the antibody to produce a "labeled" antibody The marker may be itself detectable (eg radioisotope marker or fluorescent label) or, in the case of an enzymatic marker, may be a chemical modification of a substrate compound or radionuclides which can serve as detectable markers include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212 and Pd-109 The marker may also be an undetectable entity, such as a toxin.

By "solid phase" is meant a nonaqueous matrix to which the antibody of the present invention can adhere Examples of solid phases contemplated herein include those which are partially or fully glass (e.g., controlled poreglass), polysaccharides (e.g. In some embodiments, depending on the context, the solid phase may comprise the well of a test plate, in others it may be a purification column (e.g., an affinity chromatography column) Solid phase of separated particles, such as those in the U.S. Patent No. 4,275,149 described.

A "liposome" is a small one Vesicles consisting of different lipid types, phospholipids and / or Surfactants composed for the delivery of a drug (such as z. A PRO polypeptide or antibody, and optionally a therapeutic agent) to a mammal. The Components of the liposome are usually similar to Lipidanordnung biological membranes arranged in a double layer.

As As used herein, the term "immunoadhesin" refers to antibody-like molecules which comprise the binding specificity of a heterologous protein (an "adhesin") combined 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 not that of the antigen recognition and binding site of an antibody (i.e., "heterologous") with the sequence a constant immunoglobulin domain. The adhesin portion of an immunoglobulin molecule is typical a coherent one Amino acid sequence which comprises at least the binding site of a receptor or ligand. The sequence of the immunoglobulin constant domain in the immunoadhesin can from each immunoglobulin, such. IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgG (including IgA-1 and IgA-2), IgE, IgD or IgM.

II. Compositions and Methods

A. Full-length PRO polypeptides

Here in Nucleotide sequences encoding polypeptides are described to be referred to as PRO in the present application. More accurate said cDNA was used for PRO polypeptides are encoded, identified and siolated as described in U.S. Pat the following examples is disclosed in more detail. It should be noted that proteins that produce in separate expression passages were able to get different PRO numbers lent that however, the UNQ number for each particular DNA and the encoded protein is unique and not changed becomes. For the sake of simplicity, in the present description the proteins, for which encode the nucleic acid sequences disclosed herein, as well as more native homologs and variants, in the above definition PRO4980 or PRO, referred to as "PRO4980" or "PRO", regardless of their origin or the method of production.

As disclosed in the examples below, cDNA clones were used in the ATCC deposited. The actual Nucleotide sequences of these clones can be sequenced by those skilled in the art of the deposited clones by means of methods used in the field of Invention are standard, easy to be determined. The predicted amino acid sequence can be determined from the nucleotide sequences by standard methods become. For the PRO polypeptides described herein and the ones coding for them nucleic acid Applicants were able to identify what was considered the reading frame It is assumed that he is the best available at the time Sequence information was identifiable.

B. PRO variants

In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO variants can be produced. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA and / or by synthesis of the desired PRO polypeptide. It will be appreciated by those skilled in the art that amino acid changes can alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring properties.

Variations in the native full-length sequence PRO or in different domains of the PRO described herein can be made, for example, using all methods and guidelines for conservative and non-conservative mutations described, for example, in U.S. Pat U.S. Patent No. 5,364,934 to be discribed. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that result in a change in the amino acid sequence of the PRO as compared to the native sequence PRO. Optionally, this variation occurs by substitution of at least one amino acid with each other amino acid in one or more domains of the PRO. Assistance in determining which amino acid residue can be inserted, substituted or deleted without negatively affecting the desired activity can be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes in regions of high homology. Amino acid substitutions can be made by replacing one amino acid with another amino acid having similar structural and / or chemical properties, for example, by replacing a leucine with a serine, ie by conservative amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The allowed variation can be determined by systematically making insertions, deletions or substitutions of amino acids into the sequence and testing the resulting variants for activity showing the full length or mature native sequence.

PRO polypeptide fragments are disclosed herein. Such fragments may be truncated at the N-terminus or C-terminus, or you can, for example, compared to a full-length native protein, located inside Remains are missing. Certain fragments lack amino acid residues, the for a desired one biological activity of the PRO polypeptide are not essential.

PRO fragments can produced by any number of conventional methods become. desirable Peptide fragments can be chemically synthesized. An alternative approach involves the Formation of PRO fragments by enzymatic digestion, e.g. B. by Treatment of the protein with an enzyme known to be proteins to split at sites defined by certain amino acid residues or by digestion of the DNA with appropriate restriction enzymes and isolating the desired one Fragment. Again another useful method isolating and amplifying a DNA fragment that is desired Polypeptide fragment encoded by polymerase chain reaction (PCR). Oligonucleotides containing the desired Termini of the DNA fragment are defined on the 5 'and 3' primers in the PCR used. Preferably, PRO polypeptide fragments share at least a biological and / or immunological activity with the native PRO polypeptide.

In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading "Preferred Substitutions." If such substitutions result in a change in biological activity, more substantial changes, referred to in Table 3 as "Exemplary Substitutions," or as discussed below Reference to amino acid classes is described in more detail, introduced and the products screened. Table 3 original exemplary preferred rest substitutions substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; bad gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) Per; ala ala His (H) asn; gln; lys; bad bad Ile (I) leu; val; met; ala; phe; norleucine leu Lei (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn bad Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

• (1) hydrophob: Norleucin, met, ala, val, leu, ile;
• (2) neutral hydrophil: cys, ser, thr;
• (3) sauer: asp, glu;
• (4) basisch: asn, gln, his, lys, arg;
• (5) Reste, die Kettenausrichtung beeinflussen: gly, pro; und
• (6) aromatisch: trp, tyr, phe.

Significant modifications in function or immunological identity of the polypeptide are made by selecting for substitutions that have an effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, for example in the form of a sheet or helical conformation, (b) the charge or Hydrophobicity of the molecule at the target site or (c) the volume of Sei tenkette significantly different. Naturally occurring residues are grouped into the following groups based on common side-chain properties:

• (1) hydrophobic: norleucine, met, ala, val, leu, ile;
• (2) neutral hydrophilic: cys, ser, thr;
• (3) acid: asp, glu;
• (4) basic: asn, gln, his, lys, arg;
• (5) residues affecting chain orientation: gly, pro; and
• (6) aromatic: trp, tyr, phe.

Non-conservative Substitutions require the exchange of a member of one of these Classes against another class. Such substituted radicals can also into the conservative substitution sites or, more preferably, into the remaining (non-conserved) posts.

The Variations can using methods known in the art, such as for example, oligonucleotide-mediated (site-directed) mutagenesis, Alanine scanning and PCR mutagenesis. Site-directed Mutagenesis (Carter et al., Nucl. Acids Res. 13, 4331 (1986); Zoller et al., Nucl. Acids Res. 10, 6487 (1987)), cassette mutagenesis (Wells et al., Gene 34, 315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317, 415 (1986)) or Other known methods can performed on the cloned DNA to form the PRO DNA variant.

Scanning amino acid analysis can also be used to identify one or more amino acids along with a contiguous sequence. Preferred scanning amino acids include relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred scanning amino acid in this group because it eliminates the side chain beyond the β-carbon and is less likely to alter the major chain conformation of the variant (Cunningham & Wells, Science 244, 1081-1085 (1989)). Typically, alanine is also preferred as it is the most abundant amino acid. Furthermore, it often becomes both hidden and off Exposing positions have been found (Creighton, The Proteins (WH Freeman & Co., NY); Chothia, J. Mol. Biol. 150, 1 (1976)). If alanine substitution does not yield adequate amounts of variants, then an isoteric amino acid may be used.

C. Modifications of PRO

covalent Modifications of PRO are within the scope of this invention involved. One type of covalent modification involves the reaction directed amino acid residues a PRO polypeptide with an organic derivatizing agent, that is able to with selected Side chains or the N- or C-terminal residues of the PRO to react. Derivatization with bifunctional agents is useful for example, to crosslink PRO with a water-insoluble support matrix or surface for use in the process of purifying anti-PRO antibodies and vice versa. Usually used crosslinkers include, for. For example, 1,1-bis (diazoacetyl) -2-phenylethane, Glutaraldehyde, N-hydroxysuccinimide ester, for example esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters, such as B. 3,3'-dithiobis (succinimidyl propionate), bifunctional maleimides, such as. Bis-N-maleinimido-1,8-octane, and means, such as. For example, methyl 3 - [(p-azidophenyl) dithio] propioimidate.

Other Modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl or aspartyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxy groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 79-86 (1983)), Acetylation of the N-terminal amine and amidation of any C-terminal carboxy group.

A Another type of covalent modification of the PRO polypeptide involves altering the native glycosylation pattern of the polypeptide. "Changing the native glycosylation pattern" means for the present Purposes of deleting one or more carbohydrate moieties, found in native sequence PRO (either by removal the underlying glycosylation site or by deletion glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites present in the native sequence PRO can not be found. About that In addition, this designation also binds qualitative changes on the glycosylation of native proteins, including a change the nature and proportions of the various existing ones Carbohydrate moieties.

The Add from glycosylation sites to the PRO polypeptide can be altered by altering the amino acid sequence respectively. The change For example, by the addition of or the substitution by one or more serine or threonine residues to or at the native sequence PRO (for O-bound Glycosylation sites) become. The PRO amino acid sequence may be subject to change changed to DNA level especially by mutation of the DNA coding for the PRO polypeptide coded, pre-selected Bases so that codons are formed which are the desired ones amino acids translate.

Another means for increasing the number of carbohydrate moieties on the PRO polypeptide is chemically or enzymatically binding glycosides to the polypeptide. Such methods are used in the field of the invention, e.g. B. in the WO 87/05330 , published September 11, 1987, and in Aplin & Wriston, CRC Crit. Rev. Biochem., 259-306 (1981).

The Removal of carbohydrate moieties present on the PRO polypeptide can be chemically or enzymatically or by mutational substitutions of codons for amino acid residues encode that as targets for Serve glycosylation performed become. Chemical deglycosylation processes are in the field of the invention are known, for example, from Hakimuddin et al., Arch. Bio chem. Biophys. 259, 52 (1987), and by Edge et al., Anal. Biochem. 118, 131 (1981). Enzymatic cleavage Carbohydrate moieties on polypeptides can be detected by use a variety of endo- and exo-glycosidases can be achieved, such as by Thotakura et al. in: Meth. Enzymol. 138, 350 (1987) becomes.

Another type of covalent modification of PRO involves binding of the PRO polypeptide to any of a variety of non-proteinaceous polymers, e.g. As polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes, in the manner described in the U.S. Patent No. 4,640,835 ; 4496689 ; 4301144 ; 4670417 ; 4791192 or 4179337 is described.

The PRO can also be modified in a way that a hybrid molecule is formed PRO fused to another heterologous polypeptide or other heterologous amino acid sequence.

Such a hybrid molecule optionally comprises a fusion of the PRO polypeptide with a Marker polypeptide which provides an epitope to which a Anti-marker antibody selective can bind. The epitope tag is generally attached to the amino or carboxy-terminus of the PRO polypeptide. The presence Such epitope-tagged forms of the PRO polypeptide may be prepared using of an antibody against the marker polypeptide are detected. The provision of the Epitope tag allows It thus also that the PRO easily by means of affinity purification using an anti-marker antibody or other type Affinity Matrix, which binds to the epitope tag, can be purified. Various Marker polypeptides and their respective antibodies are in the field of Invention known. Examples include poly-histidine (poly-his-) or poly-histidine-glycine (poly-his-gly) marker; the flu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol. 8, 2159-2165 (1988)); the c-myc marker and antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 (Evan et al., Molecular and Cellular Biology 5, 3610-3616 (1985)); and the herpes simplex virus glycoprotein D (gD) marker and its antibody (Paborsky et al., Protein Engineering 3 (6), 547-553 (1990)). Other marker polypeptides include the flag peptide (Hopp et al., BioTechnology 6, 1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science 255, 192-194 (1992)); an α-tubulin epitope peptide (Skinner et al., J. Biol. Chem. 266, 15163-15166 (1991)); and the T7 gene 10 protein peptide marker (Lutz-Freyermuth et al., Proc Natl Acad Sci USA 87, 6393-6397 (1990)).

The hybrid molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin. For a divalent form of the hybrid molecule (also referred to as an "immunoadhesin"), such a fusion could be formed to the Fc region of an IgG molecule The Ig fusions preferably comprise the substitution of a soluble form (deleted or inactivated transmembrane domain) of a PRO molecule. In one particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3 or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule further information on the production of immunoglobulin fusions see also U.S. Patent No. 5,428,130 , issued on June 27, 1995.

D. Preparation of PRO Polypeptides

The The following description relates primarily to the production of PRO by culturing cells containing a PRO nucleic acid-containing vector are transformed or transfected. Of course, it is considered alternative Processes known in the art for manufacturing can be used by PRO. For example, you can the PRO sequence or parts thereof by direct peptide synthesis under Use of solid phase processes are prepared (see, for. Stewart et al., Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc. 85, 2149-2154 (1963)). In vitro protein synthesis can be performed using manual or automated methods. Automated synthesis can be carried out using, for example, a Applied Biosystems Peptide Synthesizer (Foster City, CA) according to instructions made by the manufacturer. Different parts of PRO can be sold separately chemically synthesized and by means of chemical or enzymatic Procedures are combined to form the full-length PRO.

a. Isolation of coding for a PRO polypeptide DNA

DNA, the for PRO can be obtained from a cDNA library, the made of fabric, which is believed to be the PRO mRNA and expressing them at a detectable level. Accordingly, human PRO DNA can be easily obtained from a cDNA library consisting of human tissue is made, as in the examples is described. That for PRO coding gene can also be obtained from a genomic library or be obtained by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the PRO polypeptide or oligonucleotides of at least about 20-80 bases) whose purpose is to identify the gene of interest or the protein encoded by that gene. Screening of the cDNA or genomic library with the selected probe can be performed using standard methods, e.g. In Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press (1989)). An alternative means of isolating the PRO coding gene is the use of the PCR method (Sambrook et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1995))).

The following examples describe methods for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently ambiguous so that false positives are minimized. The oligonucleotide is preferably labeled so that it can be detected by hybridization to DNA in the library to be screened. Labeling methods are known in the art and include the use of radioactive labels such as e.g. ³² P-labeled ATP, biotinylation or enzyme label. Hybridization conditions, including moderate stringency and high stringency, are described in Sambrook et al., Supra.

sequences identified in such library screening procedures, can with other known sequences used in public databases like z. GenBank or other private sequence databases and accessible are to be compared and compared. Sequence identity (either on Amino acid- or nucleotide plane) within defined regions of the molecule or via the entire full-length sequence This may be accomplished by methods known in the art and as herein be determined described.

Nucleic acid with Protein coding sequence can be determined by screening selected cDNA or genomic libraries using the first herein Time revealed, deduced amino acid sequence and, if necessary, using conventional Primer extension procedure as in Sambrook et al., S. o., described, forerunner and to detect processing intermediates of mRNA that may be were not reverse transcribed into cDNA, can be recovered.

b. Selection and transformation of host cells

host cells with expression or cloning vectors herein for PRO production be described, transfected or transformed and in conventional broth cultivated for inducing promoters, for the selection of Transformants or amplification of the genes responsible for the desired Encoding sequences is suitably modified. The culture conditions, such as As medium, temperature, pH and the like, can from Professionals without excessive experimentation selected become. In general, you can Principles, working instructions and practical maximization techniques productivity of Cell Cultures in Mammalian Cell Biotechnology: A Practical Approach, M. Butler (ed.), IRL Press (1991), and in Sambrook et al., P. o., to be found.

Methods for eukaryotic cell transfection and prokaryotic cell transformation are known to those of ordinary skill in the art, e.g. As CaCl ₂ method, CaPO ₄ method, liposome-mediated method and electroporation. Depending on the host cells used, transformation is performed using standard techniques suitable for the cells in question. Calcium treatment with calcium chloride as described in Sambrook et al., Supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used to transform certain plant cells, such as Shaw et al., Gene 23, 315 (1983), and the WO 89/05859 , published June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham & van der Eb, Virology 52, 456-457 (1978), can be used. General aspects of mammalian cell host system transfections are described in U.S. Patent No. 4,399,216 described. Yeast transformations are typically performed according to the method of Van Solingen et al., J. Bact. 130, 946 (1977), and Hsiao et al., Proc. Natl. Acad. Sci. (USA) 76, 3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g. As polybrene, polyornithine, are used. For various methods of transforming mammalian cells, see Keown et al., Methods in Enzymology 185, 527-537 (1990), and Mansour et al., Nature 336, 348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vector (s) herein include prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria, e.g. B. gram-negative or gram-positive organisms, such as Enterobacteriaceae such. E. coli. Various E. coli strains are publicly available, such as. E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and E. coli strain K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescans, and Shigella and Bacilli such. BB subtilis and B. licheniformis (e.g., BB licheniformis 41P, disclosed in U.S. Pat DD 266,710 , published April 12, 1989), Pseudomonas, such as BP aeruginosa, and Streptomyces. These examples are illustrative and not restrictive. Strain W3110 is a particularly preferred host or host since it is a common host strain for fermentation of recombinant DNA products. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to effect a genetic mutation in the genes encoding the host endogenous proteins, examples of such hosts being E. coli W3110 strain 1A2 having the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r ; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7ilvG kan ^r ; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain with mutated periplasmic protease disclosed in U.S. Pat U.S. Patent No. 4,946,783 , issued on August 7, 1990, are. Alternatively, in vitro cloning procedures, e.g. PCR or other nucleic acid polymerase reactions.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a commonly used, low-eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach & Nurse, Nature 290, 140 (1981); EP 139,383 , published May 2, 1985); Kluyveromyces hosts ( U.S. Patent No. 4,943,529 ; Fleer et al., Bio / Technology 9, 968-975 (1991)) such as. BK lactis (MW98-8C, CBS683, CBS4574, Louvencourt et al., J. Bacteriol 154 (2), 737-742 (1983)), K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K Wickeramii (ATCC 24.178), K.Waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906, Van den Berg et al., Bio / Technology 8, 135 (1990)), K. thermotolerans and K. marxianus; yarrowia ( EP 402,226 ); Pichia pastoris ( EP 183,070 ; Sreekrishna et al., J. Basic Microbiol. 28, 265-278 (1988)); Candida; Trichoderma reesia ( EP 244,234 ); Neurospora crassa (Case et al., Proc Natl Acad Sci USA 76, 5339-5363 (1979)); Schwanniomyces such. B. Schwanniomyces occidentalis ( EP 394,538 , published October 31, 1990); and filamentous fungi such. Neurospora, Penicillium, Tolypocladium ( WO 91/00357 , published on January 10, 1991) and Aspergillus hosts such. BA nidulans (Ballance et al., Biochem Biophys Res. Commun 112, 284-289 (1983); Tilburn et al., Gene 26, 205-221 (1983); Yelton et al., Proc. Natl. Acad Sci USA 81, 1470-1474 (1984)) and A. niger (Kelly & Hynes, EMBO J. 4, 475-479 (1985)). Methylotrophic yeasts are suitable herein and include, but are not limited to, yeast capable of growing on methanol selected from the genera of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula. A list of specific species exemplary of this class of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

suitable Host cells for The expression of glycosylated PRO are from multicellular organisms derived. examples for Invertebrate cells include insect cells such. B. Drosophila S2 and Spodoptera Sf9 as well as plant cells. Examples of useful Mammalian host cell lines include Chinese Hamster Ovary (CHO) and COS cells. More specific Examples include monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells, subcloned for growth in suspension culture, Graham et al., J. Gen virol. 36, 59 (1977)); Chinese hamster ovary cells / -DHFR (CHO, Holiday & Chasin, Proc. Natl. Acad. Sci. USA 77, 4216 (1980)); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23, 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). It is considered that the selection of the appropriate host cell is within the scope of the invention falls.

c. Selection and use of a replicable vector

The nucleic acid (eg, cDNA or genomic DNA) encoding PRO can be cloned (Amplification of the DNA) or for expression in a replicable Vector to be inserted. Different vectors are public available. For example, the vector may be in the form of a plasmid, cosmid, viral particles or phages. The appropriate nucleic acid sequence can be inserted into the vector by numerous different methods become. In general, DNA is inserted into a suitable restriction endonuclease site (s). by means of methods known in the art. Vector components generally include, but are not limited to one or more signal sequences, an origin of replication or multiple marker genes, an enhancer element, a promoter and a transcription termination sequence. In the construction of suitable Vectors containing one or more of these components are used conventional Ligation methods used, which are known to those skilled in the art.

The PRO can be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide containing a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector or may be part of the DNA encoding PRO that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leaders. For yeast secretion, the signal sequence z. Yeast invertase leader, α-factor leader (including Saccharomyces and Kluyveromyces α-factor leader, the latter being in the U.S. Patent No. 5,010,182 or acid phosphatase leader, the C. albicans glucoamylase leader ( EP 362,179 , published on 4 April 1990) or in the WO 90/13646 , published November 15, 1990, described signal. For mammalian cell expression, mammalian signal sequences can be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or a related species, as well as viral secretory leaders.

Either Expression as well as cloning vectors contain a nucleic acid sequence, which makes it possible that the vector is in one or more of the secreted host cells replicated. Such sequences are for many different bacteria, yeasts and viruses known. The origin of replication from the plasmid pBR322 is for most gram-negative bacteria suitable, the 2μ plasmid origin is for Yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are for Cloning vectors in mammalian cells useful.

expression and cloning vectors typically contain a selection gene, which is also referred to as a selectable marker. Typical selection genes code for Proteins that are (a) resistant to antibiotics or others Toxins, e.g. Ampicillin, neomycin, methotrexate or tetracycline, confer, (b) auxotrophic deficiencies fix or (c) essential nutrients, which are not available from complex medium, z. As that for D-alanine racemase for Bacilli encode gene.

One example for suitable selectable marker for mammalian cells are those that enable the identification of cells that are able the for PRO encoding nucleic acid to record, such. B. DHFR or thymidine kinase. A suitable Host cell, if wild-type DHFR is used, is the CHO cell line DHFR activity is missing and as described by Urlaub et al., Proc. Natl. Acad. Sci. USA 77, 4216 (1980), prepared and propagated. A suitable one Selection gene for use in yeast is the trp1 gene found in the yeast plasmid YRp7 is present (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Chemper et al., Gene 10, 157 (1980)). The trp1 gene provides a selection marker for a mutated yeast strain, the ability lacking to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)).

Expression and cloning vectors usually contain a promoter operably linked to the PRO coding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by many different potential host cells are known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., Nature 275, 615 (1978), Goeddel et al., Nature 281, 544 (1979)), alkaline Phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res. 8, 4057 (1980); EP 36,776 ) and hybrid promoters such. The tac promoter (deBoer et al., Proc Natl Acad Sci USA 80, 21-25 (1983)). Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence which is operably linked to the PRO-encoding DNA.

Examples for suitable Promoter 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 (Ness et al., J. Adv. Enzyme Reg. 7, 149 (1968); Holland, Biochemistry 17, 4900 (1978)) such. Enolase, glyceraldehyde-3-phosphate dehydrogenase, Hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters which are inducible promoters and have the added advantage of being driven by growth conditions in their transcription are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation 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 become more apparent EP 73,657 described.

PRO transcription from vectors in mammalian host cells is carried out, for example, by promoters controls that are derived from the genomes of viruses such. B. Polyomavirus, fowlpox virus ( UK 2,211,504 , published July 5, 1989), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, 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 that such promoters are compatible with the host cell systems.

transcription a DNA for the PRO coded by higher ones Eukaryotes can be made by inserting an enhancer sequence into the vector be increased. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp that act on a promoter that its Transcription is increased. Numerous enhancer sequences are from mammalian genes known (globin, elastase, albumin, α-fetoprotein and insulin). typically, however, an eukaryotic cell virus enhancer is used. Examples include the SV40 enhancer on the late side of the replication origin (bpp 100-270), the early cytomegalovirus promoter enhancer, the polyoma enhancer at the late Side of the replication origin and adenovirus enhancer. The enhancer can be spliced into the vector at a position 5 'or 3' to the PRO coding sequence, However, it is preferably arranged at a position 5 'of the promoter.

Expression vectors in eukaryotic host cells (yeast, fungus, insect, plant, Animal, human or nucleated cells from other multicellular Organisms) also contain sequences that are used for Completion of transcription and stabilization of mRNA required are. Such sequences are common from the untranslated 5'- and optionally 3 'regions eukaryotic or viral DNA or cDNA. These regions contain Nucleotide segments that are polyadenylated fragments in the untranslated Section of for Be transcribed PRO coded mRNA.

Other methods, vectors and host cells suitable for adaptation to the synthesis of PRO in recombinant vertebrate cell culture are described in Gething et al., Nature 293, 620-625 (1981); Mantei et al., Nature 281, 40-46 (1979); EP 117,060 ; and EP 117,058 described.

d. Detection of gene amplification / expression

Gene Amplification and / or -expression may be based on those provided herein Sequences are measured directly in a sample, z. B. by conventional Southern blotting, Northern blotting to quantitate transcription of mRNA (Thomas, Proc Natl Acad Sci., USA 77, 5201-5205 (1980)), dot blotting (DNA analysis) or In situ hybridization using a suitable labeled probe. alternative can do this antibody used, the specific Duplices, including DNA Duplices, RNA Duplices and DNA-RNA hybrid duplexes or DNA protein duplexes. The antibody turn can be marked, and the test can be performed when the duplex to a surface is bound so that when forming duplex on the surface of the Presence of antibody, which is bound to the duplex can be detected.

gene expression may alternatively by immunological methods, such as immunohistochemical staining of cells or tissue sections and tests of cell culture or body fluids, can be measured to quantify the expression of gene product directly. Antibody, the for immunohistochemical staining and / or testing of sample fluids useful are, can either monoclonal or polyclonal and can be in any mammal getting produced. In a simple way, the antibodies can be used against a native sequence PRO polypeptide or against a synthetic peptide based on those provided herein DNA sequences, or against exogenous sequence, fused to PRO DNA and for a specific antibody epitope to be produced.

e. Purification of a polypeptide

to shape from PRO can be obtained from a culture medium or from host cell lysates. If membrane bound, it may be prepared using a suitable surfactant solution (eg Triton-X 100) or by enzymatic cleavage from the membrane be released. Cells used for the expression of PRO can, can by means of various physical or chemical means, such as z. B. freeze-thaw cycling, sonication, mechanical digestion or cell lysing agents, to be disrupted.

It may be desirable to purify PRO from recombinant cell proteins or polypeptides. The following are examples of suitable purification procedures: fractionation on an ion exchange column; Ethanol precipitation; Reverse phase HPLC; Chromatography on silica or on a cation exchange resin such. B. DEAE; chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; Gel filtration under Using, for example, Sephadex G-75; Protein A Sepharose columns for removal of contaminants such as IgG; and metal chelator columns for binding epitope-tagged forms of the PRO. Various methods of protein purification can be used and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The selected cleaning step (s) depends, for example, on the nature of the manufacturing process used and the particular PRO produced.

E. Amplification of genes encoding PRO polypeptides encode, in tumor tissue and cell lines

The present invention is based on identification and characterization of genes that are amplified in certain cancer cells.

The Genome of prokaryotic and eukaryotic organisms is apparently two contradictory requirements subjected. One of them is the preservation and propagation of DNA as genetic information in its original form to a stable Inheritance over to ensure several generations. On the other hand Cells and organisms will be able to sustain sustainable environmental changes adapt. The adaptation mechanisms can be qualitative or quantitative Modifications of the genetic material include. qualitative Modifications include DNA mutations involving coding sequences changed be what happens in a structurally and / or functionally different way Protein results. Gene amplification is a quantitative change which causes the actual Number of complete coding sequence, d. h., a gene increases, resulting in an increased number available Template for the transcription, an elevated Number of translatable transcripts and ultimately to an increased amount of the protein encoded by the amplified gene.

The phenomenon gene amplification and its underlying mechanisms in vitro in several prokaryotic and eukaryotic culture systems been examined. The best characterized. Example of Gene amplification includes the culture of eukaryotic cells in medium, the variable concentrations of the cytotoxic drug methotrexate (MTX) contains. MTX is a folic acid analogue and disturbs DNA synthesis by blocking the enzyme dihydrofolate reductase (DHFL). While of the initial one Exposure to Low concentrations of MTX kill most cells (> 99.9%). A small Number of cells survives and is capable to grow in increasing MTX concentrations by taking large amounts produced on DHFR RNA and protein. The basis of this overproduction is the amplification of the single DHFR gene. The additional Copies of the gene are found as extra-chromosomal copies in the form of small, supernumerary chromosomes (Minimal chromosomes) or as integrated chromosomal copies.

The Gene amplification is most common in the development of resistance to cytotoxic drugs (Antibiotics for Bacteria and chemotherapeutic agents for eukaryotic cells) and neoplastic transformation encountered. The transformation of a eukaryotic cell as a spontaneous event or due to viral or chemical / environmental insults is typically associated with changes linked to the genetic material of this cell. One of the most common genetic changes, in human malignancies observed are mutations of the p53 protein. p53 controlled the transition of cells from the stationary one (G1-) in the replicative (S) phase and prevents this transition in the presence of DNA damage. In other words, one of the main consequences is deactivating p53 mutations accumulation and increase DNA damage, d. H. genetic changes. Usual types genetic changes in neoplastic cells, in addition to point mutations, Amplifications and strong structural changes, such. B. translocations.

The amplification of DNA sequences may indicate a specific functional requirement, as illustrated by the experimental DHFR system. Therefore, the amplification of certain oncogenes in malignancies indicates a causative role of these genes in the process of malignant transformation and maintenance of the transformed phenotype. This hypothesis has been confirmed in recent studies. For example, it has been found that the bcl-2 protein is amplified in certain types of non-Hodgkin's lymphoma. This protein inhibits apoptosis and leads to the progressive accumulation of neoplastic cells. It has been shown that elements of the growth factor receptor gene family are amplified in different cancers, suggesting that overexpression of these receptors could make neoplastic cells less susceptible to limiting levels of available growth factor. Examples include amplification of the androgen receptor in recurrent prostate cancer during androgen deficiency therapy and amplification of the growth factor receptor homologue ERB2 in breast carcinoma. Finally, genes involved in intracellular signaling and control of cell cycle progression can be involved are undergoing amplification during malignant transformation. This is illustrated by the amplification of the bclI and ras genes in various epithelial and lymphoid neoplasms.

These earlier Studies illustrate the feasibility the identification of amplified DNA sequences in neoplasms, since this approach can identify genes responsible for malignant transformation are important. The case of ERB2 also demonstrates its feasibility from a therapeutic standpoint, as transforming proteins are new and specific goals for could represent the tumor therapy.

Several different techniques can be used to detect amplified genomic sequences. The classic cytogenetic analysis of chromosomal regions made from cancer cells is used to identify major structural changes, such as: B. Translocations, deletions and inversions appropriate. Amplified genomic regions can only be visualized if they comprise large regions with a high copy number or are present as extrachromosomal material. Although cytogenetics has been the first technique to demonstrate the consistent association of specific chromosomal alterations with certain neoplasms, it is unsuitable for the identification and isolation of manageable DNA sequences. The recently developed technique of comparative genomic hybridization (CGH) has illustrated the widespread phenomenon of genomic amplification in neoplasms. Tumor and normal DNA are simultaneously hybridized to metaphases of normal cells and the entire genome can be screened by image analysis for DNA sequences present in the tumor at increased frequency. ( WO 93/18186 ; Gray et al., Radiation Res. 137, 275-289 (1994)). As a screening method, this type of analysis has revealed a large number of repeating amplicons (a segment of amplified DNA) in a variety of human neoplasms. Although CGH is more sensitive to the identification of amplified DNA segments than classical cytogenetic analysis, it does not allow rapid identification and isolation of coding sequences within the amplicon by standard molecular genetic techniques.

The most sensitive methods for detecting gene amplification Tests based on polymerase chain reaction (PCR). These tests use very small amounts of tumor DNA as starting material extremely sensitive, provide DNA for another Analysis, such as B. Sequencing is accessible and are for high-throughput analysis suitable.

The mentioned above Close tests are not mutually exclusive, but are often used in combination to identify amplifications in neoplasms. During cytogenetic Analysis and CGH screening procedures represent the entire genome to examine for amplified regions are PCR-based Tests for the final Identifying coding sequences, d. H. Genes in amplified regions, maximum suitable.

According to the present invention, such genes are isolated 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, kidney, pancreatic, spleen, thymus, testicular, ovarian, uterine, etc. tumors or tumor cell lines have been identified with pooled DNA from healthy donors. Quantitative PCR was performed using a TaqMan ^™ instrument (ABI).

Gene-specific Primers and fluorogenic probes were based on those for the DNA designed coding sequences.

Human lung carcinoma cell lines include A549 (SRCC768), Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775). , H522 (SRCC832) and H810 (SRCC833), all available from ATCC. Primary tumor human cells are usually derived from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small cell carcinomas, small cell carcinomas and bronchoalveolar carcinomas, and include, for example, SRCC724 (adenocarcinoma, abbreviated as "AdenoCa") (LT1), SRCC725 (squamous cell carcinoma, abbreviated as "SqCCa") (LT1a ), SRCC726 (adenocarcinoma) (LT2), SRCC727 (adenocarcinoma) (LT3), SRCC728 (adenocarcinoma) (LT4), SRCC729 (squamous cell carcinoma) (LT6), SRCC730 (adeno / squamous cell carcinoma) (LT7), SRCC731 (adenocarcinoma) ( LT9), SRCC732 (squamous cell carcinoma) (LT10), SRCC733 (squamous cell carcinoma) (LT11), SRCC734 (adenocarcinoma) (LT12), SRCC735 (adeno / squamous cell carcinoma) (LT13), SRCC736 (squamous cell carcinoma) (LT15), SRCC737 (squamous cell carcinoma) (LT16), SRCC738 (squamous cell carcinoma) (LT17), SRCC739 (squamous cell carcinoma) (LT18), SRCC740 (squamous cell carcinoma) (LT19), SRCC741 (lung cell carcinoma, abbreviated as "LCCa") (LT21), SRCC811 (adenocarcinoma) (LT22), SRCC825 (adenocarcinoma) (LT8), SRCC886 (adenocarcinoma) (LT25), SRCC887 (squamous cell carcinoma) (LT26), SRCC888 (adeno-BAC carcinoma) (LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell carcinoma) (LT29), SRCC891 (adenocarcinoma) (LT30), SRCC892 (squamous cell carcinoma) (LT31 ), SRCC894 (adenocarcinoma) (LT33). Also included are human lung tumors identified as SRCC1125 [HF-000631], SRCC1127 [HF-000641], SRCC1129 [HF-000643], SRCC1133 [HF-000840], SRCC1135 [HF-000842], SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230 [HF-001294], SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233 [HF-001297], SRCC1235 [HF-001299] and SRCC1236 [HF-001300] ,

colon cancer cell lines include, for example, the ATCC cell lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (a strong mucin producing variant of the ATCC colon adenocarcinoma cell line, SRCC780, obtained from Dr. med. Robert Warren, UCSF), CaWiDr (adenocarcinoma, SRCC781), HCT116 (carcinoma, SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403 (Adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), Colo205 (carcinoma, SRCC828), HCT15 (carcinoma, SRCC829), HCC2998 (carcinoma, SRCC830) and KM12 (carcinoma, SRCC831). Colon primary tumors include colon adenocarcinomas, those with CT2 (SRCC742), CT3 (SRCC743), CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17 (SRCC750), CT1 (SRCC751), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT11 (SRCC757), CT18 (SRCC-758), CT19 (adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21 (adenocarcinoma, SRCC908), CT22 (adenocarcinoma, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (Adenocarcinoma, SRCC911), CT25 (adenocarcinoma, SRCC912), CT26 (adenocarcinoma, SRCC913), CT27 (adenocarcinoma, SRCC914), CT28 (adenocarcinoma, SRCC915), CT29 (adenocarcinoma, SRCC916), CT30 (adenocarcinoma, SRCC917), CT31 (Adenocarcinoma, SRCC 918), CT32 (adenocarcinoma, SRCC919), CT33 (adenocarcinoma, SRCC920), CT35 (adenocarcinoma, SRCC921) and CT36 (adenocarcinoma, SRCC922). Also human colon tumor centers, described as SRCC1051 [HF-000499], SRCC1052 [HF-000539], SRCC1053 [HF-000575], SRCC1054 [HF-000698], SRCC1059 [HF-000755], SRCC1060 [HF-000756], SRCC1142 [HF-000762], SRCC1144 [HF-000789], SRCC1146 [HF-000795] and SRCC1148 [HF-000811] are included.

human Breast carcinoma cell lines include, for example, HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468 (SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3 (SRCC767) and human breast tumor centers identified as SRCC1057 [HF-000545] are designated are included. Furthermore, they are human Breast tumors identified as SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1100, SRCC 1101 and a human Breast met-lung nucleic acid tumor called SRCC893 [LT 32] is included.

human Rumen cancer tumors include SRCC981 [HF-000550] and SRCC982 [HP-000551].

human Kidney tumor centers include SRCC989 [HF-000611] and SRCC1014 [HF-000613].

human Testicular tumor centers include SRCC1001 [HF-000733] and testicular SRCC999 [HF-000716].

human Parathyroid tumors include [HF-000831] and SRCC1003 [HF-000832].

human Lymph node tumors include SRCC1004 [HF-000854], SRCC1005 [HF-000855] and SRCC1006 [HF-000856]

F. Tissue distribution

The Results of the gene amplification assays herein may be confirmed by further investigation, such as B. by determining the mRNA expression in different human tissues are verified.

As noted previously, gene amplification and / or gene expression in various tissues can be determined by conventional Southern blotting, Northern blotting to quantitate transcription of mRNA (Thomas, Proc Natl Acad Sci., USA 77, 5201-5205 (1980)). , Dot-blotting (DNA analysis) or in situ hybridization using a suitably labeled probe based on the sequences provided herein. Alternatively, antibodies can be used which detect specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes can.

The Gene expression in different tissues can alternatively be done by means of immunological methods, such. B. immunohistochemical staining of Tissue sections and testing of cell culture or body fluids be measured to directly quantify the expression of the gene product. For immunohistochemical To dye and / or testing of sample fluids appropriate antibodies can either monoclonal or polyclonal and can be produced in any animal become. Conveniently the antibodies against a native sequence PRO polypeptide or against a synthetic peptide based on the herein provided DNA sequences or against an exogenous sequence fused to PRO DNA is and for a specific antibody epitope encoded. General techniques for generation of antibodies and special protocols for Northern blotting and in situ hybridization are provided herein below.

G. Chromosome mapping

If the amplification of a given gene is functionally relevant, then this gene should be amplified more are called neighboring genomic regions responsible for survival of the tumor are not important. To test this, the gene may be against a particular chromosome, e.g. B. by irradiation hybrid analysis be mapped. The amplification level is then identified on the Determined location, as well as adjacent genomic regions. The selective or preferred amplification at the genomic region, to which the gene has been mapped is consistent with the possibility that the observed gene amplification growth or survival promotes the tumor. Chromosome mapping involves scaffold and epicenter mapping. For further For details see z. Stewart et al., Genome Research 7, 422-433 (1997).

H. Antibody Binding Studies

The Results of the gene amplification study can be obtained by antibody binding studies be further verified, testing the ability of anti-PRO antibodies is the expression of PRO polypeptides on tumor (cancer) cells to inhibit. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugated antibodies, their preparation will be described hereinafter.

Antibody binding studies can be carried out in any known test method, such as Competitive binding assays, direct and indirect sandwich assays and immunoprecipitation tests. Zola, Monoclonal Antibodies: A Laboratory Manual of Techniques, CRC Press Inc., pp. 147-158 (1987).

competitive Binding tests are based on the ability of a labeled standard, with the test sample analyte attached to the binding with a limited amount of antibodies to compete. The amount of target protein (encoded by an in a tumor cell amplified gene) in the test sample is reversed proportional to the amount of standard bound to the antibodies becomes. To facilitate the determination of the amount of bound standard, become the antibodies preferably insolubilized before or after the competition reaction, so that the standard and analyte bound to the antibodies are light separated from the unbound standard and analyte can.

Sandwich assays involve the use of two antibodies, both of which are capable of binding to another immunogenic portion or to another immunogenic epitope of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody immobilized on a solid support, and thereafter a second antibody binds to the analyte, forming an insoluble tripartite complex. See, for example, B. U.S. Patent No. 4,376,110 , The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one of the sandwich test types is an ELISA test, in which case the detectable moiety is an enzyme.

For immunohistochemistry For example, the tumor sample may be fresh or frozen or may be in Paraffin embedded and with a preservative such. B. be fixed formalin.

I. Cell-based tumor assays

On Cell-based tests and animal models for tumors (eg carcinomas) can used to verify the findings of the gene amplification test and the relationship between the genes identified herein and the development and pathogenesis of neoplastic cell growth to understand better. The role of gene products identified herein in the development and pathology of tumors or cancers be tested by primary tumor cells or cell lines that have been recognized to be used that they amplify the genes. Such cells include, for example Breast, colon and lung carcinoma cells and cell lines, the above enumerated are.

In Another approach is to cells of a cell type known to be attached to a cell involved in certain tumor transfected with the cDNAs herein, and it becomes the ability of these cDNAs analyzed, overgrowth trigger. Suitable cells include, for example, stable tumor cell lines, such as The B104-1-1 cell line (stable NIH-3T3 cell lines transfected with the neu-proto-oncogene) and ras-transfected NIH-3T3 cells, with the desired Gene transfected and observed for tumorigenic growth can. Such transfected cell lines can then be used about the ability of poly- or monoclonal antibodies or antibody compositions to test the tumor-like cell growth by exerting cytostatic or cytotoxic activity on the growth of transformed cells or by mediating Antibody-dependent cell toxicity (ADCC) to inhibit. With the coding sequences of those identified herein Gene transfected cells can continue to be used to drug candidates for cancer treatment to identify.

In addition, primary cultures, derived from tumors in transgenic animals (as described below), in the cell-based assays herein though stable cell lines are preferred. Techniques for the derivation of continuous Cell lines from transgenic animals are within the scope of the invention well known (see, for example, Small et al., Mol. Cell Biol., 5: 642-648 (1985)).

J. Animal Models

A variety of well-known animal models can be used to better understand the role of the genes identified herein in the development and pathogenesis of tumors and the efficacy of candidate therapeutic agents, including antibodies and other antagonists of native polypeptides, including small antagonist molecules testing. The in vivo character of such models makes them particularly predictive of responses in human patients. Animal models of tumors and carcinomas (eg, breast carcinoma, colon carcinoma, prostate carcinoma, lung carcinoma) include non-recombinant as well as recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g. B. mouse models. Such models can be constructed by introducing tumor 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. B. in colon tissue implanted colon carcinoma cells. (See, e.g., PCT Publication No. WO 97/33551 , published September 18, 1997).

The probably the most common Animal species used in oncological studies are immunodeficient Mice and especially nude mice. The observation that nude mice with hypo- / aplasia successful as hosts for human tumor xenografts can act has led to its widespread use for this purpose. The autosomal recessive nu gene is in a very large number of different congenic strains of nude mice introduced for example in ASW, A / He, AKR, BALB / c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I / st, NC, NFR, NFS, NFS / N, NCB, NZC, NZW, P, RIII and SJL. Furthermore is a wide variety of other animals that are not nude mice, bred with inherited immunological defects and as a recipient of Tumor xenoimplants have been used. For more details see z. B. The Nude Mouse in Oncology Research, E. Boven and B. Winograd (Ed.), CRC Press Inc. (1991).

The introduced into such animals Cells can come from known tumor / carcinoma cell lines, such. B. of any the above enumerated Tumor cell lines and, for example, from the B104-1-1 cell line (stable NIH 3T3 cell lines transfected with the neu proto-oncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-37); a moderately well-differentiated human Grade II colon adenocarcinoma cell lines, HAT-29 (ATCC HTB-38); or of tumors and carcinomas. Samples of tumor or cancer cells can out Patients undergoing surgery are under treatment from standard conditions, freezing and storage in liquid nitrogen (Karmali et al., Br. J. Cancer 48, 689-696 (1983)).

tumor cells can by means of a variety of methods in animals, such. B. nude mice introduced. The subcutaneous (s.c.) space is particularly suitable for tumor implantation. Tumors can s.c. as massive blocks, as needle biopsies using a hollow needle or as cell suspensions be transplanted. For the massive block or hollow needle implantation become tumor tissue fragments suitable size in the subcutaneous space introduced. Cell suspensions become primary tumors or stable tumor cell lines freshly prepared and injected subcutaneously. Tumor cell lines can also be injected as subdermal implants. At this point The inoculum is between the lower part of the skin connective tissue and the subcutaneous tissue. Boven and Winograd (1991), so.

animal models of breast carcinoma for example, by implanting rat neuroblastoma cells (from which the neu-oncogen originally isolated) or neu-transformed NIH-3T3 cells in nude mice, in Essentially as described by Drebin et al., PNAS USA 83, 9129-9133 (1986). be generated described.

Similarly, animal models of colon carcinoma can be generated by allowing passage of colon carcinoma cells in animals, e.g. B. Nude mice is performed, resulting in the occurrence of tumors in these animals. An orthotopic graft model of human colon carcinoma in nude mice has been described, for example, by Wang et al., Cancer Research 54, 4726-4728 (1994) and Too et al., Cancer Research 55, 681-684 (1995). This model is based on the so-called "METAMOUSE ^™ " sold by Anticancer Inc. (San Diego, California).

tumors which arise in animals can be removed and cultured in vitro. Cells from in vitro cultures can then be passaged to animals. Such tumors can as Goals for serve further testing or drug screening. Alternatively can the tumors resulting from the passage isolated and RNA from Pre-passage cells and cells isolated after one or more passage rounds, analyzed for differential expression of genes of interest become. Such passage techniques can be used with any known Tumor or cancer cell lines are performed.

For example, Meth A, CMS4, CMS5, CMS21 and WEHI-164 are chemically-induced fibrosarcomas of female BALB / c mice (DeLeo et al., J. Exp. Med. 146, 720 (1977)), which is a very well-controllable model system Assaying the antitumor activities of various agents (Palladino et al., J. Immunol., 138: 4023-4032 (1987)). In summary, tumor cells are propagated in vitro in cell culture. Before injection into the animals, the cell lines are washed and suspended in buffer at a cell density of about 10 × 10 ⁶ to 10 × 10 ⁷ cells / ml. The animals are then subcutaneously infected with 10 to 100 μl of the cell suspension and the appearance of a tumor is waited for one to three weeks.

In addition, can the Lewis lung (3LL) carcinoma of the mice, which is one of the most thorough investigated experimental tumors is, as a research tumor model be used. The effectiveness of this tumor model is advantageous Effects have been correlated in the treatment of human patients, who have been diagnosed with small cell lung carcinoma (SCCL) are. This tumor can be injected by injecting tumor fragments affected mouse or cultured cells into normal Mice are introduced (Zupi et al., Br. J. Cancer 41, Supplement 4, 309 (1980)). The results point out that tumors even from the injection of a single Cell triggered can be and that a very high proportion of infected tumor cells survive. For further information about this tumor see Zacharski, Haemostasis 16, 300-320 (1986)).

One way of assessing the effectiveness of a test compound in an animal model on an implanted tumor is to measure the size of the tumor before and after treatment. Conventionally, the size of implanted tumors has been measured with a vernier caliper in two or three dimensions. The measurement limited to two dimensions can not accurately reflect the size of the tumor, and therefore it is usually converted to the appropriate volume by using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effects of a drug candidate can be better described as a treatment-induced growth retardation and specific growth retardation. Another important variable in describing tumor growth is tumor volume doubling time. Computer programs for calculating and describing tumor growth are also available, such as: For example, that described by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng (Ed.), Basel, 301 (1989). It is noted, however, that necrosis and inflammatory reactions after treatment, at least initially, may also result in an increase in tumor size. Therefore, these changes must be carefully observed, through a combination of morphometri method and a flow cytometric analysis.

Recombinant (transgenic) animal models can be constructed using standard techniques for producing transgenic animals by introducing the coding portion of the genes identified herein into the genome of the animals of interest. Animals that can serve as targets 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 for introducing a transgene into such animals known in the art include pronuclear microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191 ); Retrovirus-mediated gene transfer in germlines (eg Van der Putten et al., Proc Natl Acad Sci USA 82, 6148-615 (1985)); Gene targeting in embryonic stem cells (Thompson et al., Cell 56, 313-321 (1989)); Electroporation of embryos (Lo, Mol. Cel. Biol. 3: 1803-1814 (1983)); Sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 (1989)). For an overview, see for example U.S. Patent No. 4,736,866 ,

To the For purposes of the present invention, transgenic animals include those which harbor the transgene in only part of their cells ("mosaic animal") .The transgene can either as a single transgene or as a con catemere, e.g. B. as Head-to-head or Be integrated head-tail tandems. The selective introduction of a Transgene in a particular cell type is also possible by, for example the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992) is followed.

The Expression of the transgene in transgenic animals may be achieved by standard techniques to be watched. For example, Southern blotting analysis or PCR amplification used to integrate the To verify transgene. The extent of mRNA expression can then using techniques such. B. in situ hybridization, Northern blot analysis, PCR or immunocytochemistry are analyzed. The animals are on signs of tumor or carcinoma development further investigated.

alternative In addition, "knockout" animals can be constructed which is a defective or modified one identified herein As a result, gene encoding PRO polypeptide the homologous recombination between the endogenous, for the polypeptide coding gene and modified, for the same Polypeptide encoding genomic DNA into an embryonic cell of the animal was introduced. For example, for a PRO polypeptide-encoding cDNA can be used to obtain established Techniques for to clone this polypeptide encoding genomic DNA. A section the for a particular PRO polypeptide-encoding genomic DNA can be deleted or by another gene, such as. B. by a for a selectable Marker encoding gene, which is used to determine the Integration can be used. Typically, the vector includes several kilobases unchanged flanking DNA (at the 5 'and 3 'ends) (see z. Thomas and Capecchi, Cell 51, 503 (1987) for a homologous description Recombination vectors). The vector becomes an embryonic stem cell line (eg, by electroporation) and it becomes those cells selected in which the imported DNA has homologously recombined with the endogenous DNA (see eg. Li et al., Cell 69, 915 (1992)). The selected cells become then into a blastocyst of an animal (eg a mouse or rat) injected to form aggregation chimeras (See, eg, Bradley, in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertsen (ed.), IRL, Oxford, pp. 113-152 (1987)). A chimeric Embryo can then enter a suitable pseudopregnant female pet implanted and born the embryo to create a "knockout" animal. Offspring carrying the homologously recombined DNA in their germ cells wear, can identified and used by standard techniques To breed animals, in which all cells of the animal contain the homologously recombined DNA. Knockout animals can for example, by their ability be characterized, to defend against certain pathological conditions and by their development of pathological conditions due to absence the PRO polypeptide.

The efficacy of antibodies specifically binding to the polypeptides identified herein and other drug candidates can also be tested in the treatment of spontaneous animal tumors. A suitable target for such studies is oral squamous cell carcinoma (SCC). Oral Cats SCC is a highly invasive malignant tumor responsible for the most common oral malignancy in cats and for over 60% of the reported oral tumors in this species. It rarely metastasizes to distant sites, although the low frequency of metastasis could only reflect the short survival times for cats with this tumor. These tumors are usually inaccessible to surgery, primarily due to the anatomy of the cat's oral cavity. There is currently no effective treatment for this tumor. Before entering the study, each cat undergoes a full clinical examination and biopsy and is scanned using computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. The tongue can as a result ei If such a tumor is paralyzed and even if the treatment kills the tumor, the animals may not be able to feed themselves. Each cat is treated repeatedly over a longer period of time. Photographs of the tumor are taken daily during the treatment period and at each subsequent follow-up visit. After treatment, each cat undergoes another computed tomography scan. Computed tomography scans and chest radiographs are scored every 8 weeks thereafter. The data are assessed for differences in survival, response and toxicity compared to control groups. A positive reaction may require evidence of tumor regression, preferably with improvement in quality of life and / or increased lifetime.

In addition, others can spontaneous animal tumors such. Fibrosarcoma, adenocarcinoma, lymphoma, chondroma, Leiomyosarcoma of dogs, cats and baboons also tested become. Of these, mammary adenocarcinoma is in dogs and cats a preferred model because of their appearance and behavior People very similar is. However, the usefulness of this model is due to the rare Occurrence of this type of tumor in animals is limited.

K. Screening tests for drug candidates

screening tests on drug candidates are designed to be compounds which are identified to those identified herein Genes encoded polypeptides bind or complex or interact of the encoded proteins interfere with other cell proteins otherwise. such Screening tests include tests that involve high-throughput screening accessible to chemical libraries, making them especially for the identification of drug candidates small molecule size suitable are. Intended small molecules include synthetic organic or inorganic compounds, including Peptides, preferably soluble Peptides, (poly) peptide-immunoglobulin fusions, and in particular antibodies, including and without restriction poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies and chimera or humanized versions of such antibodies and fragments, as well as human antibody and antibody fragments. The tests can be performed in a variety of formats, including as Protein-protein binding assays, biochemical screening tests, immunoassays and cell-based assays that are within the scope of the invention are well characterized.

All Tests have in common that they are contacting the drug candidate with a nucleic acid encoded by the nucleic acid identified herein Require polypeptide under conditions and for a time that is sufficient to allow the interaction of these two components.

at Bonding tests is the interaction the bond and educated Complex can be isolated from the reaction mixture and detected become. In a special embodiment is the polypeptide encoded by the gene identified herein or the drug candidate to a solid phase, z. B. on a microtiter plate immobilized by covalent or non-covalent attachments. The non-covalent attachment is generally by coating the solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized Antibody, z. B. a for the polypeptide of specific monoclonal antibody to be immobilized used to anchor the polypeptide to a solid surface. The test is carried out, by placing the non-immobilized component with a detectable marker may be labeled to the immobilized component, for. B. the anchored component containing coated surface added becomes. When the reaction is over, the unreacted components become for example, removed by washing and anchored to the solid surface Complexes detected. If the originally not immobilized Component carries a detectable marker, shows the detection of on the surface immobilized marker that complexation has occurred. If the original not immobilized component none. Carries marker, can a complexation, for example by using a marked antibody be detected, specific to the immobilized complex binds.

If the candidate compound interacts with but does not bind to the particular PRO polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be tested by methods well known for the detection of protein-protein interactions. Such tests include conventional approaches such. Cross-linking, co-immunoprecipitation and co-purification via gradients or chromatographic columns. In addition, protein-protein interactions can be observed by using a yeast-based genetic system as described by Fields and coworkers (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 Nathan, Proc Natl Acad Sci USA 89, 5789-5793 (1991)). Many transcriptional activators, such as Yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain, while the other acts as a transcriptional activation domain. The yeast expression system (commonly referred to as a "two-hybrid system") described in the previous publications exploits the advantage of this property and employs two hybrid proteins, one in which the target protein fuses to the DNA-binding domain of GAL4 and another in which activating candidate proteins are fused to the activation domain The expression of a GAL1-lacZ reporter gene under the control of a GAL4-activated promoter depends on the restoration of GAL4 activity via protein-protein interaction. Colonies containing interacting proteins are detected with a chromogenic substrate for β-galactosidase A complete set (MATCHMAKER ^™ ) for the identification of protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech This system can also be extended to provide protein domains which are involved in specific protein interactions, as well as to pinpoint amino acid residues that are critical to this interaction.

Links, which identifies the interaction of a PRO coding gene herein Genes interfering with other intracellular or extracellular components, such as to be tested: usually becomes a reaction mixture which amplifies the product of Contains gene and the intra or extracellular component, under Conditions and for made a time showing the interaction and bonding of the two Products possible. To the ability testing a test compound to inhibit binding will Reaction carried out in the absence and presence of the test compound. In addition, can a placebo may be added to a third reaction mixture to serve as a control. The binding (complex formation) between test compound and the intracellular or extracellular Compound present in mixture will be as above described observed. The formation of a complex in the control reaction (s), but not in the test compound-containing reaction mixture indicates that the test compound is the interaction of the test compound and their reactants.

M. Antibody

1. Polyclonal antibodies

method for the production of polyclonal antibodies are known in the art. Polyclonal antibodies can in a mammal be prepared, for example by one or more injections an immunizing agent and, if desired, an adjuvant. typically, the immunizing agent and / or adjuvant is replaced by multiple subcutaneous or intraperitoneal injections into the mammal injected. The immunizing agent may be the PRO polypeptide or include a fusion protein thereof. It may be useful to do that immunizing agents to conjugate to a protein to be immunized in Mammalian known to be immunogenic is. Examples of such immunogenic proteins include but are not limited on keyhole limpet hemocyanin, Serum albumin, bovine thyroglobulin and soybean trypsin inhibitor. examples for Adjuvants that can be employed include Freund's complete Adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic Trehalose dicorynomycolate). The immunization protocol can be from one skilled in the art without undue experimentation chosen become.

2. Monoclonal antibodies

The Anti-PRO antibodies can alternatively be monoclonal antibodies. Monoclonal antibodies may be added Use of hybridoma methods, such. Eg those of Kohler and Milstein, Nature 256, 495 (1975). In a hybridoma procedure, a mouse, a hamster or another suitable host animal typically with an immunogenic agent immunized to elicit lymphocytes producing antibodies or capable of doing so are, antibodies which bind specifically to the immunizing agent. Alternatively, you can the lymphocytes are immunized in vitro.

The immunizing agent will typically comprise the PRO polypeptide, including fragments, or a fusion protein of such protein or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells if non-human mammalian sources are desired The lymphocytes are then probed with an immortalized cell line using a suitable fusing agent, such as e.g. B. Polyethylene glycol fused to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). Immortalized cell lines are more common wise transformed mammalian cells, especially rodent, bovine or human myeloma cells. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable culture medium which preferably contains one or more substances which inhibit the growth or survival of unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium will typically comprise hypoxanthine, aminopterin, and thymidine ("HAT medium"), which inhibits the growth of HGPRT-deficient cells.

preferred immortalized cell lines are those that fuse efficiently, a stable expression of the antibody by the selected Antibody-producing Upright cells obtained and against a medium such. B. HAT medium sensitive are. More preferred immortalized cell lines are mouse myeloma lines, for example 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 are more human for production monoclonal antibody as well (Kozbor, J. Immunol., 133, 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker Inc., New York, pp. 51-63 (1987)).

The Culture medium in which the hybridoma cells are cultured can then to the presence of antibodies tested against PRO. Preferably the binding specificity the monoclonal antibody produced by the hybridoma cells immunoprecipitation or by an in vitro binding assay, such as. B. radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and tests are within the scope of the invention known. The binding affinity of the monoclonal antibody can be detected, for example, by means of Scatchard analysis by Munson and Pollard, Anal. Biochem. 107, 220 (1980).

After this the desired Hybridoma cells have been identified, the clones can by limiting dilution subcloned 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, you can the hybridoma cells in vivo as ascites in a mammal be bred.

The Monoclonal antibodies secreted by the subclones can pass through conventional Immunoglobulin purification method, such. Protein A-Sepharose, Hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography isolated or purified from the culture medium or ascites.

The monoclonal antibodies can also be prepared by recombinant DNA methods, such as. B. those in the U.S. Patent No. 4,816,567 are described. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies) become. The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed in expression vectors which can then be transformed into host cells, e.g. Simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that otherwise do not produce immunoglobulin protein to achieve the synthesis of monoclonal antibodies in the recombinant host cells. The DNA may also be modified, for example, by substituting the heavy and light chain constant domain coding sequence for the homologous mouse sequences ( U.S. Patent No. 4,816,567 ; Morrison et al., Supra) or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. The constant domains of an antibody of the invention or the variable domains of one of the antigen-combining sites of an antibody of the invention may be replaced with such a non-immunoglobulin polypeptide to produce a chimeric bivalent antibody.

The antibody can monovalent antibodies be. Methods for producing monovalent antibodies well known in the art. For example, includes one of the methods the recombinant expression of the immunoglobulin light chain and the modified heavy chain. The heavy chain is generally on truncated anywhere in the Fc region to heavy chain crosslinking to prevent. Alternatively, the relevant cysteine residues with one at their amino acid residue substituted or deleted to prevent cross-linking.

In vitro methods are also suitable for producing monovalent antibodies. The digestion of Antibodies to produce fragments thereof, particularly Fab fragments, can be obtained using routine techniques known in the art.

3. Human and humanized antibody

The anti-PRO antibodies may also include humanized or human antibodies. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F (ab') _2, or other antigen-binding subsequences of antibodies) contain a non-human immunoglobulin-derived minimal sequence. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues of a complementarity-determining-region (CDR) of the recipient are represented by residues of a CDR of a non-human species (donor antibody), such as a donor antibody. As mouse, rat or rabbit with the desired specificity, affinity and capacity are replaced. In some cases, Fv framework regions of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises 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 the non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin Consensus sequence. The humanized antibody also optimally comprises at least part 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 (1990); 1988); and Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from a variable "import" domain. Humanization can be performed essentially according to the method of Winter et al. (Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332, 323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)) the corresponding sequences of a human antibody are substituted by rodent CDRs or CDR sequences. Accordingly, such "humanized" antibodies are chimeric antibodies ( U.S. Patent No. 4,816,567 ), wherein substantially less than one intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be prepared using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227, 381 (1991); Marks et al. J. Mol. Biol. 222, 581 (1991)). The techniques of Cole et al. and Boerner et al. are also available for the production 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 (1995); 1991)). Likewise, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g. In mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon exposure, a production of human antibodies is observed that closely approximates those observed in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This approach is used, for example, in the U.S. Patent Nos. 5,545,807 ; 5545806 ; 5569852 ; 5625126 ; 5633425 ; 5661016 and in the following scientific publications: Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, intern. Rev. Immunol. 13, 65-93 (1995).

5. Bispecific antibodies

bispecific antibody are monoclonal, preferably human or humanized, antibodies binding specificities for at least have two different antigens. In the present case, one of the binding specificities for the PRO, the other is for any other antigen, and preferably for one Cell surface protein or receptor or a receptor subunit.

Methods of preparing bispecific antibodies are known in the art. Conventionally, the production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chains / light chain pairs, wherein the two heavy chains have different specificities (Milstein & Cuello, Nature 305, 537-539 (1983)). Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are in the WO 93/08829 , published May 13, 1993, and in Traunecker et al., EMBO J. 10, 3655-3659 (1991).

variable Antibody domains with the desired one binding specificities (Antibody-antigen combining sites) can on immunoglobulin constant domain sequences be merged. The fusion is preferably carried out with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CH1) contains the site that for light chain binding, present in at least one of the mergers, is required. DNAs that for the Immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and are co-transfected into suitable host organisms. For more details for the production of bispecific antibodies see for example Suresh et al., Methods in Enzymology 121, 210 (1986).

According to another approach, in the WO 96/27011 For example, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least part of the CH3 region of a constant antibody domain. In this method, one or more short amino acid side chains from the interface of the first antibody molecule are replaced by longer side chains (eg, tyrosine or tryptophan). Compensation "cavities" of identical or similar size to the long side chain (s) are created at the interface of the second antibody molecule by replacing the long amino acid side chains with shorter ones (eg, alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer as compared to undesired end products such as homodimers.

Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, bispecific F (ab ') ₂ antibodies). Methods for producing bispecific antibodies from antibody fragments have already been described in the literature. For example, bispecific antibodies can be made using chemical linkage. Brennan et al., Science 229, 81 (1985), describe a method wherein intact antibodies are proteolytically cleaved to form F (ab ') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and to inhibit intermolecular disulfide formation. The resulting Fab 'fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The produced bispecific antibodies can be used as means for the selective immobilization of enzymes.

Fab 'fragments can be recovered directly from E. coli and chemically bound to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175, 217-225 (1992) describe the production of a fully humanized bispecific F (ab ') ₂ antibody molecule. Each Fab 'fragment was separately secreted from E. coli and subjected to directional chemical binding in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor as well as to normal human T cells, and could enhance the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, bispecific antibodies were prepared using leucine zipper. Kostelny et al., J. Immunol. 148 (5), 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used to prepare antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448 (1993), provides an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a variable Heavy chain domain (V _H ) linked by a linker to a variable light chain domain (V _L ) that 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 form pairs with the complementary V _L and V _H domains of another fragment, thereby forming two antigen binding sites. Another procedure for the preparation of bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has already been described. See Gruber et al., J. Immunol. 152, 5368 (1994).

Antibodies with more than two valences are also considered. For example can tispecific antibodies getting produced. Tutt et al., J. Immunol. 147, 60 (1991).

exemplary bispecific antibodies can bind to two different epitopes on a given polypeptide bind the present invention. Alternatively, an anti-polypeptide arm be combined with an arm that connects to a trigger molecule at one Leukocytes such as a T cell receptor molecule (e.g. CD2, CD3, CD28 or B7) or Fc receptors for IgG (FcγR), such as. B. FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), binds to cellular Defense mechanisms are focused on the cell that the particular Polypeptide expressed. Bispecific antibodies can also be used to locate cytotoxic agents on cells that have a specific Express polypeptide. These antibodies have a binding arm and an arm containing a cytotoxic agent or a radionuclide chelator binds, such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds the polypeptide and further binds tissue factor ( "Tissue factor ", TF).

6. Heteroconjugated antibodies

Heteroconjugated antibodies are composed of two covalently bound antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells [ U.S. Patent No. 4,676,980 ] as well as for the treatment of HIV infection [ WO 91/00360 ; WO 92/200373 ; EP 03089 ]. It is contemplated that the antibodies can be produced in vitro using methods known in the art of synthetic protein chemistry, including those that incorporate crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate as well as those reagents described, for example, in U.S. Pat U.S. Patent No. 4,676,980 are disclosed.

7. Effector function editing

It may be desirable be the antibody of the invention with regard to the effector function. For example, you can one or more cysteine residues are introduced into the Fc region, whereby the formation of disulfide bonds between the chains in this region allows becomes. The homodimeric antibody thus formed can have improved internalizing ability and / or enhanced complement-mediated cell killing and antibody-mediated cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176, 1191-1195 (1992), and Shopes, J. Immunol. 148, 2918-2922 (1992). Homodimeric antibodies with enhanced Anti-tumor activity can also be prepared using heterobifunctional crosslinkers, as described in Wolff et al., Cancer Research 53, 2560-2565 (1993). Alternatively, an antibody may be used be edited so that it has dual Fc regions and thereby increased via Complement lysis and ADCC capabilities feature can. See Stevenson et al., Anti-Cancer Drug Design 3, 219-230 (1989).

8. Immunoconjugates

The Invention also optionally employs immunoconjugates comprising an antibody, the to a cytotoxic agent such as a chemotherapeutic Agent, toxin (eg an enzymatically active toxin or toxin of bacterial, plant or animal origin or even of Fungi or fragments thereof or a small molecule toxin) or to a radioactive isotope (i.e., a radio-conjugate) is.

Chemotherapeutic agents useful for preparing such immunoconjugates have been described above. Enzymatically active protein toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, cholera toxin, botulinum toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Modeccin A chain, α-sarcin, aleurites-fordii proteins, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotine, sapaonaria -officinalis Inhibitor, Gelonin, Saporin, Mitogellin, Restricto cin, phenomycin, enomycin and the tricothecenes. Small molecule toxins include, for example, calicheamincines, maytansinoids, palyoxin and CC1065. Many different radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Conjugates of the antibody and the cytotoxic agent are prepared using a variety of bifunctional protein binding agents, such as e.g. N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (such as toluene-2,6- diisocyanate) and bis-active fluoro compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin may be prepared as described in Vitetta et al., Science 238, 1098 (1987). C-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotide to the antibody. See the WO 94/11026 ,

In another embodiment can the antibody to a "receptor" (like streptavidin) be conjugated for use in tumor pretargeting, wherein the Antibody-receptor conjugate administered to the patient, followed by removal unbound Conjugate from the bloodstream using a clarifier and the subsequent one Administration of a "ligand" (eg avidin), which is conjugated to a cytotoxic agent (eg, a radionucleotide) is.

9. Immunoliposomes

The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, as described, for example, in Epstein et al., Proc. Natl. Acad. Sci. USA 82, 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77, 4030 (1980); and the U.S. Patent Nos. 4,485,045 and 4544545 to be discribed. Liposomes with increased circulation time are in the U.S. Patent No. 5,013,556 disclosed.

Especially useful Liposomes can by the reverse phase evaporation method with a lipid composition, the phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are filtered filtered of defined pore size to Liposomes with the desired To give diameter. Fab 'fragments of the antibody of the present invention to the liposomes as described in Martin et al., J. Biol. Chem. 257, 286-288 (1982), described conjugated by means of a disulfide exchange reaction become. A chemotherapeutic agent (such as doxorubicin) is optionally contained in the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19), 1484 (1989).

Q. Diagnosis and prognosis of tumors

While cell surface proteins, such as B. overexpressed in certain tumors Growth receptors are excellent targets for drug candidates or Tumor (eg cancer) treatment, find the same proteins in common with secreted, encoded by the genes amplified in tumor cells Proteins further applications in the diagnosis and prognosis of Tumors. For example, you can Antibody, those against protein products of genes amplified in tumor cells are used as tumor diagnostics or prognostic drugs become.

For example can Antibody, including Antibody fragments used to express the expression of the amplified genes encoded proteins ("marker gene products") qualitatively or to prove quantitatively. The antibody is preferably with a detectable, z. B. fluorescent markers, and the binding can be done by light microscopy, flow cytometry or other techniques known in the art become. These techniques are particularly suitable when amplified Gene for a cell surface protein, z. B. encodes a growth factor. Such binding tests are essentially as described in section 5 above.

The in situ detection of antibody binding to the marker gene products can be carried out, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient and a labeled antibody is applied to it, preferably by overlaying the biological specimen with the antibody. This method allows the determination of the distribution of the marker gene product in the examined tissue. For the qualified professional is of It is evident that a wide variety of histological techniques are readily available for in situ detection.

The The following examples are for illustrative purposes only provided and in no way intend to limit the Scope of the present invention.

EXAMPLES

in the Commercially available Reagents referred to in the Examples were Unless otherwise specified, according to the manufacturer's instructions used. The source of those cells, in the following examples and throughout the specification by ATCC accession numbers are the American Type Culture Collection, Manassas, VA. All original deposits referenced in the present application Is made in accordance with the rules of the Budapest Contract about the international recognition of the deposit of microorganisms for the Purposes of Patent Procedure and the Provisions Thereof (Budapest Contract). This ensures the preservation of a viable culture the deposit for 30 years from the date of deposit. The deposits are made by the ATCC in accordance with the provisions of the Budapest Contract and according to one Agreement between Genentech, Inc., and ATCC made available, the permanent and unrestricted Availability the progeny of the culture of deposit for the public upon issue of the relevant US patent or disclosure to the public either US or foreign Patent application, depending on what comes first, guaranteed and that the availability the progeny for someone by the president of the United States Patent Office in accordance with 35 USC § 122 and the valid Provisions (including 37 CFR § 1.14 with special reference to 886 OG 638).

Provided Unless otherwise stated, the present invention uses standard methods the recombinant DNA technology, such. Those mentioned hereinabove and in the following textbooks Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989); 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); M.J. Gait, Oligonucleotide Synthesis, IRL Press, Oxford (1984); R.I. Freshney, Animal Cell Culture (1987); Coligan et al., Current Protocols in Immunology (1991).

EXAMPLE 1

Screening of extracellular domain homology for the identification of new polypeptides and cDNA coding therefor

The extracellular domain (ECD) sequences (including the secretory signal sequence, if any) from about 950 known secreted proteins from the publicly available Swiss-Prot database were used to search EST databases. The EST databases included public databases (eg Dayhoff, GenBank) and private databases (eg LIFESEQ ^™ , Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266, 460-480 (1996)) in the form of a comparison of the ECD protein sequences with a 6-raster translation of the EST sequences , These comparisons with a BLAST score of 70 (or in some cases 90) or more that did not code for known proteins were grouped and consensused into the program "phrap" (Phil Green, University of Washington, Seattle, WA) Assembled DNA sequences.

Under Use of this homology screen of the extracellular domains were Consensus DNA sequences in relation to the others identified EST sequences arranged using phrap. Furthermore were the obtained consensus DNA sequences frequently (but not always) by means of repetitive cycles from BLAST or BLAST-2 and phrap extended to the consensus sequence as far as possible using the sources of previously explained EST sequences.

On the basis of the consensus sequences obtained as described above, oligonucleotides were subsequently synthesized and used to PCR-identify a cDNA library containing the sequence of interest, and were also used as probes to construct a clone of the full length coding sequence for a cDNA library Isolate PRO polypeptide. Forward and reverse PCR primers generally have 20 to 30 nucleotides and are often designed to be a PCR product of about 100-1,000 bp result. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is longer than about 1-1.5 kbp. To screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification as described by Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were by means of conventional Method using commercially available reagents such as those constructed by Invitrogen, San Diego, CA. The cDNA was digested with oligo-dT, that contained a NotI site, primed, hemicinased at the blunt end SalI adapters bound, cleaved with NotI, in an appropriate manner classified by gel electrophoresis and in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D, which does not contain the SfiI site; see Holmes et al., Science 253, 1278-1280 (1991)) in the unique XhoI and NotI sites.

EXAMPLE 2

Isolation of cDNA clones using of signal algorithm analysis

Various nucleic acid sequences encoding polypeptides have been generated by applying a proprietary signal sequencing algorithm developed by Genentech, Inc., (South San Francisco, CA) to ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and / or private (LIFE-SEQ ^®, Incyte Pharmaceuticals, Inc., Palo Alto, CA) databases identified. The signal sequence algorithm calculates a secretion signal value based on the property of the DNA nucleotides surrounding the first and, if appropriate, the second methionine codon (ATG) at the 5 'end of the sequence under investigation or the sequence fragment examined. The nucleotides that attach to the first ATG must code for at least 35 unique amino acids without any stop codons. If the first ATG has the required amino acids, the second is not investigated. If neither meets the requirements, the candidate sequence is not rated. To determine if the EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are evaluated using a set of seven sensors (evaluation parameters) known to associate with secretion signals , The use of this algorithm has led to the identification of numerous polypeptide-encoding nucleic acid sequences.

EXAMPLE 3

Isolation of cDNA clones encoding human Code PRO4980

A DNA starting sequence referred to herein as DNA81573 was identified by a yeast screen, in a human cDNA library, which preferably represents the 5 'ends of the primary cDNA clones. This cDNA was then treated with ESTs from public databases (eg., GenBank) and a private EST database (LIFESEQ ^®, Incyte Pharmaceuticals, Palo Alto, CA, USA) are compared, wherein the computer program BLAST or was used BLAST2 (Altschul et al , Methods in Enzymology 266, 460-480 (1996)). The ESTs were clustered and assembled into a consensus DNA sequence by the program "phrap" (Phil Green, University of Washington, Seattle, Washington) .This consensus sequence is referred to herein as DNA90613.

PCR primers (forward and reverse primers) were synthesized from the DNA90613 sequence for use as probes in the isolation of a clone of the PRO4980 full-length coding sequence from a human aortic endothelial cell cDNA library:

RNA to construct the cDNA libraries were from human aortic endothelial cell tissue isolated. The cDNA libraries used to isolate the cDNA clones were, by means of conventional Method Produced Using Commercially Available Reagents as available, for example, from Invitrogen, San Diego, CA, USA. The cDNA was primed with oligo-dT containing a NotI site, blunt-ended bound to hemi-integrated SalI adapters, cleaved with NotI, on classified by gel electrophoresis and in a defined manner Alignment into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D, which does not contain the SfiI site; see Holmes et al., Science 253, 1278-1280 (1991)) in the unique XhoI and NotI digits cloned.

The full-length DNA97003-2649 clone obtained from this screen is in 1 (SEQ ID NO: 1) and contains a single open reading frame with a unique translation initiation site at nucleotide positions 286-288 and a unique stop codon at nucleotide positions 1900-1902. The predicted polypeptide precursor is 538 amino acids long ( 2 ). This in 2 The full length PRO4980 protein shown has an estimated molecular weight of about 59,268 daltons and a pI of about 8.94. An analysis of in 2 The full-length PRO4980 sequence (SEQ ID NO: 2) shown demonstrates the presence of many different important polypeptide domains, wherein the positions indicated for those important polypeptide domains, as described above, are only approximate. An analysis of in 2 The full-length PRO4980 polypeptide shown demonstrates the presence of the following elements: a signal peptide from about amino acid 1 to about amino acid 36; Transmembrane domains from about amino acid 77 to about amino acid 95, from about amino acid 111 to about amino acid 133, from about amino acid 161 to about amino acid 184, from about amino acid 225 to about amino acid 248, from about amino acid 255 to about amino acid 273, from about amino acid 299 to about amino acid 314, from about amino acid 348 to about amino acid 373, from about amino acid 406 to about amino acid 421, from about amino acid 435 to about amino acid 456, and from about amino acid 480 to about amino acid 497; an N-glycosylation site from about amino acid 500 to about amino acid 504; a cAMP and a cGMP dependent protein kinase phosphorylation site from about amino acid 321 to about amino acid 325; N-myristoylation sites from about amino acid 13 to about amino acid 19, from about amino acid 18 to about amino acid 24, from about amino acid 80 to about amino acid 86, from about amino acid 111 to about amino acid 117, from about amino acid 118 to about amino acid 124, of about Amino acid 145 to about amino acid 151, from about amino acid 238 to about amino acid 244, from about amino acid 251 to about amino acid 257, from about amino acid 430 to about amino acid 436, from about amino acid 433 to about amino acid 439, such as amino acid 448, for example, amino acid 454, from about amino acid 458 to about amino acid 464, from about amino acid 468 to about amino acid 474, from about amino acid 475 to about amino acid 481, from about amino acid 496 to about amino acid 502, and from about amino acid 508 to about amino acid 514; and a prokaryotic membrane lipoprotein-lipid attachment site from about amino acid 302 to about amino acid 313. Clone DNA97003-2649 was deposited with the ATCC on May 11, 1999 and assigned ATCC accession number PTA-43.

An analysis of the Dayhoff database (version 35.45 SwissProt 35) using a WU-BLAST2 sequence assembly analysis of in 2 The full-length sequence shown (SEQ ID NO: 2) demonstrated significant homology between the PRO4980 amino acid sequence and the following Dayhoff sequences: SC59_YEAST, S76857, CELF31F4_12, AC002464_1, NU5M_CHOCR, S59109, SAY10108_2, AF055482_2, F69049, and G70433.

EXAMPLE 4

Gene Amplification

This example demonstrates that the PRO coding genes in the genome of certain human lung, Colon and / or breast carcinoma cell lines are amplified. The amplification is associated with the overexpression of the gene product, indicating that the polypeptides have useful therapeutic intervention targets in certain carcinomas, such as, for example, carcinomas. Colon, lung, breast or other carcinomas. Therapeutic agents may take the form of antagonists of PRO polypeptides, for example mouse-human chimeric, humanized or human antibodies to a PRO polypeptide.

The starting material for the screening was genomic DNA isolated from a number of carcinomas. The DNA is z. B. quantified exactly by fluorimetry. As a negative control, DNA was isolated from the cells of ten normal healthy individuals pooled and used as test controls for gene copy number in healthy individuals (not shown). The 5'-nuclease assay (for example, TaqMan ^™ ) and the quantitative real-time PCR (for example, ABI Prizm 7700 Sequence Detection System ^™ (Perkin Elmer, Applied Biosystems Division, Foster City, CA)) were used to detect genes which may be amplified in certain carcinomas. The results were used to determine if PRO-encoding DNA was over-represented in any of the screened primary lung or colon carcinomas or carcinoma cell lines or breast carcinoma cell lines. The primary lung carcinomas were obtained from individuals with tumors of the type and stage listed in Table 6. An explanation of the abbreviations used for the designation of the primary tumors listed in Table 6 and the primary tumors and cell lines, referred to throughout this example, has been given above.

The results of the TaqMan ^™ test are given in delta (Δ) Ct units. One unit corresponds to one PCR cycle or approximately two times amplification compared to the normal, two units correspond to a fourfold, three units to an eightfold amplification and so on. Quantitation was achieved using primers and a TaqMan ^™ fluorescent probe derived from the PRO coding gene. PRO regions that are most likely to contain unique nucleic acid sequences and are least likely to have spliced out introns are preferred for deriving primer and probe, e.g. A 3 'untranslated region. The sequences for the primers and probes (forward, backward and probe) used for the PRO gene amplification analysis were as follows: PRO4980 (DNA97003-2649):

The 5'-nuclease assay is a PCR-based fluorescence technique demonstrating the 5 'exonuclease activity of the Taq DNA polymerase enzyme used to observe the amplification in real time. It will used two oligonucleotide primers to give one typical for a PCR reaction To produce amplicon. A third oligonucleotide or probe is constructed to be between the two PCR primers Prove sequence. The probe is from the Taq DNA polymerase enzyme not renewable and is with a reporter fluorescent dye and a quencher fluorescent dye marked. A laser-induced emission from the reporter dye is quenched by the quench dye when the two dyes lie close to each other, as is the case with the probe. During the Amplification reaction, the Taq DNA polymerase enzyme cleaves the probe on template-dependent Wise. The resulting probe fragments dissociate in solution and the signal from the released reporter dye is free of the quenching effect of the second fluorophore. A molecule of the reporter dye each newly synthesized molecule is released and the detection The unquenched reporter dye provides the basis for the quantitative Interpretation of the data ready.

The 5'-nuclease procedure is performed on a quantitative real-time PCR device, such as. B. the ABI Prism 7700 ^TM Sequence Detection. The system consists of a thermal cycler, laser, a camera with a high-resolution pixel array (CCD camera) and a computer. The system amplifies samples in 96-well format on the thermal cycler. During amplification, the laser-induced fluorescence signal is collected in real time through fiber optic lines for all 96 wells and detected on the CCD camera. The sys It includes software for operating the device and for data analysis.

5'-Nucleasetestdaten be initial expressed as Ct or threshold cycle. This is as that cycle defines the reporter signal over the background level of fluorescence accumulated out. The ΔCt values be used as a quantitative measure of the relative number of initial copies a particular target sequence in a nucleic acid comparing carcinoma DNA results used with DNA results of normal human DNA.

Table 6 describes the stage, T-stage and N-stage of various primary tumors used to screen the PRO compound of the invention. Table 6 Primary Lung and Colon Tumor Profiles primary tumor Sta Other Dukes- T- N- dium stage Stm. Stm. Stm. Human lung tumor adeno (SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725) [LT1a] IIB T3 N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB N0 Human lung tumor AdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung tumor AdenoCa (SRCC728) [LT4] IB T2 N0 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 N0 Human lung tumor Aden / SgCCa (SRCC730) [LT7] IA T1 N0 Human lung tumor AdenoCa (SRCC731) [LT9] IB T2 N0 Human lung tumor SqCCa (SRCC732) [LT10] IIB T2 N1 Human lung tumor SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumor AdenoCa (SRCC734) [LT12] IV T2 N0 Human lung tumor AdenoSqCCa (SRCC735) [LT13] IB T2 N0 Human lung tumor SgCCa (SRCC736) [LT15] IB T2 N0 Human lung tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumor SqCCa (SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18] IB T2 N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human lung tumor LCCa (SRCC741) [LT21] IIB T3 N1 Human Lung AdenoCa (SRCC811) [LT22] 1A T1 N0 Human Colon AdenoCa (SRCC742) [CT2] M1 D pT4 N0 Human Colon AdenoCa (SRCC743) [CT3] B pT3 N0 Human Colon AdenoCa (SRCC744) [CT8] B T3 N0 Human Colon AdenoCa (SRCC745) [CT10] A pT2 N0 Human Colon AdenoCa (SRCC746) [CT12] MO, R1 B T3 N0 Human Colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pN0 Human Colon AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2 Human Colon AdenoCa (SRCC749) [CT16] PMO B pT3 pN0 Human Colon AdenoCa (SRCC750) [CT17] C1 pT3 pN1 Human colon adenoCa (SRCC751) [CT1] MO, R1 B pT3 N0 Human Colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human Colon AdenoCa (SRCC753) [CT5] G2 C1 pT3 pN0 Human Colon AdenoCa (SRCC754) [CT6] pMO, RO B pT3 pN0 Human Colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0 Human Colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human Colon AdenoCa (SRCC757) [CT11] B T3 N0 Human Colon AdenoCa (SRCC758) [CT18] MO, RO B pT3 pN0

DNA Preparation:

DNA was from cultured cell lines, primary tumors and normal human Made of blood. The insulation was made using cleaning kit, Buffer Set and Protease (all from Quiagen) according to the instructions of the Manufacturer and the description below.

Zellkulturlyse:

Cells were washed at a concentration of 7.5 x 10 ⁸ per spike and trypsinized and pelleted by centrifugation at 1,000 rpm for 5 minutes at 4 ° C followed by repeated washing with ½ volume PBS and further centrifugation. The pellets were washed a third time, the suspended cells collected and washed 2x with PBS. The cells were then suspended in 10 ml PBS. Buffer C1 was equilibrated at 4 ° C. Quiagen protease # 19155 was diluted in 6.25 ml cold ddH ₂ O to a final concentration of 20 mg / ml and equilibrated at 4 ° C. 10 ml of G2 buffer was prepared by diluting Quiagen RNAse A stock solution (100 mg / ml) to a final concentration of 200 μg / ml.

Buffers C1 (10 ml, 4 ° C) and ddH ₂ O (40 ml, 4 ° C) were then added to the 10 ml cell suspension, mixed by tilting and incubated on ice for 10 minutes. The nuclei were pelleted by centrifugation in a Beckman swing bucket rotor at 2,500 rpm at 4 ° C for 15 minutes. The supernatant was discarded and the nuclei were vortexed in 2 ml buffer C1 (at 4 ° C) and 6 ml ddH ₂ O, followed by a second centrifugation at 2500 rpm for 15 minutes at 4 ° C. The cores were then resuspended in residual buffer using 200 μl per peak. G2 buffer (10 ml) was added to the suspended nuclei with gentle vortexing. After complete addition of buffer was vigorously shaken by vortex for 30 seconds. Quiagen protease (200 μl, prepared as indicated above) was added and incubated at 50 ° C for 60 minutes. The incubation and centrifugation were repeated until the lysates were clear (eg, incubating for an additional 30-60 minutes, pelleting at 3,000 x g for 10 minutes, 4 ° C).

Production and lysis of a human massive tumor sample:

Tumor samples were weighed and placed in 50 ml conical tubes and kept on ice. The processing was limited to no more than 250 mg tissue per preparation (1 tip / dissection). The Protease solution was made fresh by dilution in 6.25 ml of cold ddH ₂ O to a final concentration of 20 mg / ml and stored at 4 ° C. G2 buffer (20 ml) was prepared by diluting DNAse A to a final concentration of 200 mg / ml (from a 100 mg / ml stock solution). The tumor tissue was homogenized in 19 ml of G2 buffer for 60 seconds using the polytron's large tip in a laminar TC bench to avoid inhalation of aerosols and kept at room temperature. Between the samples, the polytron was purified by centrifugation at 2 x 30 seconds each in 2 L of ddH ₂ O followed by G2 buffer (50 mL). If there was still tissue on the generator tip, the apparatus was disassembled and cleaned.

Qiagen protease (prepared as above, 1.0 ml) was added, followed by vortexing and incubating at 50 ° C for 3 hours. The incubation and centrifugation was repeated until the lysates were clear (e.g. B. Incubate for another 30-60 Minutes, pellet at 3,000 x g for 10 Minutes, 4 ° C).

Production and lysis of human blood

Blood was collected from volunteer healthy donors using standard protocols of infectious agents and titrated in 10 ml samples per tip. Quiagen protease was made fresh by dilution in 6.25 ml of cold ddH ₂ O to a final concentration of 20 mg / ml and stored at 4 ° C. G2 buffer was prepared by diluting RNAse A to a final concentration of 200 μg / ml from a stock solution at 100 mg / ml. The blood (10 ml) was added to a 50 ml conical tube and 10 ml of C1 buffer and 30 ml of ddH ₂ O were added (both previously equilibrated to 4 ° C) and the components mixed by tilting and for 10 minutes kept on ice. The cores were pelleted with a Beckman swing bucket rotor at 2,500 rpm, 4 ° C for 15 minutes and the supernatant discarded. The nuclei were vortexed in 2 ml of C1 buffer (4 ° C) and 6 ml ddH ₂ O (4 ° C). The vortexing was repeated until the pellet was white. The cores were then suspended in the residual buffer using a 200 ml tip. G2 buffer (10 ml) was added to the suspended pellets with gentle vortexing, followed by vigorous vortexing for 30 seconds. Quiagen protease was added (200 μl) and incubated at 50 ° C for 60 minutes. The incubation and centrifugation was repeated until the lysates were clear (eg, incubating for an additional 30-60 minutes, pelleting at 3,000 x g for 10 minutes, 4 ° C).

Purification of clarified lysates:

(1) Isolation of genomic DNA

genomic DNA was equilibrated with 10 ml of QBT buffer (1 sample per maxi-tip preparation). QF elution buffer was equilibrated at 50 ° C. The samples were for Vortex for 30 seconds, then on equilibration tips abandoned and emptied by gravity. The tips were with 2 × 15 Washed ml of QC buffer. The DNA was silanized, autoclaved 30 ml Cortex-tube with 15 ml QF buffer (50 ° C) eluted. Isopropanol (10.5 ml) was added to each sample, the tube sealed with paraffin and mixed by repeated tilting, until the DNA failed. The samples were added by centrifugation in the SS-34 rotor 15,000 rpm for 10 minutes at 4 ° C pelletized. The location of the pellet was marked, the supernatant discarded and 10 ml of 70% ethanol (4 ° C) added. The samples were again by centrifuging on the SS-34 rotor at 10,000 rpm for 10 minutes at 4 ° C pelletized. The location of the pellet was marked and the supernatant discarded. The tubes were then placed in a drying rack on the side and 10 Minutes at 37 ° C dried, being careful not to over-sample to dry.

To On drying, the pellets were dissolved in 1.0 ml TE (pH 8.5) and for 1-2 hours stored at 50 ° C. The samples were over Night at 4 ° C held, dissolving continued. The DNA solution was then placed in 1.5 ml tubes transferred to a tuberculin syringe with a No. 26 injection needle. The overpass was Repeated 5 times to shear the DNA. The samples were then for 1-2 hours at 50 ° C kept.

(2) quantification of genomic DNA and Production for the gene amplification test

DNA levels in each tube were determined by standard A ₂₆₀ , A ₂₈₀ spectrophotometry at 1:20 dilution (5 μl DNA + 95 μl ddH ₂ O) using the 0.1 ml quartz cuvettes in the Beckman Spectrophotometer DU640 quantified. The A ₂₆₀ / A ₂₈₀ ratios ranged from 1.8-1.9. Each DNA sample was then further diluted to approximately 200 ng / ml in TE (pH 8.5). If the starting material was highly concentrated (about 700 ng / μl), the material was stored at 50 ° C for several hours until resuspension.

A Fluorimetric DNA quantification was then performed on the diluted material (20-600 ng / ml), the manufacturer's guidelines modified as indicated below were used. This was achieved by using a fluorimeter DyNA Quant 200 from Hoeffer for warmed up for about 15 minutes has been. The Hoechst Dye Working Solution (# H33258, 10 μl, prepared within 12 hours of use) was in 100 ml of 1 × TNE buffer diluted. A 2 ml cuvette was using the fluorimeter solution filled, in the device given and the device adjusted to zero. pGEM 3Zf (+) (2 μl, Lot # 360851026) to 2 ml of fluorimeter solution added and calibrated to 200 units. Another 2 μl of pGEM 3Zf (+) DNA were then tested and the measurement confirmed at 400 +/- 10 units. each Sample was then measured at least in triplicate. If 3 samples within 10%, their mean was taken and this value used as quantification value.

The fluorimetrically determined concentration was then used to dilute each sample to 10 ng / μl in ddH ₂ O. This was done simultaneously on all templated samples for a single TaqMan ^™ plate test and with sufficient material to perform 500-1000 tests. Trials were run in triplicate with TaqMan ^™ primers and B-actin and GAPDH probes on a single panel of normal human DNA and non-template controls. The diluted samples were used on the assumption that the Ct value of normal human DNA subtracted from the test DNA was +/- 1 Ct. The diluted, lot-qualified genomic DNA was stored in 1 ml aliquots at -80 ° C. Aliquots to be subsequently used in the gene amplification assay were stored at 4 ° C. Each 1 ml aliquot was sufficient for 8-9 plates or 64 tests.

Genamplifikationstest:

The PRO compounds of the invention were screened in the following primary tumors, and the resulting ΔCt values are given in Table 7. Table 7 ΔCt values in lung and colon primary tumor models primary tumor PRO4980 HF-000840 1.01 HF-001294 1.14 HF-001296 1.87 HF-001299 1.12

PRO4980 (DNA97003-2649):

The ΔCt values for DNA97003-2649 in different tumors are summarized in Table 7. One ACt value> 1 typically as a threshold for evaluation of amplification used, since this value represents a doubling of gene copy number. Table 7 indicates that significant amplification of nucleic acid DNA97003-2649 used for PRO4980 encodes in lung primary tumors occurred: HF-000840, HF-001294, HF-001296 and HF-001299.

EXAMPLE 5

In situ hybridization

The In-situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences in cell or Tissue preparations. For example, it may be convenient to sites of gene expression to identify the tissue distribution of the transcription, Virus infection to identify and locate changes to track specific mRNA synthesis and chromosome mapping to support.

In situ hybridization will be performed according to an optimized version of the protocol of Lu and Gillett, Cell Vision 1, 169-176 (1994) using PCR generated ³³ P-labeled riboprobes. In summary, formalin-fixed, paraffin-embedded human tissues are cut deproteinized proteinase K (20 g / ml) for 15 minutes at 37 ° C and further processed for in situ hybridization as described by Lu and Gillett, supra. A [ ³³ P] -UTP-labeled antisense riboprobe is generated from a PCR product and hybridized at 55 ° C overnight. The slides are dipped in Kodak NTB2 core emulsion and exposed to film for 4 weeks.

³³ P-ribosondensynthesis

6.0 μL (125 mCi) ³³ P-UTP (Amersham BF 1002, SA <2,000 Ci / mmol) are dried by Speed-Vac. To each tube containing ³³ P-UTP dried, the following ingredients are added:
2.0 μl 5 × transcription buffer
1.0 μl DTT (100 mM)
2.0 μl NTP mixture (2.5 mM: 10 μl, 10 mM each of GTP, CTP and ATP + 10 μl H ₂ O)
1.0 μl UTP (50 μM)
1.0 μl RNAsin
1.0 μl DNA template (1 μg)
1.0 μl H ₂ O
1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually)

The tube be at 37 ° C for 1 hour incubated. 1.0 μl RQ1 DNase is added, followed by incubation at 37 ° C for 15 minutes. 90 μl TE (Tris pH 7.6 / 1mM EDTA pH 8.0) are added and the mixture pipetted on DE81 paper. The remaining solution is transferred to a Microcon 50 ultrafiltration unit and centrifuged using program 10 (6 minutes). The filtration unit is over a second tube inverted and centrifuged using program 2 (3 minutes). To 100 μl of TE are added to the last centrifugation for recovery. 1 μl of Final product is pipetted on DE81 paper and in 6 ml biofluor II counted.

The probe is run on a TBE / urea gel. 1-3 μl of the probe or 5 μl of RNA Mrk III are added to 3 μl of loading buffer. After heating in a heating block at 95 ° C for three minutes, the gel is immediately put on ice. The wells of the gel are rinsed, the sample is abandoned and run at 180-250 volts for 45 minutes. The gel is wrapped in a plastic film (trademark SARAM ^™ ) and exposed at -70 ° C in the freezer for one hour to overnight to XAR film with an intensifying screen.

³³ P hybridization

A. Pretreatment of frozen sections

The slides are removed from the freezer, placed on aluminum trays and thawed at room temperature for 5 minutes. The dishes are placed in an incubator for 5 minutes at 55 ° C to reduce condensation. The slides are fixed in 4% paraformaldehyde on ice for 10 min in a fume hood and washed in 0.5x SSC for 5 min at room temperature (25 ml 20x SSC + 975 ml SQ-H ₂ O). After deproteination in 0.5 μg / ml proteinase K for 10 minutes at 37 ° C (12.5 μl of a 10 mg / ml stock solution in 250 ml prewarmed RNase-free RNAse buffer), slice into 0.5 × SSC washed for 10 minutes at room temperature. The sections are dehydrated for 2 minutes in 70%, 95%, 100% ethanol.

B. Pretreatment of embedded in paraffin sections

The slides are paraffin-free, placed in SQ-H ₂ O and rinsed twice in 2X SSC at room temperature for 5 minutes each. The sections are resuspended in 20 μg / ml proteinase K (500 μl of a 10 mg / ml solution in 250 ml RNase-free RNase buffer, 37 ° C, 15 minutes) in a human embryo or 8 × proteinase K (100 μl in 250 ml RNase buffer, 37 ° C, 30 minutes) on formalin tissue. Subsequent rinsing in 0.5 x SSC and dewatering are performed as described above.

C. Prehybridization

The slides are placed in a plastic box lined with box buffer (4xSSC, 50% formamide) -saturated filter paper. The tissue is covered with 50 μl hybridization buffer (3.75 g dextran sulfate + 6 ml SQ-H ₂ O), vortexed and heated in a microwave for 2 minutes with loosened caps. After cooling on ice, 18.75 ml of formamide, 3.75 ml of 20 × SSC and 9 ml of SQ-H ₂ O are added, the Ge Prepare tissue well and incubate at 42 ° C for 1-4 hours.

D. Hybridization

1.0 x 10 ⁶ cpm probe and 1.0 μl tRNA 850 mg / ml stock) per slide are heated at 95 ° C for 3 minutes. The slides are cooled on ice and 48 μl hybridization buffer per slide is added. After vortexing, 50 μl of ³³ P mix is added to 50 μl pre-hybridization on the slide. The slides are incubated overnight at 55 ° C.

E. washes

The washing is carried out for 2 × 10 minutes with 2 × SSC, EDTA at room temperature (400 ml 20 × SSC + 16 ml 0.25 M EDTA, V _f = 4 L), followed by RNAseA treatment at 37 ° C. for 30 Minutes (500 μl of a 10 mg / ml solution in 250 ml Rnase buffer = 20 μg / ml). The slides are washed 2 x 10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions are as follows: 2 hours at 55 ° C, 0.1X SSC, EDTA (20mL 20x SSC + 16mL EDTA, V _f = 4L).

EXAMPLE 6

Use of PRO as hybridization probe

The The following procedure describes the use of one for a PRO polypeptide encoding nucleotide sequence as a hybridization probe.

DNA, which encodes the coding sequence of a full length or mature "PRO" polypeptide, such as disclosed herein and / or comprises fragments thereof, may be used as a probe can be used to target homologous DNAs (such as those that are naturally occurring Variants of PRO) in human cDNA libraries or to screen human genomic tissue genome libraries.

The Hybridization and washing of filters, one of the two Library DNAs is performed under the following stringency conditions. The Hybridization of radiolabeled, derived from PRO Probe to the filter is dissolved in a solution of 50% formamide, 5 × 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 carried out. The washing of the filters is carried out in an aqueous solution of 0.1 × SSC and 0.1% SDS at 42 ° C carried out.

DNAs which the desired sequence identity with the for the full-length native sequence PRO can have coding DNA then using standard techniques that are in the field of the invention are identified.

EXAMPLE 7

Expression of PRO Polypeptides in E. coli

This Example illustrates the preparation of an unglycosylated form of PRO by recombinant expression in E. coli.

The for the PRO polypeptide of interest encoding DNA sequence is initially under Use of selected PCR primer amplified. The primers should be restriction enzyme sites having the restriction enzyme sites on the selected expression vector correspond. It can be used a variety of expression vectors become. An example of a suitable vector is pBR322 (from E. coli; see Bolivar et al., Gene 2, 95 (1977)), Gene for ampicillin and contains tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably comprise sequences coding for an antibiotic resistance gene, a trp promoter, a poly-his-leader (including the first six STII codons, poly-his-sequence and enterokinase cleavage site) sequence coding for PRO Region, λ transcriptional terminator and encode 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 due their ability to grow on LB plates and then antibiotic-resistant colonies are selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

selected Clones can overnight in liquid medium, such as B. supplemented with antibiotics Bred LB broth become. The overnight culture can subsequently used to inoculate a larger scale culture. The Cells are then to a desired bred optical density, meanwhile the Expression promoter is turned on.

To cultivating the cells for several more hours can the cells are harvested by centrifugation. The by centrifugation obtained cell pellet can be made using various, in the field Solubilized the invention known means and solubilized PRO protein can then be prepared using a metal chelating agent Column under Conditions are purified, which is the firm binding of the protein enable.

A PRO protein can be successfully expressed in E. coli in poly-his-labeled form using the following procedure. The DNA encoding the PRO is first amplified using selected PCR primers. The primers contain restriction enzyme sites corresponding to the restriction enzyme sites on the selected expression vector and other convenient sequences that provide for efficient and reliable translation initiation, rapid purification on a metal chelating column and proteolytic removal with enterokinase. The PCR-amplified, poly-his-tagged sequences are then ligated into an expression vector, which is then used to generate E. coli based on strain 52 (W3110fuhA (tonA) ion galE rpoHts (htpTrs) clpP (lacIq)) -Wirt transform. Transformants are first cultured in LB containing 50 mg / ml carbenicillin at 30 ° C with shaking until an OD600 of 3-5 is reached. The cultures are then 50-100 fold in CRAP medium (prepared by mixing 3.57 g (NH ₄ ) ₂ SO ₄ , 0.71 g sodium citrate 2H ₂ O, 1.07 g KCl, 5.36 g Difco- Yeast extract, 5.36 g of Sheffield Hycase SF in 500 ml water, and 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO ₄ ), and diluted for about 20-30 Bred for hours at 30 ° C with shaking. Samples are taken to verify expression by SDS-PAGE analysis and most of the culture is centrifuged to pellet the cells. Cell pellets are frozen until they are cleaned and refolded.

E. coli paste from fermentations of 0.5 to 1 L (6-10 g pellets) is resuspended in 10 volumes (w / v) of 7 M guanidine, 20 mM Tris buffer, pH8. Solid sodium sulfite and sodium tetrathiosulfate are added until a final concentration of 0.1 M or 0.02 M is reached, and the solution is stirred overnight at 4 ° C. This step results in a denatured protein in which all cysteine residues are blocked by sulfitolation. The solution is centrifuged at 40,000 rpm in a Beckman ultracentrifuge for 30 minutes. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered for clarification through 0.22 μm filters. The clarified extract is applied to a 5 ml Qiagen-Ni ²⁺ -NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. The desired protein-containing fractions are pooled and stored at 4 ° C. The protein concentration is estimated by absorbance at 280 nm using the extinction coefficient calculated based on its amino acid sequence.

The protein is refolded by diluting the sample slowly in freshly prepared refolding buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA exists. Refolding volumes are chosen so that the final protein concentration is between 50 and 100 micrograms / ml. The refolding solution is gently stirred at 4 ° C for 12-36 hours. The refolding reaction is quenched by addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 μm filter and acetonitrile is added to a final concentration of 2-10%. The refolded protein is chromatographed on a Poros R1 / H reverse phase column using a mobile buffer of 0.1% TFA eluted with a gradient of acetonitrile from 10 to 80%. Aliquots of A ₂₈₀ absorbance fractions are analyzed on SDS-polyacrylamide gels and fractions containing homogenous refolded protein are pooled. In general, the properly folded species of most proteins are eluted at the lowest acetonitrile concentrations since those species are the most compact and their hydrophobic interior is shielded from interaction with the reverse phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to separating misfolded protein forms from the desired shape, the reverse phase step also removes endotoxin from the samples.

Fractions containing the desired folded PRO are combined and the acetonitrile with a light, directed to the solution nitrogen stream removed. Proteins are formulated in 20mM Hepes, pH 6.8, with 0.14M sodium chloride and 4% mannitol by dialysis or gel filtration using G25 superfine resins (Pharmacia) equilibrated in formulation buffer and sterile filtered.

EXAMPLE 8

Expression of PRO in mammalian cells

This Example illustrates the preparation of a possibly glycosylated Form of PRO by recombinant expression in mammalian cells.

The vector pRK5 (see EP 307,247 , published March 15, 1989) is used as an expression vector. Optionally, the PRO DNA in pRK5 is ligated with selected restriction enzymes to facilitate insertion of the PRO DNA using ligation techniques, such as. For example, those described in Sambrook et al., Supra. The resulting vector is called pRK5-PRO4980.

In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are transfected into tissue culture plates in medium, e.g. B. bred with fetal calf serum and, optionally, with components and / or antibiotics supplemented DMEM to confluency. About 10 μg of pRK5-PRO4980 DNA is mixed with about 1 μg of DNA encoding the VA RNA gene (Thimmappaya et al., Cell 31, 543 (1982)) and mixed in 500 μl of 1 mM Tris-HCl. 0.1 M EDTA, 0.227 M CaCl ₂ dissolved. To this mixture is added drop by drop 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ and allowing precipitate to form for 10 minutes at 25 ° C. The precipitate is suspended and added to the 293 cells and allowed to settle for approximately four hours at 37 ° C. The culture medium is aspirated and 2 ml of 20% glycerol in PBS are added for 30 seconds. The 293 cells are then washed with serum-free medium, fresh medium added and the cells incubated for approximately 5 days.

Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium replaced, containing 200 uCi / ml ³⁵ S-cysteine and 200 Ci / ml ³⁵ S-methionine. After incubation for 12 hours, the conditioned medium is collected, concentrated on a centrifuge filter and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to a film for a selected period of time to reveal the presence of the PRO polypeptide. The cultures containing transfected cells may be subjected to further incubation (in serum-free medium) and the medium tested in selected bioassays.

In In an alternative technique, PRO DNA can be prepared using the method described in US Pat Somparyrac et al., Proc. Natl. Acad. Sci. USA 12, 7575 (1981) Dextran sulfate process temporarily introduced into 293 cells become. 293 cells are maximized in a centrifuge bottle Density bred, and it will be 700 micrograms pRK5-PRO4980 DNA added. The cells are first centrifuged from the centrifuge bottle and washed with PBS. The DNA-dextran precipitate is at the cell pellet for incubated for four hours. The cells are treated with 20% glycerol for 90 seconds washed, washed with tissue culture medium and returned to the tissue culture medium, 5 μg / ml Bovine insulin and 0.1 μg / ml Bovine transferrin containing centrifuge bottle introduced. To about four days, the conditioned medium is centrifuged and filtered, around cells and debris to remove. The sample containing PRO can then be probed with any chosen Method, such. As dialysis and / or column chromatography become.

In another embodiment, PRO can be expressed in CHO cells. The pRK5-PRO4980 vector can be prepared using known reagents, such as e.g. B. CaPO ₄ or DEAE-dextran be transfected into CHO cells. As described above, the cell cultures can be incubated and the medium replaced by culture medium (alone) or culture medium containing a radioactive marker, such. B. ^{35 contains} S-methionine. After detection of the presence of the PRO polypeptide, the culture medium may be replaced with serum-free medium. Preferably, the cultures are incubated for about 6 days and then the conditioned medium is harvested. The medium containing the expressed PRO can then be concentrated and purified by any chosen method.

Epitope-tagged PRO can also be expressed in host CHO cells. The PRO can be subcloned from the pRK5 vector. The subclone insert may be subjected to a PCR to generate in-frame with a selected epitope tag, such as. To fuse a poly-his marker in a baculovirus expression vector. The poly-his tagged PRO insert can then be subcloned into an SV40 driven vector be a selection marker, such as. B. DHFR for the selection of stable clones. Finally, the CHO cells can be transfected with the SV40 driven vector (as described above). The labeling may be performed as described above to verify expression. The culture medium containing the poly-his-tagged PRO may then be removed by any chosen method, such as a. For example, Ni ²⁺ chelate affinity chromatography can be concentrated and purified. Expression in CHO and / or COS cells may also be by a transient expression method.

One PRO protein can be obtained by a stable expression method or by a temporary one Methods are expressed in CHO cells. The stable expression in CHO cells is performed using the following procedure. The Proteins are expressed as an IgG construct (immunoadhesin) in which the coding sequences for the soluble ones Forms (eg extracellular domains) the respective proteins to a IgG1 sequence of the constant region are fused, which contains the hinge, CH2 and CH2 domains, and / or in a poly-his-labeled form.

To PCR amplification becomes the respective DNAs in a CHO expression vector subcloned, using standard techniques, the in Ausubel et al., Current Protocols in Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are engineered to be compatible restriction sites 5 'and 3' of the DNA of interest to provide the appropriate shuttling of cDNAs. The vector used for expression in CHO cells is described in Lucas et al., Nucl. Acids Res. 24, 9 (1774-1779) (1996) and uses the early ones SV40 promoter / enhancer to express the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression allows selection for stable maintenance of the plasmid after Transfection.

Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect ^® (Quiagen), Dosper ^® or Fugene ^® (Boehringer Mannheim) were introduced. The cells are grown as described in Lucas et al., Supra. Approximately 3 x 10 cells are frozen in an ampule for further growth and production described below.

The ampoules containing the plasmid DNA are thawed by placing them in a water bath and vortexing. The contents are pipetted into a centrifuge tube containing 10 ml of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 ml of selective medium (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 ml centrifuge bottle containing 90 ml of selective medium. After 1-2 days, the cells are transferred to a 250 ml centrifuge bottle filled with 150 ml of selective growth medium and incubated at 37 ° C. After a further 2-3 days, a 250 ml, 500 ml and 2,000 ml centrifuge bottle is inoculated at 3 × 10 ⁵ cells / ml. The cell medium is replaced by Zentri Fugation and resuspension in production medium through fresh medium. Although any suitable CHO medium can be used, one issued June 16, 1992 U.S. Patent No. 5,122,469 described production medium preferred. A 3 liter production centrifuge bottle is inoculated at 1.2 x 10 ⁶ cells / ml. On day 0, cell number and pH are determined. On day 1, samples are taken from the centrifuge bottle and the ventilation started with filtered air. On day 2, samples are taken from the centrifuge bottle, the temperature is changed to 33 ° C and 30 ml of a solution of 500 g / l glucose and 0.6 ml of 10% antifoam (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade emulsion) was added. Throughout the production time, the pH is adjusted as needed to maintain a pH of about 7.2. After 10 days or when viability drops below 70%, the cell culture is harvested by centrifugation and filtration through a 0.22 μm filter. The filtrate is either stored at 4 ° C or immediately applied to columns for purification.

For the poly-his tagged constructs, the proteins are purified using a Ni ²⁺ NTA column (Qiagen). Prior to purification, imidazole is added to the conditioned medium to a concentration of 5 mM. The conditioned medium is pumped at a flow rate of 4-5 ml / min at 4 ° C onto a Ni ²⁺ -NTA column packed with 0.3 mM NaCl and 20 mM Hepes buffer, pH 7, containing 5 mM imidazole , 4, is equilibrated. After loading, the column is washed with additional equilibration buffer and the protein is eluted with 0.25 M imidazole-containing equilibration buffer. The most purified protein becomes. then desalted with a G25 Superfine column (Pharmacia) into storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, and stored at -80 ° C.

Immunoadhesin (Fc containing) constructs are purified from the conditioned medium as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) dissolved in 20 mM Natri Umphosphatpuffer, pH 6.8, has been equilibrated. After loading, the column is washed extensively with equilibration buffer before being eluted with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions in 275 μl M Tris buffer, pH 9, containing tubes. The highly purified protein is then desalted in storage buffer as described above for the poly-his tagged proteins. Homogeneity is verified by SDS-polyacrylamide gels and N-terminal amino acid sequencing by Edman degradation.

EXAMPLE 9

Expression of PRO in yeast

The The following procedure describes the recombinant expression of PRO in yeast.

First be expression vectors for intracellular production or secretion PRO constructed from the ADH2 / GAPDH promoter. Coding for PRO DNA and the promoter are placed in appropriate restriction enzyme sites in the chosen one Plasmid inserted to control the intracellular expression of PRO. to Secretion can for PRO-encoding DNA together with DNA coding for the ADH2 / GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide or, for example, a yeast α-factor or Invetase secretion signal / leader sequence and linker sequences (if needed) for expression of PRO in the chosen plasmid.

yeast cells such as B. yeast strain AB110 can then transformed with the expression plasmids described above and in selected Fermentation be cultivated. The supernatants of the transformed yeasts can by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie blue dye to be analyzed.

recombinant PRO can subsequently by removing the yeast cells from the fermentation medium by means of Centrifugation and subsequent Concentrate the medium using selected cartridge filters isolated and be cleaned.

The PRO containing concentrate can continue using selected column chromatography resins getting cleaned.

EXAMPLE 10

Expression of PRO in Baculovirus-infected insect cells

The The following procedure describes the recombinant PRO expression in Baculovirus-infected insect cells.

The for PRO coding sequence is upstream of an epitope marker, which in one Baculovirus expression vector is included, fused. such Epitope markers include poly-his-markers and immunoglobulin markers (such as z. Fc regions of IgG). It can be a variety of plasmids be used, including Plasmids obtained from commercially available Plasmids, such as. B. pVL1393 (Novagen). In summary will the for PRO or the desired one Section of the coding sequence of PRO (such as that for the extracellular domain of a Transmembrane protein coding sequence or that for the mature protein coding sequence, if the protein is extracellular) Sequence amplified by PCR with primers that are complementary to the 5 'and 3' regions. The 5'-primer can flanking (selected) restriction enzyme sites. The product is then digested with these chosen restriction enzymes and subcloned into the expression vector.

A recombinant baculovirus is prepared by co-transfecting the above plasmid and BaculoGold ^™ viral DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using Lipofectin (commercially available from GIBCO-BRL After 4-5 days of incubation at 28 ° C, the released viruses are harvested and used for further amplifications Virus infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford, Oxford University Press (1994).

Expressed poly-his-tagged PRO can then be purified, for example, by Ni ²⁺ chelate affinity chromatography as follows. Extracts from recombinant, virus-infected Sf9 cells such as Rupert et al., Nature 362, 175-179 (1993). In summary, Sf9 cells are washed in sonication buffer (25 ml Hepes, pH 7.9, 12.5 mM MgCl ₂ , 0.1 mM EDTA, 10% glycerol, 0.1% NP-40, 0.4 M KCl). resuspended and sonicated twice for 20 seconds on ice. The sonicated samples are clarified by centrifugation and the supernatant diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni ²⁺ -agarose column (commercially available from Quiagen) is prepared with a bed volume of 5 ml, washed with 20 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed to baseline A ₂₈₀ with loading buffer, at which time fraction collection begins. Next, the column is washed with a second wash buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6.0) to elute non-specifically bound protein. After reaching the A ₂₈₀ baseline again, the column is developed with a 0-500 mM imidazole gradient in the second wash buffer. 1 ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ²⁺ -NTA-conjugated alkaline phosphatase (Quiagen). Fractions containing eluted His _10- labeled PRO are pooled and dialyzed against loading buffer.

alternative this may involve purification of the IgG-labeled (or Fc-labeled) PRO using known chromatographic techniques, which include, for example, protein A or protein G chromatography.

Even though the expression actually in a scale from 0.5 to 2 l it can easily be scaled up for larger (eg 8 l) preparations. The proteins are expressed as an IgG construct (immunoadhesin) in which the extracellular Protein region fused to an IgG1 sequence of the constant region which contains the hinge, CH2 and CH3 domains, and / or as poly-his tagged To shape.

After PCR amplification, the respective coding sequences are subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and BaculoGold baculovirus ^® DNA (Pharmingen) are co-transfected into 105 Spondoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using Lipofectin (Gibco BRL). Pb.PH.IgG and pb.PH.His are modifications of the art Commercially available baculovirus expression vector pVL1393 (Pharmingen) with modified polylinker regions comprising His or Fc marker sequences The cells are grown in Hink TNM-FH medium supplemented with 10% FBS (Hyclone). The cells are incubated for 5 days at 28 ° C. The supernatant is harvested and then amplified for the first viral amplification by infecting Sf9 cells in Hink TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. The cells are used for 3 Days incubated at 28 ° C. The supernatant is harvested and expression of the constructs detected in the baculovirus expression vector by batch binding of 1 ml of supernatant to 25 ml of NI-NTA beads (Qiagen) for histidine-labeled proteins or Protein A-Sepharose CL-4B -Pearls (Pharmacia) for IgG-labeled proteins, followed by SDS-PAGE analysis and comparison with a known concentration of protein standard by Coomassie blue staining.

The supernatant The first viral amplification is used to prepare a centrifuge bottle culture (500 ml) cultured in ESF-921 medium (Expression Systems LLC) Sf9 cells at approximate MOI of 0.1 to infect. The cells are incubated for 3 days at 28 ° C. The supernatant is harvested and filtered. The batch binding and SDS-PAGE analysis will be repeated as necessary until the expression of the centrifuge bottle culture approved is.

The conditioned medium from the transfected cells (0.3 to 3 l) is harvested by centrifugation to remove the cells and through 0.22 μm filters filtered. For the poly-his-tagged constructs become the protein constructs by means of Ni-NTA column (Quiagen) cleaned. Before cleaning, the conditioned medium Imidazole added to a concentration of 5 mM. The conditioned Medium is at a flow rate of 4-5 ml / min at 4 ° C to a 6 ml Ni-NTA column pumped, containing 20 mM in 0.3 M NaCl and 5 mM imidazole Hepes buffer, pH 7.4, equilibrated was. After loading, the column is washed with additional equilibration buffer and the protein with 0.25 M imidazole-containing equilibration buffer eluted. The highest purified protein is then probed with a G25 Superfine (Pharmacia) column of 25 ml in a 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 8, containing Storage buffer desalted and stored at -80 ° C.

Immunoadhesin (Fc-containing) constructs of proteins are purified from the conditioned medium as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) equilibrated in 20 mM Na phosphate buffer, pH 8. After loading, the column was extensively with Equilibration buffer was washed before being eluted with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into 275 1M Tris pH 9 buffer containing tubes. The highly purified protein is then desalted in storage buffer as described above for the poly-his tagged proteins. The homogeneity of the proteins is verified by SDS-PAGE and N-terminal amino acid sequencing by Edman degradation.

alternative For this a modified baculovirus method can be used, incorporated the high-5 cells. In this procedure, the for the desired sequence encoding DNA with suitable systems, such. B. Pfu (Stratagene), amplified or upstream of an epitope tag (5 'of it) within one Baculovirus expression vector is included, merged. Such epitope tags include poly-his tags and immunoglobulin labels (such as Fc regions of IgG). numerous Plasmids can can be used, including Plasmids obtained from commercial Plasmids such. B. plE1-1 (Novagen) derived. The plE1-1 and plE1-2 vectors are more recombinant for constitutive expression Proteins from the baculovirus ie1 promoter in stably transformed Insect cells (1) designed. The plasmids differ only in the alignment of multiple cloning sites and contain all Promoter sequences for that they are known for being ie1-mediated gene expression in uninfected insect cells important, as well as the hr5 enhancer element. plE1-1 and plE1-2 include the translation initiation site and can be used be used to produce fusion proteins. Short summary will be the desired Sequence or the desired Section of the sequence (such as the sequence coding for the extracellular domain of a encoded transmembrane protein) by PCR with primers that are complementary to the 5 'and 3' regions, amplified. The 5 'primer may incorporate flanking (selected) restriction enzyme sites. The product is then combined with those selected restriction enzymes digested and subcloned into the expression vector. Derivatives of plE1-1 for example the Fc region of human IgG (pb.PH.IgG) or an 8-histidine (pb.PH.His) label downstream of the desired Sequence (3 'of it) include. Preferably, the vector construct is sequenced for confirmation.

High-5 cells are grown to 50% confluence under the conditions: 27 ° C, no CO ₂ , no pen / strep; bred. For each 150 mm plate, 30 μg of PlE-based vector containing the sequence is mixed with 1 ml of Ex-Cell medium (medium: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences No. 14401-78P (Note: this medium is photosensitive)) and in a separate tube 100 μl of CellFectin (CellFECTIN (GibcoBRL No. 10362-010) (mixed by centrifugation)) is mixed with 1 ml of Ex-Cell medium. The two solutions are combined and allowed to incubate at room temperature for 15 minutes. 8 ml of Ex-Cell medium is added to 2 ml of DNA / CellFECTIN mixture and this is layered on high-5 cells that were previously washed once with Ex-Cell medium. The plate is then incubated in darkness for 1 h at room temperature. The DNA / CellFECTIN mixture is then aspirated and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell medium is added and the cells are incubated for 3 days at 28 ° C , The supernatant is harvested and the expression of the sequence in the baculovirus expression vector is determined by batch binding of 1 ml of supernatant to 25 ml of Ni ²⁺ NTA beads (QIAGEN) for histidine-labeled proteins or protein A-Sepharose CL-4B Beads (Pharmacia) for IgG-labeled proteins, followed by SDS-PAGE analysis, which compares to a known concentration of protein standard by Coomassie blue staining.

The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by centrifugation to remove the cells and filtered through 0.22 μm filters. For the poly-his tagged constructs, the protein comprising the sequence is purified using a Ni ²⁺ NTA column (Qiagen). Prior to purification, imidazole is added to the conditioned medium at a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni ²⁺ -NTA column, which is equilibrated in 20 mM Hepes buffer, pH 7.4 containing 0.3 M NaCl and 5 mM imidazole at a flow rate of Pumped 4-5 ml / min at 48 ° C. After loading, the column is washed with additional equilibration buffer and the protein is eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is then desalted in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine column (Pharmacia) and at -80 ° C stored.

(Fc-containing) immunoadhesin constructs of proteins are purified from the conditioned medium as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before eluting with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions in tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purified protein is then desalted in storage buffer as previously described for the poly-his tagged proteins. The homogeneity of the sequence is determined by SDS-polyacrylamide gels and by Sequencing of N-terminal amino acids by Edman degradation and other analytical methods, as needed and desired.

EXAMPLE 11

Production of antibodies that bind PRO

This Example illustrates the preparation of monoclonal antibodies which can specifically bind to PRO.

method for the production of monoclonal antibodies are in the field of Invention are known and, for example, in Goding, s. o., Described. Immunogens that can be used include purified ones PRO, fusion proteins that contain PRO, and cells that are recombinant PRO on the cell surface express. The selection of the immunogen can be made by those skilled in the art without undue experimentation.

Mice, like z. B. Balb / c are immunized with the PRO immunogen in complete Freund's adjuvant emulsified and subcutaneously or intraperitoneally in an amount of 1 to 100 μg is injected. Alternatively, the immunogen is added to MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) emulsified and in the balls of the feet the hind legs the animals injected. The immunized mice will then be 10 to 12 days later with additional Immunogen selected in the Adjuvant emulsified, boosted. After that, the mice can also be used for several weeks with additional Immunization injections are boosted. Serum samples can be used mice by retro-orbital blood sampling for testing by ELISA detection for detection of anti-PRO antibodies at periodic intervals be removed.

After this a suitable antibody titer animals with "positive" antibody levels may receive a final intravenous injection administered by PRO. Three or four days later, the Mice killed and the spleen cells harvested. The spleen cells are then selected Mouse myeloma cell line such. P3X63AgU.1, available from the ATCC, No. CRL 1597, available is fused (using 35% polyethylene glycol). The Fusions form hybridoma cells, which are then transformed into 96-well tissue culture plates HAT (hypoxanthine, aminopterin and thymidine) medium, plated become proliferation of unfused cells, myeloma hybrids and spleen cell hybrids.

The Hybridoma cells are tested for reactivity in an ELISA PRO screened. Determination of "positive" hybridoma cells, the desired ones monoclonal antibody secrete against PRO is within the scope of the invention.

The positive hybridoma cells can Intraperitoneally injected into syngeneic Balb / c mice to ascites to produce the monoclonal anti-PRO antibodies. Alternatively, you can the hybridoma cells are grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies, which are produced in ascites can be followed using ammonium sulfate precipitation of gel exclusion chromatography. Alternatively, it can affinity based on binding of antibodies be used on protein A or protein G.

Deposit of material

The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, (ATCC): material ATCC. deposit date DNA97003-2649 PTA-43 May 11, 1999

This deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Provisions Thereunder (Budapest Treaty). This ensures the preservation of a viable culture of deposit for 30 years from the date of deposit. The deposits are made available by the ATCC under the terms of the Budapest Treaty and pursuant to an agreement between Genentech, Inc., and ATCC, the permanent and unrestricted availability of the progeny of the culture of the deposit to the public when the relevant US patent is issued or disclosure to the public, either the US or a foreign patent application, whichever comes first, and guarantees the availability of the progeny to any person appointed by the President of the United States Patent Office in accordance with 35 USC § 122 and the relevant provisions ( including 37 CFR § 1.14 with special reference to 886 OG 638).

Of the Assignee of the present application has agreed that, provided a culture of the deposited materials under appropriate Conditions were cultured, die or lost or destroyed should be, the materials immediately after notification be replaced by new ones of the same kind. Availability of the deposited material is not a license to perform the invention in contradiction with the under the authority any government according to theirs Patent laws guaranteed rights to understand.

The the above written description is considered sufficient to: Professionals the opportunity to give, to carry out the invention. The present invention is intended to be limited in scope by the present invention Do not construct as restricted apply, as the deposited embodiment solely as an illustration of certain aspects of the invention understand, and any constructs that are functionally equivalent, are within the scope of this invention. The deposit Material herein does not constitute an admission that the herein written description is inadequate, the practical execution of any Aspect of the invention, including the best embodiment of it, to enable nor is it as a limitation the scope of the claims to understand the specific illustrations that it represents.

sequence Listing

Claims (20)

A method of diagnosing a tumor in a mammal, the method comprising detecting the level of expression of a PRO4980 polypeptide-encoding gene in (a) a sample of mammalian tissue cells and (b) a control sample of known normal cell tissue of the same cell type, wherein Higher expression level in the sample compared to the control sample indicates the presence of a tumor in the mammal from which the test tissue cells were obtained, and wherein the PRO4980 polypeptide has a polypeptide with at least 80% amino acid sequence identity with: (i) the in 2 represented amino acid sequence (SEQ ID NO: 2), (ii) the in 2 represented amino acid sequence (Seq.-ID No. 2) without signal peptide, (iii) an extracellular domain of in 2 represented amino acid sequence (SEQ ID NO: 2) with the signal peptide, (iv) an extracellular domain of in 2 and (v) the amino acid sequence encoded by the full-length PRO4980 coding sequence of DNA97003-2649 deposited under ATCC accession number PTA-43, wherein is the amino acid sequence shown in SEQ. ID Nucleic acid or amino acid sequence identity is determined using the ALIGN-2 computer program. The method of claim 1, wherein the degree of identity is at least 90%. The method of claim 2, wherein the degree of identity is at least 95%. The method of claim 3, wherein the PRO4980 polypeptide comprises a polypeptide having (i) the in 2 represented amino acid sequence (SEQ ID NO: 2), (ii) the in 2 represented amino acid sequence (Seq.-ID No. 2) without signal peptide, (iii) an extracellular domain of in 2 represented amino acid sequence (SEQ ID NO: 2) with the signal peptide, (iv) an extracellular domain of in 2 and (v) the amino acid sequence encoded by the full length PRO4980 coding sequence of DNA97003-2649 deposited under ATCC accession number PTA-43. A method according to any one of the preceding claims wherein the signal peptide is selected from amino acids 1 to 36 ± 5 2 (SEQ ID NO: 2). A method according to any one of the preceding claims wherein the extracellular domain with the signal peptide is selected from amino acids 1 to 76 ± 5 2 (SEQ ID NO: 2) or the extracellular domain without signal peptide consists of the amino acids 37 ± 5 to 76 ± 5 2 (SEQ ID NO: 2) to amino acids 134 ± 5 to 160 ± 5 2 (SEQ ID NO: 2) to amino acids 249 ± 5 to 254 ± 5 2 (SEQ ID NO: 2) to the amino acids 315 ± 5 to 347 ± 5 2 (SEQ ID NO: 2) to amino acids 422 ± 5 to 434 ± 5 2 (SEQ ID NO: 2) or amino acids 498 ± 5 to 538 ± 5 2 (SEQ ID NO: 2). A method of diagnosing a tumor in a mammal, the method comprising (a) contacting an antibody with (i) a sample of tissue cells from the mammal, and (ii) a control sample of known normal tissue cells of the same cell type and (b) detecting the formation of a A complex of the antibody and a PRO4980 polypeptide according to any one of claims 1 to 6 in the samples; wherein the formation of a greater amount of complexes in the sample indicates the presence of a tumor in the mammal, and wherein the antibody is for specific binding to a polypeptide having: (i) the in 2 represented amino acid sequence (SEQ ID NO: 2), (ii) the in 2 represented amino acid sequence (Seq.-ID No. 2) without signal peptide, (iii) an extracellular domain of in 2 represented amino acid sequence (SEQ ID NO: 2) with the signal peptide, (iv) an extracellular domain of in 2 and (v) the amino acid sequence encoded by the full length PRO4980 coding sequence of DNA97003-2649 deposited under ATCC accession number PTA-43. The method of claim 7 wherein the signal peptide is selected from amino acids 1 to 36 ± 5 2 (SEQ ID NO: 2). The method of claim 7 or claim 8, wherein the extracellular domain with the signal peptide is selected from amino acids 1 to 76 ± 5 2 (SEQ ID NO: 2) or the extracellular domain without signal peptide consists of the amino acids 37 ± 5 to 76 ± 5 2 (SEQ ID NO: 2) to amino acids 134 ± 5 to 160 ± 5 2 (SEQ ID NO: 2) to amino acids 249 ± 5 to 254 ± 5 2 (SEQ ID NO: 2) to the amino acids 315 ± 5 to 347 ± 5 2 (SEQ ID NO: 2) amino acids 422 ± 5 to 434 ± 5 2 (SEQ ID NO: 2) or amino acids 498 ± 5 to 538 ± 5 2 (SEQ ID NO: 2). The method of claim 7, wherein the antibody is detectable is marked. The method of claim 7, wherein the sample is from tissue cells comes from an individual who is believed to have neoplastic (s) cell growth or proliferation. Cancer Diagnostic Kit Containing an Antibody Claim 7 and a carrier in a suitable packaging. Set according to claim 12, further instructions for Use of the antibody for detecting the presence of a polypeptide according to any one of claims 1 to 6 in a sample suspected of containing it. antibody according to one of the claims 7 to 9 for use in an in vivo diagnosis. antibody according to claim 14, wherein the diagnosis is that of a tumor. antibody according to claim 15, wherein the diagnosis is that of a lung tumor. antibody according to claim 14, which is a monoclonal antibody. antibody Claim 17, which is a nonhuman complementarity determining Region (CDR) or a human framework region (FR). antibody The claim 17, wherein the antibody is a humanized antibody. antibody according to claim 14, which is an antibody fragment or a single chain one antibody is.