ES2341257T3 - SECRETED AND TRANSMEMBRANE POLIPEPTIDES AND NUCLEIC ACIDS THAT CODE THEM. - Google Patents

Abstract

Method for detecting the presence of a tumor in a mammal, said method comprising comparing the level of expression of a gene encoding a PRO1865 polypeptide in (a) an analysis sample of esophageal or skin tissue cells obtained from said mammal and (b) in a control sample of known normal tissue cells of the same type of cells, in which a higher or lower level of the expression of said gene encoding a PRO1865 polypeptide in said esophageal or skin tissue analysis sample in comparison with the control sample it is indicative of the presence of esophageal or melanoma tumor, respectively, in said mammal, in which the PRO1865 polypeptide is a polypeptide that has at least 80% identity in the amino acid sequence with : (a) the amino acid sequence shown in SEQ ID No. 132; (b) the amino acid sequence encoded by the full length coding sequence of the DNA deposited under accession number ATCC 203543; (c) the amino acid sequence of the polypeptide shown in SEQ ID No. 132, which lacks its associated signal peptide; (d) the amino acid sequence of an extracellular domain of the polypeptide shown in SEQ ID No. 132, with its associated signal peptide; or (e) the amino acid sequence of an extracellular domain of the polypeptide shown in SEQ ID No. 132, which lacks its signal peptide, and in which a higher expression is indicative of the presence of an esophageal or melanoma tumor.

Description

Secreted and transmembrane polypeptides and acids Nuclei that encode them.

Field of the Invention

The present invention relates generally to the identification and isolation of new and production DNA recombinant of new polypeptides.

Background of the invention

Extracellular proteins have functions important in, among other things, training, differentiation and maintenance of multicellular organisms. The fate of many individual cells, for example, proliferation, migration, differentiation, or interaction with other cells, is governed usually by the information received from other cells and / or the immediate medium. This information is frequently transmitted by secreted polypeptides (e.g., mitogenic factors, survival factors, cytotoxic factors, differentiation, neuropeptides, and hormones) which, in turn, is received and interpreted by various cell receptors or membrane bound proteins. These secreted polypeptides or signaling molecules normally pass through the mechanism cell secretor to reach its site of action in the middle extracellular

Secreted proteins have several industrial applications, including pharmaceutical, diagnostic, biosensors and bioreactors. Most of the currently available protein drugs, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and several other cytokines, are secretory proteins. Its receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts are being made both at the industry and academic levels to identify new native secreted proteins. Many efforts focus on the screening of recombinant mammalian DNA libraries to identify the coding sequences of new secreted proteins. Examples of screening procedures and techniques are described in the literature [see, for example, Klein et al., Proc. Natl Acad. Sci . 93: 7108-7113 (1996); U.S. Patent No. 5,536,637)]

Membrane-bound proteins and receptors they can have important functions in, among other things, the training, differentiation and maintenance of organisms multicellular The fate of many individual cells, for example, proliferation, migration, differentiation, or interaction with other cells, is usually governed by information received from other cells and / or the immediate medium. This information is frequently transmitted by secreted polypeptides (by example, mitogenic factors, survival factors, factors cytotoxic, differentiation factors, neuropeptides, and hormones) which, in turn, is received and interpreted by various receivers of membrane-bound cells or proteins. Among those proteins Membrane-bound and cell receptors are included, but not limited to, cytokine receptors, receptor kinases, phosphatases receptors, receptors involved in interactions cell-cell, and cell adhesin molecules, such as selectins and integrins. For example, signal transduction that regulate cell growth and differentiation is regulated in part by the phosphorylation of several proteins cell phones. Tyrosine protein kinases, enzymes that catalyze that process can also act as receptors for the factors of growth Examples include the receiver of the fibroblast growth factor and receptor factor nerve growth

Membrane bound proteins and molecules receivers have several industrial applications, including as Pharmaceutical and diagnostic agents. Immunoadhesins receptors, for example, can be used as agents therapeutic to block interactions receptor-ligand. Membrane bound proteins they can also be used to screen the potential peptide or small molecules that inhibit receptor / ligand interaction relevant.

Efforts are being made both at the level of industry as an academic to identify new proteins linked to native membrane or receptors. Many efforts focus on the screening of mammalian recombinant DNA libraries for identify the coding sequences of new proteins bound to membrane or receptors

Summary Description of the Invention

The present invention relates to procedures for using PRO polypeptides as described in the present invention for a set of uses Diagnostics based on data from functional biological tests presented in the following examples, as defined in the claims.

In one embodiment, the present invention uses an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide.

In one aspect, the nucleic acid molecule isolated comprises a nucleotide sequence that has so less about 80% identity in the acid sequence nucleic, alternatively at least about 81% of identity in the nucleic acid sequence, alternatively by at least about 82% identity in the sequence of nucleic acids, alternatively at least about a 83% identity in the nucleic acid sequence, alternatively at least about 84% identity in the nucleic acid sequence, alternatively at least approximately 85% identity in the acid sequence nucleic, alternatively at least about 86% of identity in the nucleic acid sequence, alternatively by at least about 87% identity in the sequence of nucleic acids, alternatively at least about a 88% identity in the nucleic acid sequence, alternatively at least about 89% identity in  the nucleic acid sequence, alternatively at least approximately 90% identity in the acid sequence nucleic, alternatively at least about 91% of identity in the nucleic acid sequence, alternatively by at least about 92% identity in the sequence of nucleic acids, alternatively at least about a 93% identity in the nucleic acid sequence, alternatively at least about 94% identity in the nucleic acid sequence, alternatively at least approximately 95% identity in the acid sequence nucleic, alternatively at least about 96% of identity in the nucleic acid sequence, alternatively by at least about 97% identity in the sequence of nucleic acids, alternatively at least about a 98% identity in the nucleic acid sequence, and alternatively at least about 99% identity in the nucleic acid sequence with (a) a DNA molecule that encodes a PRO polypeptide that has an amino acid sequence full length as described here, a sequence of amino acids lacking the signal peptide as described herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as described herein or any other fragment specifically defined amino acid sequence length complete as described here, or (b) the complement of the DNA molecule of (a).

In other aspects, the nucleic acid molecule comprises a nucleotide sequence that has at least approximately 80% identity in the acid sequence nucleic, alternatively at least about 81% of identity in the nucleic acid sequence, alternatively by at least about 82% identity in the sequence of nucleic acids, alternatively at least about a 83% identity in the nucleic acid sequence, alternatively at least about 84% identity in the nucleic acid sequence, alternatively at least approximately 85% identity in the acid sequence nucleic, alternatively at least about 86% of identity in the nucleic acid sequence, alternatively by at least about 87% identity in the sequence of nucleic acids, alternatively at least about a 88% identity in the nucleic acid sequence, alternatively at least about 89% identity in the nucleic acid sequence, alternatively at least approximately 90% identity in the acid sequence nucleic, alternatively at least about 91% of identity in the nucleic acid sequence, alternatively by at least about 92% identity in the sequence of nucleic acids, alternatively at least about a 93% identity in the nucleic acid sequence, alternatively at least about 94% identity in the nucleic acid sequence, alternatively at least approximately 95% identity in the acid sequence nucleic, alternatively at least about 96% of identity in the nucleic acid sequence, alternatively by at least about 97% identity in the sequence of nucleic acids, alternatively at least about a 98% identity in the nucleic acid sequence, and alternatively at least about 99% identity in the nucleic acid sequence with (a) a DNA molecule that comprises the coding sequence of a PRO polypeptide cDNA of full length as described here, the sequence coding for a PRO polypeptide that lacks the signal peptide such as described here, the coding sequence of a domain extracellular of a transmembrane PRO polypeptide, with or without the signal peptide, as described herein or the coding sequence of any other specifically defined fragment of the sequence of full length amino acids as described herein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the present invention uses an isolated nucleic acid molecule comprising a nucleotide sequence that has at least about one 80% identity in the nucleic acid sequence, alternatively at least about 81% identity in the nucleic acid sequence, alternatively at least approximately 82% identity in the acid sequence nucleic, alternatively at least about 83% of identity in the nucleic acid sequence, alternatively by at least about 84% identity in the sequence of nucleic acids, alternatively at least about a 85% identity in the nucleic acid sequence, alternatively at least about 86% identity in the nucleic acid sequence, alternatively at least approximately 87% identity in the acid sequence nucleic, alternatively at least about 88% of identity in the nucleic acid sequence, alternatively by at least about 89% identity in the sequence of nucleic acids, alternatively at least about a 90% identity in the nucleic acid sequence, alternatively at least about 91% identity in the nucleic acid sequence, alternatively at least approximately 92% identity in the acid sequence nucleic, alternatively at least about 93% of identity in the nucleic acid sequence, alternatively by at least approximately 94% identity in the sequence of nucleic acids, alternatively at least about a 95% identity in the nucleic acid sequence, alternatively at least about 96% identity in the nucleic acid sequence, alternatively at least approximately 97% identity in the acid sequence nucleic, alternatively at least about 98% of identity in the nucleic acid sequence, and alternatively at least about 99% identity in the sequence of nucleic acids with (a) a DNA molecule that encodes the same mature polypeptide encoded by any human protein cDNA deposited with the ATCC as described here, or (b) the complement of the DNA molecule of (a).

Another aspect of the present invention uses an isolated nucleic acid molecule comprising a sequence nucleotide encoding a PRO polypeptide that has been deleted the transmembrane domain or the inactivated transmembrane domain, or is complementary to said sequence of coding nucleotides, in which the domain or domains Transmembrane of said polypeptide are described herein. Thus, soluble extracellular domains of the PRO polypeptides described here.

Another embodiment relates to fragments of a PRO polypeptide coding sequence, or complement of the same, which can be useful for example, as hybridization probes, to encode fragments of a PRO polypeptide that can encode optionally a polypeptide comprising a binding site for a anti-PRO antibody or as probes of antisense oligonucleotides. Such nucleic acid fragments they are usually at least about 20 nucleotides of length, alternately of at least about 30 nucleotides in length, alternately at least approximately 40 nucleotides in length, alternately by at least about 50 nucleotides in length, alternatively of at least about 60 nucleotides of length, alternately of at least about 70 nucleotides in length, alternately at least approximately 80 nucleotides in length, alternately by at least about 90 nucleotides in length, alternatively of at least about 100 nucleotides of length, alternately at least about 110 nucleotides in length, alternately at least approximately 120 nucleotides in length, alternately of by at least about 130 nucleotides in length, alternatively of at least about 140 nucleotides of length, alternately at least about 150 nucleotides in length, alternately at least approximately 160 nucleotides in length, alternately of at least about 170 nucleotides in length, alternatively of at least about 180 nucleotides of length, alternately at least about 190 nucleotides in length, alternately at least approximately 200 nucleotides in length, alternately of at least about 250 nucleotides in length, alternatively of at least about 300 nucleotides of length, alternately at least about 350 nucleotides in length, alternately at least approximately 400 nucleotides in length, alternately of at least about 450 nucleotides in length, alternatively of at least about 500 nucleotides in length, alternately at least about approximately 600 nucleotides in length, alternately of at least about 700 nucleotides in length, alternatively of at least about 800 nucleotides of length, alternately of at least about 900 nucleotides in length, alternately at least approximately 1000 nucleotides in length where in this context the term "approximately" means the length of the nucleotide sequence referred to more or less 10% of the reference length. It should be noted that new fragments of a nucleotide sequence encoding the PRO polypeptide can be determined routinely by the alignment of the nucleotide sequence encoding the PRO polypeptide with other known nucleotide sequences by using any of the programs of known sequence alignment and determining what fragment or fragments of nucleotide sequence encoding the PRO polypeptide are new. All such nucleotide sequences encoding the PRO polypeptide are contemplated herein. invention. PRO polypeptide fragments are also contemplated encoded by these fragments of nucleotide molecules, preferably those PRO polypeptide fragments that comprise a binding site for an antibody anti-PRO.

In another embodiment, the present invention uses an isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences identified above here.

In a certain aspect, the invention is refers to an isolated PRO polypeptide, which comprises a sequence of amino acids that have at least 80% identity in the amino acid sequence, alternatively at least approximately 81% identity in the amino acid sequence, alternatively at least about 82% identity in the amino acid sequence, alternatively at least approximately 83% identity in the amino acid sequence, alternatively at least about 84% identity in the amino acid sequence, alternatively at least approximately 85% identity in the amino acid sequence, alternatively at least about 86% identity in the amino acid sequence, alternatively at least approximately 87% identity in the amino acid sequence, alternatively at least about 88% identity in the amino acid sequence, alternatively at least approximately 89% identity in the amino acid sequence, alternatively at least about 90% identity in the amino acid sequence, alternatively at least approximately 91% identity in the amino acid sequence, alternatively at least about 92% identity in the amino acid sequence, alternatively at least approximately 93% identity in the amino acid sequence, alternatively at least about 94% identity in the amino acid sequence, alternatively at least approximately 95% identity in the amino acid sequence, alternatively at least about 96% identity in the amino acid sequence, alternatively at least approximately 97% identity in the amino acid sequence, alternatively at least about 98% identity in the amino acid sequence and alternatively at least approximately 99% identity in the amino acid sequence with a PRO polypeptide that has an amino acid sequence of full length as described here, a sequence of amino acids lacking the signal peptide as described herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as described herein or any other fragment specifically defined amino acid sequence length Complete as described here.

In a further aspect, the present invention even isolated PRO polypeptide comprising a sequence is referred to of amino acids that have at least 80% identity in the amino acid sequence, alternatively at least approximately 81% identity in the amino acid sequence, alternatively at least about 82% identity in the amino acid sequence, alternatively at least approximately 83% identity in the amino acid sequence, alternatively at least about 84% identity in the amino acid sequence, alternatively at least approximately 85% identity in the amino acid sequence, alternatively at least about 86% identity in the amino acid sequence, alternatively at least approximately 87% identity in the amino acid sequence, alternatively at least about 88% identity in the amino acid sequence, alternatively at least approximately 89% identity in the amino acid sequence, alternatively at least about 90% identity in the amino acid sequence, alternatively at least approximately 91% identity in the amino acid sequence, alternatively at least about 92% identity in the amino acid sequence, alternatively at least approximately 93% identity in the amino acid sequence, alternatively at least about 94% identity in the amino acid sequence, alternatively at least approximately 95% identity in the amino acid sequence, alternatively at least about 96% identity in the amino acid sequence, alternatively at least approximately 97% identity in the amino acid sequence, alternatively at least about 98% identity in the amino acid sequence and alternatively at least approximately 99% identity in the amino acid sequence with an amino acid sequence encoded by any of the Human protein cDNA encoded with the ATCC as described here.

In a specific aspect, the present invention uses an isolated PRO polypeptide without the signal sequence N-terminal and / or initiation methionine and is encoded by a nucleotide sequence encoding said amino acid sequence as described above in the present invention The processes for its production They are also described in the present invention, where these processes comprise the culture of a host cell comprising a vector which comprises the nucleic acid molecule encoding suitable under appropriate conditions for polypeptide expression PRO and recovery of the PRO polypeptide from cell culture.

Another aspect of the present invention uses a isolated PRO polypeptide to which the domain has been removed transmembrane or, the transmembrane domain has been inactivated. The processes for its production are also described in the present invention, where these processes comprise the cultivation of a host cell comprising a vector which comprises the suitable coding nucleic acid molecule under conditions appropriate for PRO polypeptide expression and recovery of the PRO polypeptide of the cell culture.

Agonists are also described here and antagonists of a native PRO polypeptide as defined herein. The agonist or antagonist can be an antibody anti-PRO or a small molecule.

A procedure of identification of agonists or antagonists to a PRO polypeptide that comprises contacting the PRO polypeptide with a molecule candidate and monitor a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.

A subject composition is also described comprising a PRO polypeptide or an agonist or antagonist of a PRO polypeptide as described herein, or an antibody anti-PRO, in combination with a carrier. Optionally, the carrier is a pharmaceutically carrier acceptable.

The use of a polypeptide is also described. PRO, or an agonist or antagonist thereof as described. earlier in the present invention, or an antibody anti-PRO, for the preparation of a medicine useful in treating a condition that is sensitive to PRO polypeptide, an agonist or antagonist thereof or a anti-PRO antibody.

Vectors comprising DNA encoding any of the polypeptides described herein are described. Host cells comprising any of said vectors are also described. By way of example, the host cells can be CHO cells, E. coli cells or yeasts. A process for the production of any of the polypeptides described herein is further described and comprises the cultivation of host cells under conditions suitable for the expression of the desired polypeptide and the recovery of the desired polypeptide from cell culture.

Chimeric molecules are also described here. comprising any of the polypeptides described in the present invention fused to a polypeptide or sequence of heterologous amino acids Examples of such molecules chimeric comprise any of the polypeptides described herein fused with a sequence of a tag epitope or an Fc region of an immunoglobulin.

In another embodiment, the present invention is refers to an antibody that binds, preferably specifically,  to any of the polypeptides described above or later. Optionally, the antibody is an antibody monoclonal, a humanized antibody, an antibody fragment or a single chain antibody.

Oligonucleotide probes are also described useful for the isolation of genomic nucleotide sequences and cDNA or as antisense probes, where these probes can derive of any of the nucleotide sequences described above or later.

Brief description of the drawings

Figure 1 shows a nucleotide sequence (SEQ Id No. 131) of a native sequence PRO1865 cDNA, where SEQ ID No. 131 is a clone designated here as "DNA81757-2512".

Figure 2 shows the amino acid sequence (Sec Id No. 132) derived from the coding sequence of Sec Id No. 131 shown in Figure 131.

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Detailed description of the preferred embodiments 1. Definitions

The terms "PRO polypeptide" and "PRO", as used here, and when they are followed immediately by a numerical denomination, they refer to various polypeptides, where the full name (ie, PRO / number) refers to specific polypeptide sequences as described here. The terms "polypeptide PRO / number "and" PRO / number "where the term" number " which is provided as a real numerical denomination as used here, encompasses native sequence polypeptides and polypeptide variants (described in detail in the present invention). PRO polypeptides described herein may isolate from various sources, such as human tissue types or from other sources, or they can be prepared by procedures recombinant or synthetic. The term "PRO polypeptide" is refers to each PRO polypeptide / individual number described herein. All the mentions in this report that refer to "PRO polypeptide" refers to each of the polypeptides individually or together. For example, the descriptions of the preparation, purification, derivation, antibody formation for or against, the administration, compositions containing, the treatment of the disease with, etc ,. refer to each polypeptide of the present invention individually. The term "PRO polypeptide" also includes variants of the polypeptides PRO / number described here.

A "native sequence PRO polypeptide" comprises a polypeptide that has the same amino acid sequence  than the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from the nature or can be produced by recombinant means or synthetic The term "native sequence PRO polypeptide" specifically covers truncated or natural secreted forms of the specific PRO polypeptide (for example, a sequence of extracellular domain), natural variant forms (for example, cut and spliced forms ("spliced" alternately) and natural allelic variants of the polypeptide. In several embodiments of the invention, sequence PRO polypeptides native described here are mature native sequence polypeptides or full length comprising amino acid sequences full length shown in the accompanying figures. Start and stop codons are shown in bold and underlined In the figures. However, while the PRO polypeptide described in the accompanying figures starts with the residues of methionine referred to herein as amino acid at position 1 in the figures, it is conceivable and possible that other methionine residues located upstream or downstream of position 1 of amino acids in the figures can be used as the residue of Starting amino acid of PRO polypeptides.

The "extracellular domain" or "ECD" of PRO polypeptide refers to a form of the PRO polypeptide that is essentially free of the transmembrane and cytoplasmic domains. Generally, an ECD of the PRO polypeptide will have less than 1% of said transmembrane and / or cytoplasmic domains and preferably, It will have less than 0.5% of those domains. It will be understood that any transmembrane domain identified for polypeptides PRO of the present invention are identified following the criteria commonly used in the art for the identification of that kind of hydrophobic domain. The exact limits of a domain transmembrane may vary, but more likely, in no more than approximately 5 amino acids at any end of the domain such as initially identified here. Optionally, therefore, a Extracellular domain of a PRO polypeptide may contain from approximately 5 or less amino acids at any end of the transmembrane domain / extracellular domain boundaries as identifies in the Examples or the memory and said polypeptides, with or without the associated signal peptide, and the nucleic acid that encodes, are contemplated by the present invention.

The approximate location of the "signal peptides" of various PRO polypeptides described herein are shown herein and / or the accompanying figures. It should be noted, however, that the C-terminal limit of a signal peptide may vary, but most likely by no more than about 5 amino acids at any end of the C-terminal limit of the signal peptide as initially identified herein. , where the C-terminal limit of the signal peptide can be identified according to the criteria routinely used in the art to identify the type of amino acid sequence element (eg, Nielsen et al ., Prot Eng, 10: 1-6 (1997) and von Heinje et al ., Nucl Acids Res, 14: 4683-4690 (1986)). In addition, it has also been recognized that, in some cases, the division of a signal sequence of a secreted polypeptide is not completely uniform, resulting in more than one secreted species. These mature polypeptides, when their signal peptide is cut in no more than about 5 amino acids by any end of the C-terminal limit of the signal peptide identified herein, and the polynucleotides encoding them, are contemplated by the present invention.

"PRO polypeptide variant" means a active PRO polypeptide as defined above or subsequently which has at least about 80% identity in the sequence with a native sequence PRO polypeptide sequence full length as described here, a sequence of PRO polypeptide lacking the signal peptide as described here, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as described herein, or any other fragment of a full length PRO polypeptide sequence such as It is described here. Such PRO polypeptide variants include, by example, PRO polypeptides wherein one or more residues of amino acid are added, or removed, at the N- or end C-terminal of the native amino acid sequence of full length Generally, a PRO polypeptide variant will have at least approximately 80% identity in the amino acid sequence, alternatively at least approximately 81% identity in the amino acid sequence, alternatively at least about 82% identity in the amino acid sequence, alternatively at least approximately 83% identity in the amino acid sequence, alternatively at least about 84% identity in the amino acid sequence, alternatively at least approximately 85% identity in the amino acid sequence, alternatively at least about 86% identity in the amino acid sequence, alternatively at least approximately 87% identity in the amino acid sequence, alternatively at least about 88% identity in the amino acid sequence, alternatively at least approximately 89% identity in the amino acid sequence, alternatively at least about 90% identity in the amino acid sequence, alternatively at least approximately 91% identity in the amino acid sequence, alternatively at least about 92% identity in the amino acid sequence, alternatively at least approximately 93% identity in the amino acid sequence, alternatively at least about 94% identity in the amino acid sequence, alternatively at least approximately 95% identity in the amino acid sequence, alternatively at least about 96% identity in the amino acid sequence, alternatively at least approximately 97% identity in the amino acid sequence, alternatively at least about 98% identity in the amino acid sequence and alternatively at least approximately 99% identity in the amino acid sequence with a sequence of a native sequence PRO polypeptide of full length as described here, a sequence of PRO polypeptide lacking the signal peptide as described here, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as described herein or any other fragment specifically defined from a PRO polypeptide sequence of full length as described here. Generally, the PRO polypeptide variants have a length of at least approximately 10 amino acids, alternatively of at least approximately 20 amino acids in length, alternately from per at least about 30 amino acids in length, alternatively of at least about 40 amino acids of length, alternately at least about 50 amino acids in length, alternately at least approximately 60 amino acids in length, alternately from per at least about 70 amino acids in length, alternatively of at least about 80 amino acids of length, alternately at least about 90 amino acids in length, alternately at least approximately 100 amino acids in length, alternately of by at least about 150 amino acids in length, alternatively of at least about 200 amino acids of length, alternately at least about 300 amino acids in length, or more.

The "percentage (%) of identity in the amino acid sequence "with respect to the sequences of the PRO polypeptide identified in the present invention is defined as the percentage of amino acid residues in a sequence candidate that are identical with amino acid residues in the PRO polypeptide sequence, after alignment of the sequences and the introduction of spaces, if necessary, to get the maximum percentage of identity in the sequence, and without consider any conservative substitution as part of identity of the sequence. The alignment with the objective of determining the percent identity in the amino acid sequence can be get in several ways that are within the technique, by example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign software (DNASTAR). Those skilled in the art can determine parameters appropriate for measuring alignment, including any algorithm necessary to achieve maximum alignment over length full of the sequences that are compared. For the purposes of the present invention, however, values in% identity  in the amino acid sequence they are generated using the program ALIGN-2 sequence comparison software, in which the complete source code for the program ALIGN-2 is provided in Table 1 below. He sequence comparison software ALIGN-2 was owned by Genentech, Inc. and the code  Source shown in Table 1 has been submitted with documentation of the user in the U.S. Copyright Office, Washington D.C. 20559, where it is registered under the U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is available publicly through Genentech Inc., South San Francisco, California or can be compiled from source code provided in Table 1 below. The program ALIGN-2 should be compiled for use in a UNIX operating system, preferably digital UNIX V4.0D. Everybody the sequence comparison parameters are set in the ALIGN-2 program and do not vary.

In the situations in which it is used ALIGN-2 for sequence comparisons of amino acids, the% identity in the amino acid sequence of a given amino acid sequence A to, with, or against a determined amino acid sequence B (which can be written alternatively as a given amino acid sequence A that has or comprises a certain% identity in the sequence of amino acids to, with, or against an amino acid sequence determined B) is calculated as indicated below:

100 times the fraction X / Y

where X is the number of residues of amino acids identified as identical pairings by the ALIGN-2 sequence alignment program in said alignment of the program of A and B, and where Y is the total number  of amino acid residues in B. It will be understood that when the amino acid sequence length A is not equal to the length of amino acid sequence B, the% identity in the sequence of amino acids from A to B will not be equal to% identity in the amino acid sequence from B to A. As examples of the calculations % identity in the nucleic acid sequence using This procedure, Tables 2 and 3 demonstrate how to calculate% of identity in the amino acid sequence designated "Protein from comparison "with the amino acid sequence called "PRO", where "PRO" represents the amino acid sequence of a hypothetical PRO polypeptide of interest, "Protein from comparison "represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest, and "X", "Y" and "Z" each represent different amino acid residues hypothetical

Unless specifically stated otherwise, all values in% identity in the amino acid sequence used in the present invention are obtained as described in the immediately preceding paragraph using the ALIGN-2 software. However, values in% identity in the amino acid sequence can also be determined using the sequence comparison program WU-BLAST-2 (Altschul et al ., Methods in Enzymology, 266: 460-480 (1996). Most WU-BLAST-2 search parameters are set to default values, values not set to default values, that is, adjustable parameters, are set according to the following values: overlap span ") = 1," overlap fraction "= 0.125, word threshold (" word threshold ") T = 11, and punctuation matrix (" scoring matrix ") = BLOSUM 62. When WU-BLAST is used -2, the% identity value in the amino acid sequence is determined by dividing (a) the number of identical amino acid residues in the match between the amino acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and amino acid sequence of comparison of interest (ie, the sequence against which the PRO polypeptide of interest that can be a PRO variant polypeptide is compared) as determined by WU-BLAST-2 between (b) the total number of amino acid residues of the polypeptide PRO of interest. For example, in the statement "a polypeptide comprising an amino acid sequence A that has at least 80% identity in the amino acid sequence with the amino acid sequence of B", amino acid sequence A is the amino acid sequence Comparison of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.

The percent identity in the amino acid sequence can also be determined using the sequence comparison program NCI-BLAST-2 (Altschul et al ., Nucleic Acids Res., 25: 3389-3402 (1997)). The NCI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov . NCI-BLAST2 or in any case can be obtained from the National Institute of Health, Bethesda, MD. NCI-BLAST2 uses various search parameters, where all these search parameters are set to the default values, including, for example, "unmask (unmasked) = yes (yes)", "strand (thread) = all (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 = BLOSUM 62".

In the situations in which it is used NCI-BLAST2 for sequence comparisons of amino acids, the% identity in the amino acid sequence of a given amino acid sequence A to, with, or against a determined amino acid sequence B (which can be written alternatively as a given amino acid sequence A that has or comprises a certain% identity in the sequence of amino acids to, with, or against an amino acid sequence determined B) is calculated as indicated below:

100 times the fraction X / Y

where X is the number of residues of amino acids identified as identical pairings by the sequence alignment program NCI-BLAST2 in said alignment of the program of A and B, and where Y is the total number  of amino acid residues in B. It will be understood that when the amino acid sequence length A is not equal to the length of amino acid sequence B, the% identity in the sequence of amino acids from A to B will not be equal to% identity in the amino acid sequence from B to TO.

The "PRO polynucleotide variant" or "PRO variant nucleic acid sequence" means a nucleic acid molecule encoding an active PRO polypeptide as defined below and it has at least approximately 80% identity in the acid sequence nucleic acids with a nucleic acid sequence that encodes a PRO polypeptide sequence of native sequence and length complete as described in the present invention, a PRO polypeptide sequence of native sequence and length complete that lacks the signal peptide as described in the present invention, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as described herein invention, or any other fragment of a sequence of full length PRO polypeptide as described in the present invention Usually, a PRO variant polynucleotide will have at least approximately 80% identity in the nucleic acid sequence, alternatively at least approximately 81% identity in the acid sequence nucleic, alternatively at least about 82% of identity in the nucleic acid sequence, alternatively by at least about 83% identity in the sequence of nucleic acids, alternatively at least about a 84% identity in the nucleic acid sequence, alternatively at least about 85% identity in  the nucleic acid sequence, alternatively at least approximately 86% identity in the acid sequence nucleic, alternatively at least about 87% of identity in the nucleic acid sequence, alternatively by at least about 88% identity in the sequence of nucleic acids, alternatively at least about a 89% identity in the nucleic acid sequence, alternatively at least about 90% identity in the nucleic acid sequence, alternatively at least approximately 91% identity in the acid sequence nucleic, alternatively at least about 92% of identity in the nucleic acid sequence, alternatively by at least approximately 93% identity in the sequence of nucleic acids, alternatively at least about a 94% identity in the nucleic acid sequence, alternatively at least about 95% identity in the nucleic acid sequence, alternatively at least approximately 96% identity in the acid sequence nucleic, alternatively at least about 97% of identity in the nucleic acid sequence, alternatively by at least about 98% identity in the sequence of nucleic acids, and alternatively at least about one 99% identity in the nucleic acid sequence with a nucleic acid sequence encoding a sequence of native and full length PRO sequence polypeptide such and as described in the present invention, a sequence of PRO full-length native sequence polypeptide lacking of the signal peptide as described in the present invention, an extracellular domain of a PRO polypeptide, with or without the peptide signal, as described in the present invention, or any another fragment of a PRO polypeptide sequence of length complete as described in the present invention. The variants do not comprise the native nucleotide sequence.

Normally, PRO variant polynucleotides They are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides of length, alternatively at least about 90 nucleotides in length, alternately at least approximately 120 nucleotides in length, alternatively by at least about 150 nucleotides in length, alternatively at least about 180 nucleotides of length, alternatively at least about 210 nucleotides in length, alternately at least approximately 240 nucleotides in length, alternatively by at least about 270 nucleotides in length, alternatively at least about 300 nucleotides of length, alternately at least about 450 nucleotides in length, alternately at least approximately 600 nucleotides in length, alternatively so less about 900 nucleotides in length, or more.

The "percentage (%) of identity in the nucleic acid sequence "with respect to the sequences of nucleic acids encoding the PRO identified herein invention is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the nucleic acid sequence encoding the PRO polypeptide of interest, after the sequence alignment and the introduction of spaces, if necessary, to achieve maximum percent identity in the sequence. The alignment with the objective of determining the percentage of identity in the sequence of nucleic acids can be achieved in several ways that are within the art, for example, using computer software publicly available, such as BLAST software, BLAST-2, ALIGN, or Megalign (DNASTAR). For the objectives of the present invention, however, the values in% of the identity in the nucleic acid sequence are obtained such as described below using the computer program ALIGN-2 sequence comparison, in which the Full source code for the ALIGN-2 program provided in Table 1 below. The computer program of sequence comparison ALIGN-2 was owned by Genentech, Inc. and the source code shown in Table 1 below has been submitted with user documentation in the U.S. Copyright Office, Washington D.C. 20559, where you are registered under the U.S. Copyright Registration No. TXU510087. The program ALIGN-2 is publicly available through Genentech Inc., South San Francisco, California or can be compiled from the source code provided in Table 1. The program ALIGN-2 should be compiled for use in a UNIX operating system, preferably digital UNIX V4.0D. Everybody the sequence comparison parameters are set in the ALIGN-2 program and do not vary.

In the situations in which it is used ALIGN-2 for acid sequence comparisons nucleic, the% identity in the nucleic acid sequence of a particular nucleic acid sequence C a, with, or against a certain nucleic acid sequence D (which can be write alternately as a nucleic acid sequence certain C that has or comprises a certain% identity in the nucleic acid sequence to, with, or against a sequence of determined nucleic acids D) is calculated as indicated by continuation:

100 times the fraction W / z

where W is the number of nucleotides identified as identical pairings by the program of alignment of ALIGN-2 sequences in said alignment of the C and D program, and where Z is the total number of D nucleotides. It will be understood that when the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, the% identity in the sequence of nucleic acids from C to D will not be equal to% identity in the nucleic acid sequence from D to C. As examples of % identity calculations in the nucleic acid sequence, Tables 4 and 5 demonstrate how to calculate% identity in the nucleic acid sequence of the nucleic acid sequence called "comparison DNA" with the acid sequence nuclei called "PRO-DNA", where "PRO-DNA" represents an acid sequence nuclei of interest encoding a hypothetical PRO polypeptide, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the molecule is compared of "PRO-DNA" nucleic acid of interest, and "N", "L" and "V" each represent different nucleotides hypothetical

Unless specifically stated otherwise, all values in% identity in the nucleic acid sequence used in the present invention are obtained as described in the immediately preceding paragraph using the ALIGN-2 software. However, values in% identity in the nucleic acid sequence can also be obtained as described below using the sequence comparison program WU-BLAST-2 (Altschul et al ., Methods in Enzymology, 266: 460-480 (1996) Most WU-BLAST-2 search parameters are set to the default values, the values not set to default values, that is, the adjustable parameters, are set according to the following values : overlap span (= overlap span) = 1, overlap fraction = 0.125, word threshold ("word threshold") T = 11, and scoring matrix ("scoring matrix") = BLOSUM 62. When WU-BLAST-2 is used, the% identity value in the nucleic acid sequence is determined by dividing (a) the number of identical nucleotides in the match between the nucleic acid sequence of the nucleic acid molecule of interest encoding the PRO polypeptide of in teres having a nucleic acid derived sequence encoding the native sequence PRO polypeptide and the comparison nucleic acid molecule of interest (i.e., the sequence against which the nucleic acid molecule encoding the PRO polypeptide of interest is compared which may be a PRO variant polynucleotide) as determined by WU-BLAST-2 between (b) the total number of nucleotides of the nucleic acid molecule encoding the PRO polypeptide of interest. For example, in the statement "an isolated nucleic acid molecule comprising a nucleic acid sequence A having at least 80% identity in the nucleic acid sequence with the nucleic acid sequence of B", the sequence of Nucleic acids A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the nucleic acid molecule of interest encoding the PRO polypeptide.

The percent identity in the nucleic acid sequence can also be determined using the sequence comparison program NCI-BLAST-2 (Altschul et al ., Nucleic Acids Res., 25: 3389-3402 (1997)). The NCI-BLAST2 sequence comparison program can be downloaded from http://www.ncbi.nlm.nih.gov . NCI-BLAST2 or in any case can be obtained from the National Institute of Health, Bethesda, MD. NCI-BLAST2 uses various search parameters, where all these search parameters are set to the default values, including, for example, "unmask (unmasked) = yes (yes)", "strand (thread) = all (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 = BLOSUM 62".

In the situations in which it is used NCBI-BLAST2 for sequence comparisons,% of identity in the nucleic acid sequence of a sequence of certain nucleic acids C to, with, or against a sequence of certain nucleic acids D (which can be written alternatively as a particular nucleic acid sequence C that has or comprises a certain% identity in the sequence of nucleic acids to, with, or against an amino acid sequence determined D) is calculated as follows:

100 times the fraction W / z

where W is the number of nucleotides identified as identical pairings by the program of NCBI-BLAST2 sequence alignment in said alignment of the C and D program, and where Z is the total number of D nucleotides. It will be understood that when the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, the% identity in the sequence of nucleic acids from C to D will not be equal to% identity in the nucleic acid sequence from D to C.

In other embodiments, polynucleotides PRO variants are nucleic acid molecules that encode a PRO active polypeptide and which are capable of hybridizing, preferably under hybridization and washing conditions astringent, to nucleotide sequences encoding a full length PRO polypeptide as described herein. The PRO variant polypeptides can be those encoded by a PRO variant polynucleotide.

"Isolated", when used to describe the various polypeptides described in the present invention, means polypeptides that have been identified and separated and / or recovered from a component of their natural environment. The contaminating components of its natural environment are materials that would normally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide (1) will be purified to a degree sufficient to obtain at least 15 residues of an N-terminal or internal amino acid sequence by using a rotary cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. The isolated polypeptide includes the polypeptide in situ inside recombinant cells, since at least one component of the natural environment of the PRO polypeptide will not be present. Normally, however, the isolated polypeptide will be prepared by at least one purification step.

An "isolated" nucleic acid encoding a PRO polypeptide or an "isolated" nucleic acid encoding another polypeptide is a nucleic acid molecule that is identified and separates from at least one nucleic acid molecule contaminant with which it is normally associated in the medium Natural nucleic acid encoding the polypeptide. A isolated nucleic acid molecule encoding the polypeptide is in a different form or composition from that found in the nature. Therefore, isolated nucleic acid molecules encoding the polypeptide differ from the acid molecule specific nucleic encoding the polypeptide as it exists in natural cells. However, a nucleic acid molecule isolate encoding a polypeptide includes acid molecules nucleic encoding the polypeptide contained in cells that normally express the polypeptide where, for example, the molecule of nucleic acid is in a different chromosomal location than that of natural cells.

The term "control sequences" is refers to DNA sequences necessary for the expression of a coding sequence operably linked in a host organism particular. The control sequences that are suitable for prokaryotes include, for example, a promoter, optionally a operating sequence, and a ribosome binding site. It's known that eukaryotic cells use promoters, signals from polyadenylation and enhancers.

A nucleic acid is "bound Operationally "when located in a functional relationship with Another nucleic acid sequence. For example, the DNA for a Presence or secretory leader sequence is operatively linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operatively linked to a coding sequence if affects sequence transcription; or a binding site to ribosome is operatively linked to a coding sequence if It is located for easy translation. Generally, "attached Operationally "means that the DNA sequences that bind are contiguous and, in the case of a secretory leader sequence, contiguous and in reading phase. However, enhancers do not They have to be contiguous. The union is done through the union at convenient restriction sites. If those sites do not exist, adapters or linkers are used synthetic oligonucleotides according to conventional practice.

The term "antibody" is used in the broader sense and specifically covers, for example, antibodies  individual anti-PRO monoclonal (including agonist, antagonist, and neutralizing antibodies), compositions of anti-PRO antibodies with specificity polyepitopic, single chain anti-PRO antibodies, and anti-PRO antibody fragments (see continuation). The term "monoclonal antibody", as used in the present invention, refers to an antibody obtained from a population of antibodies substantially homogeneous, that is, the individual antibodies comprising the population are identical except for possible mutations natural that may be present in small quantities.

The "astringency" of hybridization reactions can easily be determined by a person skilled in the art, and is generally an empirical calculation dependent on the length of the probe, the washing temperature, and the salt concentration. In general, longer probes require higher temperatures for more correct hybridization, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to be rehybridized when complementary chains are present in a medium below their melting temperature. The higher the degree of homology desired between the probe and the hybridization sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more astringent, while lower temperatures not so much. For additional details and explanations of the astringency of hybridization reactions, see Ausubel et al ., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Astringent conditions" or "conditions of high astringency ", as defined herein invention, can be identified by those who: (1) use a low ionic strength and high temperature for washing, by example, 0.015 M sodium chloride / 0.0015 M sodium citrate / dodecyl 0.1% sodium sulfate at 50 ° C; (2) use during hybridization a denaturing agent, such as formamide, for example, 50% formamide (v / v) with 0.1% bovine serum albumin / Ficol 0.1% / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) use 50% formamide, 5 x SSC (0.75 M NaCl, sodium citrate 0.075 M), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,  5 x Denhardt solution, sonic salmon sperm DNA (50 mug / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, with washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by an astringency wash elevated consisting of 0.1 x SSC containing EDTA at 55 ° C.

The "conditions moderately astringent "can be identified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of a wash solution and hybridization conditions (for example, temperature, ionic strength and% SDS) less astringent than described above. An example of moderately conditions astringent is overnight incubation at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x solution Denhardt, 10% dextran sulfate, and salmon sperm DNA denatured fragmented 20 mg / ml, followed by washing 1 x SSC filters at approximately 35-50 ° C. He subject matter expert will know how to adjust the temperature, strength ionic, etc. necessary to accommodate factors, such as the probe length and the like.

The term "epitope labeled", when used in the present invention, refers to a polypeptide chimeric comprising a PRO polypeptide fused to a "tag polypeptide". The tag polypeptide has residues enough to provide an epitope against which you can make an antibody, although it is short enough, so which does not interfere with the activity of the polypeptide to which it is fused. The tag polypeptide is also preferably quite unique, so that the antibody does not react substantially crossed with other epitopes. Suitable tag polypeptides they generally have at least six amino acid residues and usually between approximately 8 and 50 amino acid residues (preferably, between about 10 and 20 residues of amino acids).

As used in the present invention, the term "immunoadhesin" designates type molecules antibody that combine the binding specificity of a protein heterologous (an "adhesin") with the effector functions of constant domains of immunoglobulin. Structurally, the immunoadhesins comprise a fusion of a sequence of amino acids with the desired binding specificity that is different of recognition and antigen binding site of an antibody (it is say, it is "heterologous"), and a constant domain sequence of immunoglobulin. The adhesin part of a molecule of immunoadhesin is usually an amino acid sequence contiguous that comprises at least one receptor binding site or a ligand. The immunoglobulin constant domain sequence in immunoadhesin can be obtained from any immunoglobulin, such as IgG-1 subtypes, IgG-2, IgG-3 or IgG-4, IgA (including IgA-1 e IgA-2), IgE, IgD or IgM.

"Active" or "activity" for the purposes of the present invention refers to a form or forms of a PRO polypeptide that retains a biological and / or immunological activity / property of a native or natural PRO polypeptide, where the "biological" activity refers to a function (inhibitor or stimulator) caused by a native or natural PRO polypeptide that is different from the ability to induce the production of an antibody against an antigenic epitope found in a native or natural PRO polypeptide and an "immunological" activity "refers to the ability to induce the production of an antibody against an antigenic epitope that is found in a native PRO polypeptide or
natural.

The term "antagonist" is used in the broader sense, and includes any molecule that blocks, partially or completely inhibits or neutralizes a biological activity of a native PRO polypeptide described herein. Similarly, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide described herein. Agonist molecules or suitable antagonists specifically include antibodies or antibody fragments, fragments or sequence variants of amino acids from native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc., as antagonists or antagonists. The procedures for identify agonists or antagonists of a PRO polypeptide can comprising contacting a PRO polypeptide with a molecule agonist or antagonist candidate and measure a detectable change in a or more biological activities normally associated with the PRO polypeptide.

"Treatment" refers to both therapeutic treatment as prophylactic or preventive measures, in which the objective is to prevent or slow down (decrease) the condition or objective pathological disorder. Among those who need the treatment includes those who already suffer from the disorder, as well such as those who are prone to the disorder or those to Those who should prevent the disorder.

The "chronic" administration refers to the administration of the agent or agents in a continuous manner in opposition to an acute mode, to maintain the effect (activity) initial therapeutic for a long period of time. The "intermittent" administration is the treatment that is not performed consecutively without interruption but it is quite Cyclic in nature.

"Mammal" for the purposes of treatment refers to any animal classified as a mammal,  including humans, domestic and farm animals, and animals of zoo, sports or company, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

Administration "combined with" one or more Additional therapeutic agents include administration simultaneous (concurrent) and consecutive in any order.

"Bearers", as used in The present invention includes carriers, excipients or pharmaceutically acceptable stabilizers that are non-toxic to the cell or mammal exposed to them in doses and concentrations used Frequently the carrier physiologically acceptable is an aqueous solution buffered in the pH Examples of physiologically acceptable carriers include include buffers, such as phosphate, citrate and other acids organic; antioxidants, including ascorbic acid; polypeptides low molecular weight (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and others carbohydrates that include glucose, mannose, or dextrins; agents chelators, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN?, polyethylene glycol (PEG) and PLURONICS ™.

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

Papain Digestion of Antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with an antigen binding site unique, and a residual "Fc" fragment, whose name reflects its ability to crystallize easily. Pepsin treatment produces a fragment (Fab ') 2 that has two sites of combination to antigen and is capable of cross-linking with the antigen.

"Fv" is the minimum antibody fragment which contains an antigen recognition and binding site full. This region consists of a dimer of a variable domain heavy chain and light chain in close association not covalent It is in this configuration that the three CDRs of each variable domain interact to define a binding site to dimer surface antigen V_ {H} -V_ {L}. Collectively, the six CDRs confer antigen binding specificity to the antibody. Without However, even a single variable domain (or half of an Fv that comprises only three CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although with a lower affinity than the complete binding site.

The Fab fragment also contains the domain light chain constant and the first constant domain (CH1) of the heavy chain. Fab fragments differ from fragments Fab 'for the addition of a few residues in the carboxy end CH1 heavy chain domain terminal including one or more cysteines of the hinge region of the antibody. Fab'-SH is the denomination here for Fab 'in which the cysteine residue or residues of the constant domains They contain a free thiol group. Antibody fragments F (ab ') 2 originally occurred as pairs of Fab 'fragments with a hinge of cysteines between them. Too other chemical junctions of fragments of antibodies

The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of the two clearly distinct types, called kappa (κ) and lambda (λ), according to the sequences of amino acids from their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins are They can assign to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these they can also divide into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2.

The "single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of the antibody, in which these domains are presented in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a linker polypeptide between the VH and VL domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pages 269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described in more detail in, for example, 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 has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of its natural environment are materials that would interfere with the diagnostic and therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous and non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to more than 95% by weight of antibody as determined by Lowry's method, and more preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by using a rotating cup sequencer, or (3) until homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, staining with silver The isolated antibody includes the antibody in situ within the recombinant cells since at least one component of the natural environment of the antibody will not be present. Normally, however, the isolated antibody will be prepared by at least one purification step.

An antibody that "specifically binds" or "is specific for" a particular polypeptide or an epitope in a particular polypeptide is one that binds to that polypeptide particular or epitope on a particular polypeptide without binding substantially to any other polypeptide or epitope of polypeptide.

The word "bookmark" when used in The present invention relates to a compound or composition detectable that conjugates directly or indirectly to the antibody to generate a "labeled" antibody. The "bookmark" can be detectable by itself (for example, radioisotope markers or fluorescent markers) or, in the case of an enzymatic marker, can catalyze the chemical alteration of a compound or composition substrate that is detectable.

"Solid phase" means a non-matrix aqueous to which the antibody of the present can adhere invention. Among the examples of solid phases that are included in the present invention includes those formed partially or totally of glass (for example, controlled pore glass), polysaccharides (for example, agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test plate; in others is a purification column (for example, a column of affinity chromatography). This term also includes a phase Discontinuous solid of discrete particles, such as described in U.S. Patent No. 4,275,149.

A "liposome" is a small vesicle composed of several types of lipids, phospholipids and / or surfactants which is useful for the release of a drug (such as a PRO polypeptide or an antibody thereto) to a mammal. The Liposome components are usually arranged in a formation bilayer, similar to the arrangement of the membranes Biological

A "small molecule" is defined in the present invention having a molecular weight below approximately 500 Daltons.

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TABLE 1

one

2

3

4

5

6

7

8

9

10

eleven

12

13

14

fifteen

16

17

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TABLE 2

18

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TABLE 3

19

TABLE 4

twenty

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TABLE 5

22

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II Compositions and methods of the invention A. Full length PRO polypeptides

The present invention provides sequences of newly identified and isolated nucleotides encoding polypeptides referred to in the present invention as PRO polypeptides. In particular, they have been identified and isolated cDNAs encoding various PRO polypeptides, as describe in more detail in subsequent examples. Fits indicate that proteins manufactured in expression cycles different can be determined different PRO numbers, but the UNQ number is unique to any given DNA and protein encoded, and will not change. However, for simplicity, in the present document the protein encoded by the molecules of full length native nucleic acid described herein  invention, as well as all additional native homologs and variants included in the previous definition of PRO, will refer as "PRO / number", regardless of its origin or mode of preparation.

As described in the Examples Subsequently, several classes of cDNA clones have been deposited with the ATCC. The actual nucleotide sequences of said clones are can easily be determined by a person skilled in the art by sequencing of the clone deposited using procedures Routine in the art. The expected amino acid sequence is can determine from the nucleotide sequence using The usual technique. For PRO polypeptides and acids coding nuclei described herein, applicants have identified what is believed to be the best reading frame identifiable with the sequence information available in the moment.

B. PRO polypeptide variants

In addition to the PRO sequence polypeptides native and full length described in the present invention, It is contemplated that PRO polypeptide variants can be prepared. PRO polypeptide variants can be prepared by introducing appropriate nucleotide changes in PRO DNA, and / or by the synthesis of the desired PRO polypeptide. Subject matter experts understand that amino acid changes can alter post-translational processes of the PRO polypeptide, such as changing the number or position of the sites of glycosylation or alteration of anchoring characteristics to the membrane

Variations in the native and full length PRO sequence polypeptide or in several domains of the PRO polypeptide described in the present invention can be made, for example, using any of the techniques and guidelines for established conservative and non-conservative mutations, for example , in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO polypeptide that results in a change in the amino acid sequence of the PRO polypeptide compared to the native sequence PRO polypeptide. Optionally, the variation is by substitution of at least one amino acid with any other amino acid in one or more of the PRO polypeptide domains. By determining which amino acid residue can be inserted, replaced or removed without adversely affecting the desired activity, a guide can be found by comparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions with high homology. Amino acid substitutions may be the result of the substitution of one amino acid for another amino acid that has a similar chemical structure and / or properties, such as the substitution of a leucine for a serine, that is, conservative amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The permitted variation can be determined by systematically inserting, eliminating or replacing amino acids in the sequence and testing the activity shown by the full-length native sequence in the resulting variants.
mature

In the present invention are provided PRO polypeptide fragments. These fragments can be truncated at the N-terminal end or C-terminal, or they may lack internal waste, for example, when compared with a native protein of length complete. Certain fragments lack amino acid residues that they are not essential for a desired biological activity of PRO polypeptide.

PRO polypeptide fragments can be prepare using any of a set of techniques conventional. The desired peptide fragments can be chemically synthesize An alternative approach involves PRO polypeptide fragment generation by digestion enzymatic, for example, by treating the protein with an enzyme known to divide proteins at sites defined by residues of specific amino acids or by digestion of DNA with suitable restriction enzymes and fragment isolation wanted. Another suitable technique involves isolation and amplification of a DNA fragment encoding a fragment of desired polypeptide by polymerase chain reaction (PCR) The oligonucleotides that define the desired ends of the DNA fragment are used in the primers of the 5 'ends and 3 'in the PCR. Preferably, PRO polypeptide fragments share at least one biological and / or immunological activity with the native PRO polypeptide described herein.

In particular embodiments, the substitutions Conservatives of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions give rise to a change in biological activity, then it introduce more substantial changes, called substitutions of example in Table 6, or as described later in reference to the amino acid classes, and the products.

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TABLE 6

2. 3

24

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Substantial changes in identity Functional or immunological polypeptide are carried out by the selection of substitutions that differ significantly in its effect of maintaining (a) the structure of the skeleton of the polypeptide in the area of substitution, for example, as conformation of sheet or propeller, (b) the load or hydrophobicity of the molecule at the target site, or (c) the chain volume side. Natural waste is divided into groups based on the Common side chain properties:

(one)

hydrophobic: norleucine, met, wing, val, leu, ile;

(2)

neutral hydrophilic: cys, ser, thr;

(3)

acid: asp, glu;

(4)

Basic: asn, gln, his, lys, arg;

(5)

residues that influence the chain orientation: gly, pro; Y

(6)

aromatic: trp, tyr, phe.

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Non-conservative substitutions will comprise the exchange of a member of one of these classes for another class. Such substituted residues can also be introduced at sites conservative substitution or, more preferably, at sites remaining (not preserved).

Variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine tracing, and PCR mutagenesis. To manufacture the PRO variant DNA, site directed mutagenesis can be carried out on the cloned DNA [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 techniques.

The amino acid analysis by tracing also can be performed to identify one or more amino acids throughout of an adjacent sequence. Among the tracking amino acids Preferred are relatively small and neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine Alanine is usually a tracking amino acid preferred among this group since it eliminates the side chain beyond  of beta carbon and is less likely to alter the conformation of the main chain of the variant [Cunninghan and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also usually preferred since it is the most common amino acid. In addition, it is frequently found both in hidden positions as exposed [Creighton, The Proteins, (W.H. Freeman and Co., N.Y.), Chothia, J. Mol. Biol., 150: 1 (1976)]. If replacing Alanine does not produce adequate amounts of variant, you can Use an isoteric amino acid.

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C. Modifications of PRO polypeptides

Covalent modifications of the polypeptide PRO are included in the scope of the present invention. One type Covalently modified includes the reaction of residues of labeled amino acids of a PRO polypeptide with an agent organic derivatizer that is capable of reacting with chains Selected sides or N- or waste C-terminals of the PRO polypeptide. Derivatization with bifunctional agents it is useful, for example, to cross-link the PRO polypeptide with a matrix or support surface insoluble in water for use in the purification procedure anti-PRO antibodies, and vice versa. Between agents Commonly used crosslinking are included, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, esters of N-hydroxysuccinimide, for example, esters with 4-salicylic acid, homobifunctional imidoesters, including esters disuccinimidilics, such as 3,3'-dithiobis (succinimidylpropionate), bifunctional maleimides, such as bis-N-maleimido-1,8-octane and agents, such as methyl-3 - [(p-azidophenyl) dithio] proprioimidate.

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

Another type of covalent modification of PRO polypeptide included in the scope of the present invention comprises the alteration of the native glycosylation pattern of the polypeptide. By "alteration of the glycosylation pattern native "for the purposes of the present invention is intended indicate the elimination of one or more carbohydrate groups found in native sequence PRO polypeptides (by removing underlying glycosylation site or by eliminating the glycosylation by chemical and / or enzymatic means), and / or the addition of one or more glycosylation sites that are not present in the native sequence PRO polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of native proteins, implying a change in the nature and proportions of various carbohydrate groups present.

The addition of glycosylation sites to PRO polypeptide can be performed by altering the sequence of amino acids. The alteration can be made, for example, by the addition of, or substitution by, one or more serine residues or threonine to the native sequence PRO polypeptide (for sites of glycosylation bound to O). The amino acid sequence of PRO can optionally altered through changes at the DNA level, particularly by mutation of the DNA encoding the PRO polypeptide in preselected bases, so that the codons that are generated will translate into amino acids desired

Other means to increase the number of groups Carbohydrate in the PRO polypeptide is by chemical coupling or enzymatic glycosides to the polypeptide. Such procedures are described in the art, for example, in WO 87/05330 published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., 259-306 (1981).

The elimination of carbohydrate groups present in the PRO polypeptide can be performed chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that act as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et. al . Arch. Biochem. Biophys., 259: 52 (1987) and by Edge, et al . Anal. Biochem., 118: 131 (1981). The enzymatic division of carbohydrate groups in the polypeptides can be achieved by using a set of endo- and exoglycosidases as described in Thotakura et al . Meth. Enzymol., 138: 350 (1987).

Another type of covalent modification of PRO polypeptides comprise the binding of the PRO polypeptide to one of a set of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes, as established in United States Patents Nos: 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

PRO polypeptides of the present invention they can also be modified in a way that forms a molecule chimeric comprising the PRO polypeptide fused to another heterologous polypeptide or amino acid sequence.

In one embodiment, said chimeric molecule comprises a fusion of the PRO polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally located at the amino or carboxyl terminus of the PRO polypeptide. The presence of such epitope tagged forms of the PRO polypeptide can be detected using an antibody against the tag polypeptide. In addition, the provision of the tag epitope allows the PRO polypeptide to be easily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the tag epitope. Several tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the HA flu tag polypeptide and its 12C45 antibody [Field et al ., Mol. Cell Biol., 8: 2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereof [Evan et al ., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and the gliprotein D (gD) tag of the Herpes Simplex virus and its antibody [Paborsky et al ., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include the Flag [Hopp et al . , BioTechnology, 6: 1204-1210 (1988)]; the KT3 epitope peptide [Martín 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 protein peptide tag of gene 10 [Lutz-Freyemuth et al ., Proc. Natl Acad. Sci. USA, 87: 6393-6397 (1990)].

In an alternative embodiment, the molecule chimeric may comprise a fusion of the PRO polypeptide with a immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as "immunoadhesin"), said fusion could be to the Fc region of an IgG molecule. Ig fusions preferably include the substitution of a soluble form (transmembrane domain removed or inactivated) of a PRO polypeptide instead of at least one variable region of an Ig molecule. In one embodiment particularly preferred, the immunoglobulin fusion includes the hinge, CH2 and CH3, or hinge, regions CH1, CH2 and CH3 of a IgG1 molecule. For the production of mergers of immunoglobulins, see also U.S. Patent No. 5,428,130 granted on June 27, 1995.

D. Preparation of PRO polypeptides

The following description mainly refers to the production of PRO polypeptides by culturing cells transformed or transfected with a vector containing PRO nucleic acid. Naturally, it is envisioned that alternative methods, which are well known in the art, can be used to prepare the PRO polypeptides. For example, the sequence of the PRO polypeptide, or portions thereof, can be produced by direct peptide synthesis using solid phase techniques [see, for example, Stewart et al ., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)]. In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Several parts of the PRO polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full length PRO polypeptide.

1. Isolation of DNA encoding a polypeptide PRO

DNA encoding the PRO polypeptide can be obtain from a cDNA library prepared from tissue that is believed to possess the mRNA of PRO and express it at a level detectable Therefore, human PRO DNA can be obtained conveniently from a cDNA library prepared to starting from human tissue, as described in the Examples. He gene encoding PRO can also be obtained from a genomic library or by synthesis procedures (for example, automated synthesis of nucleic acids).

Libraries can be screened with probes (such as antibodies to the PRO polypeptide or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening of the cDNA or genomic library with the selected probe can be performed using standard procedures, as described in Sambrook et al ., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding the PRO polypeptide is to use the PCR methodology [Sambrook et al., Supra ; Dieffenbach et al ., PCR First: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The following Examples describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled so that it can be detected after hybridization to DNA in the library being screened. Labeling procedures are well known in the art, and include the use of radiolabels such as 32 P-labeled ATP, biotinylation or enzymatic labeling. Hybridization conditions, including moderate astringency and elevated astringency, are provided in Sambrook et al., Supra .

The sequences identified in said library screening procedures can be compared and align with other known sequences deposited and available in public databases, such as the Gene Bank or other databases of private sequence data. The sequence identity (at the level amino acid or nucleotide) in the defined regions of the molecule or along the full length sequence you can determine using procedures known in the art and such and as described in the present invention.

The nucleic acid having the protein coding sequence can be obtained by screening the selected cDNA or genomic libraries using the deduced amino acid sequence described in the present invention for the first time, and, if necessary, using conventional methods of extension with primers as described in Sambrook et al., supra , to detect precursors and mRNA processing intermediates that have not been reverse transcribed into cDNA.

2. Selection and transformation of host cells

Host cells are transfected or transformed with expression or cloning vectors described in the present invention for the production of the PRO polypeptide and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the sequences. desired. Cultivation conditions, such as the medium, temperature, pH and the like, can be selected by one skilled in the art without undue experimentation. In general, the principles, protocols and practical techniques to maximize cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra .

The methods of transfection of eukaryotic cells and transformation of prokaryotic cells are known to those skilled in the art, for example, CaCl2, CaPO4, mediated by liposomes and electroporation. Depending on the host cell used, the transformation is performed using standard techniques appropriate to said cells. Calcium treatment using calcium chloride, as described in Sambrook et al., Supra , or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for the transformation of certain plant cells, as described by Shaw et al ., Gene, 23: 315 (1983) and WO 89/05859 published on June 29, 1989. For mammalian cells without said cell walls, the precipitation procedure with calcium phosphate from Graham and van der Eb, Virology, 52: 456-457 (1978) can be used. General aspects of transfections of mammalian host cell systems have been described in US Patent No. 4,399,216. Yeast transformations are usually carried out according to the procedure 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 can also be used to introduce DNA into cells, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, for example, polybrene, polynithin. For various techniques for transforming mammalian cells, see Keown et al ., Methods in enzymology, 185: 527-537 (1990) and Manssur et al ., Nature, 336. 348-352 (1988).

Suitable host cells for cloning or expression of DNA in the vectors of the present invention include prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as E. coli. Several strains of E. coli are publicly available, such as E. coli strain K12 MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27.325) and E. coli strain K5772 (ATCC 53.635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia , for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella , for example, Salmonella typhimurium, Serratia , for example, Serratia marcescans , and Shigella , as well as Bacilli , such as B. subtilis and B. licheniformis (for example, B. licheniformis 41P described in DD 266,710 published on April 12, 1989), Pseudomonas , such as P. aeruginosa , and Streptomyces . These examples are more illustrative than limiting. Strain W3110 is a particularly preferable parental host or host since it is a usual 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 perform a genetic mutation in the genes encoding endogenous proteins to the host, with examples of such hosts including E. coli strain W3110 1A2, which has the complete genotype ton A; E. coli strain W3110 9E4, which has the complete genotype ton A ptr3 ; E. coli strain W3110 27C7 (ATCC 55.244), which has the complete genotype ton A ptr3 phoA E15 (argF-lac) 169 degP ompT kon ' ; E. coli strain W3110 37D6, which has the complete genotype ton A ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan ' ; E. coli strain W3110 40B4, which is a 37D6 strain with a degP deletion mutation not resistant to kanamycin; and a strain of E. coli having mutant periplasmic protease described in US Patent No. 4,946,783 issued August 7, 1990. Alternatively, in vitro cloning procedures are suitable, for example, PCR or other reactions of nucleic acid polymerase.

In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for vectors encoding PRO. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and 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, for example, K. lactis (MW98-8C, CBS683, CBS574; Louvencourt et al . J. Bacteriol., 737 [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 recia (EP 244,234); Neurospora crassa (Case et al ., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 [1979]); Schwanniomyces, such as Schwanniomyces occidentalis (EP 394,538, published October 31, 1990); and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91/00357 published January 10, 1991), and Aspergillus hosts, such as A. 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 and Hynes, EMBO J., 4: 475-479 [1985]). Methylotropic yeasts are suitable in the present invention and include, but are not limited to, yeast capable of growth in methanol selected from the genus consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis , and Rhodotorula . A list of specific species that are examples of this kind of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO polypeptides are derived from multicellular organisms. Examples of invertebrate cells include insect cells, such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. Some more specific examples include the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryo kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al ., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells / -DHFR (CHO), Urlaub and 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). The selection of the appropriate host cell is estimated to be within the art.

3. Selection and use of a replicable vector

The nucleic acid (for example, cDNA or DNA genomic), which encodes the PRO polypeptide can be inserted into a replicable vector for cloning (DNA amplification) or for The expression. There are several publicly available vectors. He vector may be, for example, in the form of plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence It can be inserted into the vector by a series of procedures.  In general, DNA is inserted into an endonuclease site or sites appropriate restriction using techniques known in the sector. The components of the vectors generally include, but they are not limited to one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. The construction of suitable vectors containing one or more of these components uses standard joining techniques that are known for An expert in the field.

The PRO polypeptide can be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide that has a specific cleavage site at the N-terminal end of the protein or polypeptide. mature. In general, the signal sequence may be a component of the vector, or it may be a part of the DNA encoding the PRO that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of thermally stable alkaline phosphatase, penicillinase, Ipp or enterotoxin II sequences. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader sequence, the alpha factor leader sequence (including the Saccharomyces and Kluyveromyces ? Factor leader sequences, the latter described in the Patent of United States No. 5,010,182), or the acid phosphatase leader sequence, the C. albicans glucoamylase leader sequence (EP 362,179 published April 4, 1990) or the signal described in WO 90/13646, published 15 November 1990. In the expression of mammalian cells, mammalian signal sequences can be used to direct the secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leader sequences.

Both expression and cloning vectors they contain a nucleic acid sequence that allows the vector replication in one or more selected host cells. These sequences are known for a set of bacteria, yeast and virus. The origin of replication of plasmid pBR322 It is suitable for most bacteria gram-negative, the origin of plasmid 2 µ is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for vectors of cloning in mammalian cells.

Cloning and expression vectors will usually contain a selection gene, also referred to as a selectable marker. The usual selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from the complex medium , for example, the gene encoding D-alanine racemase for Bacilli .

An example of selectable markers suitable for mammalian cells are those that allow the identification of competent cells to capture the nucleic acid encoding PRO, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is used is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al ., Proc. Natl Acad. Sci. USA, 77: 4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the YRp7 yeast plasmid [Stinchcomb et al ., Nature, 282: 39 (1979); Kingsman et al ., Gene, 7: 141 (1979); Tschemper et al ., Gene, 10: 157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast that lacks the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Cloning and expression vectors usually contain a promoter operably linked to the nucleic acid sequence encoding PRO to direct mRNA synthesis. Promoters recognized by a set of 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 as the tac promoter [deBoer et al., Proc. Natl Acad. Sci. USA , 80: 21-25 (1983)]. Promoters for use in bacterial systems will also contain a Shine-Dalgamo (SD) sequence operatively linked to the DNA encoding the PRO polypeptide.

Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem ., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al. ., J. Adv. Enzyme Reg ., 7: 149 (1968); Holland, Biochemistry , 17: 4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosaphosphate isomerase, phosphate isomerase, phosphate and glucoqui-
pot.

Other yeast promoters, which are inducible promoters that have the additional advantage of a transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with metabolism of nitrogen, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose Vectors and promoters suitable for use in yeast expression are described in detail in EP 73,657.

Transcription of the PRO polypeptide from vectors in mammalian host cells is controlled by for example, by promoters obtained from virus genomes, such as polyoma virus, avian smallpox virus (UK Patent 2,211,504 published July 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, sarcoma virus avian, cytomegalovirus, a retrovirus, virus hepatitis-B and simian virus 40 (SV40), from heterologous mammalian promoters, for example, the promoter of actin or an immunoglobulin promoter, and from promoters of thermal shock, as long as said promoters are compatible with host cell systems.

The transcription of a DNA can be increased which encodes the PRO polypeptide by higher eukaryotes by the insertion of an enhancer sequence in the vector. The enhancers are elements that act in cis of DNA, usually approximately 10 to 300 bp, which act in a promoter for Increase your transcript. Many sequences are currently known of mammalian gene enhancers (globin, elastase, albumin,  α-fetoprotein and insulin). Habitually, however, a cell virus enhancer will be used eukaryotic Examples include the SV40 enhancer in late face of origin of replication (bp 100-270), the early promoter enhancer of cytomegalovirus, the polyoma enhancer on the late face of origin of replication, and adenovirus enhancers. He enhancer can be cut and spliced ("splice") in the vector in a 5 'or 3' position with respect to the coding sequence of PRO, but is preferably located at a 5 'site with respect to the promoter.

Expression vectors used in cells eukaryotic hosts (yeast, fungi, insects, plants, animals, humans, or nucleated cells of other organisms multicellular) will also contain sequences necessary for the termination of transcription and for mRNA stabilization. Such sequences are usually available from the regions. not translated 5 'and, occasionally 3', of eukaryotic DNAs or cDNAs or viral. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated part of the mRNA encoding the PRO polypeptide.

In Gething et al., Nature , 293: 620-625 (1981); Mantei et al., Nature , 281: 40-46 (1979); EP 117.060 and EP 117.058 further describe other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell cultures.

4. Detection of amplification / expression of genes

The amplification and / or expression of the genes can be measured in a sample directly, for example, by conventional Southern blotting, conventional Northern blotting to quantify mRNA transcription [Thomas, Proc. Natl Acad. Sci. USA , 77: 5201-5205 (1980)], point transfer (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided in the present invention. Alternatively, antibodies that can recognize specific double strands can be used, including double strands of DNA, double strands of RNA, double strands of DNA-RNA or double strands of DNA-protein. The antibodies in turn can be labeled and the assay can be carried out when the double chain is attached to a surface, so that after the formation of the double chain on the surface, the presence of antibodies bound to the surface can be detected. double chain

Gene expression, alternatively, can be measure by immunological procedures, such as staining immunohistochemistry of cells or tissue sections and the assay of culture of cells or body fluids, to quantify directly the expression of the gene product. Antibodies useful for immunohistochemical staining and / or fluid testing Sample can be monoclonal or polyclonal, and can be Prepare in any mammal. Conveniently, the antibodies are they can prepare against a native sequence PRO polypeptide or against a synthetic peptide based on DNA sequences provided in the present invention or against a sequence exogenous fused to PRO DNA and encoding an epitope of specific antibody

5. Purification of polypeptide

PRO polypeptide forms can be recover from culture medium or host cell lysates. If it is membrane bound, it can be released from the membrane using a suitable detergent solution (for example, Triton X-100) or by enzymatic division. The cells used in the expression of the PRO polypeptide can be broken by various physical or chemical means, such as cycles of freeze-thaw, sonication, destruction mechanics, or agents to lyse cells.

It may be desired to purify the PRO polypeptide at from recombinant cell proteins or polypeptides. The following procedures are examples of procedures of suitable purification: by fractionation in a column of ion exchange; ethanol precipitation; Reverse phase HPLC; chromatography on silica or a cation exchange resin, such as DEAE; "chromatofocusing"; SDS-PAGE; precipitation with ammonium sulfate; gel filtration using, for example, Sephadex G-75; protein columns A sepharose to remove contaminants, such as IgG, and columns metal chelators to join epitope-tagged forms of the PRO polypeptide. Several purification methods can be used of proteins and such methods are known in the art and are describe, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). Stage o Selected purification steps will depend, for example, on the nature of the production process used and the PRO polypeptide concrete produced.

E. Uses of PRO

Nucleotide sequences (or their complementary) coding PRO has several applications in the discipline of molecular biology, including uses as probes of hybridization, in the mapping of chromosomes and genes and in the generation of RNA and antisense DNA. PRO nucleic acid will also be useful for the preparation of PRO polypeptides by techniques recombinants described in the present invention.

The native sequence and length PRO gene complete, or parts thereof, can be used as probes of hybridization for a cDNA library to isolate the cDNA from PRO full length or to isolate even other cDNAs (for example, those encoding natural variants of PRO or PRO of other species) that have a desired sequence identity with the native PRO sequence described here. Optionally, the probe length will be approximately 20 to approximately 50 bases Hybridization probes can be derived from fewer partially new regions of the nucleotide sequence full length native in which such regions can be determine without excessive experimentation or sequence genomics that include promoters, enhancer elements and introns of native sequence PRO. As an example, a screening procedure will comprise the isolation of the region PRO gene encoder using the known DNA sequence to synthesize a selected probe of approximately 40 bases. Hybridization probes can be marked with a series of markers, including radionuclides, such as 32 P or 35 S, or enzymatic markers, such as alkaline phosphatase coupled to the probe through the coupling systems avidin / biotin. Marked probes that have a sequence complementary to that of the PRO gene of the present invention is can use to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of those libraries are hybridize the probe. Hybridization techniques are described with more detail in the following Examples.

Any EST sequence described herein request can be used similarly as probes, using the procedures described herein invention.

Other useful fragments of PRO nucleic acids include antisense and sense oligonucleotides comprising a single chain nucleic acid sequence (RNA or DNA) capable of binding to target PRO (mRNA) or PRO (antisense) mRNA sequences. ). The antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the DNA coding region of PRO. Said fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or sense oligonucleotide, based on a cDNA sequence encoding a particular protein is described in, for example, Stein and Cohen ( Cancer Res ., 48: 2659, 1988) and van der Krol et al . ( BioTechniques 6: 958, 1988).

The binding of sense and antisense oligonucleotides to target nucleic acid sequences results in the formation of double chains that block transcription or translation of the target sequence by one of the different means, which include increased degradation of the double chains, the premature termination of transcription or translation, or by any other means. Thus, antisense oligonucleotides can be used to block the expression of PRO proteins. The antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester skeletons (or other sugar bonds, such as those described in WO 91/06629) and in which said sugar bonds are resistant to endogenous nucleases. Such oligonucleotides with binding to resistant sugars are stable in vivo (i.e., capable of resisting enzymatic degradation) but maintaining sequence specificity to be able to bind to target nucleotide sequences.

Among other examples of sense oligonucleotides or antisense include those nucleotides that are bound covalently to organic groups, such as those described in WO 90/10048, and other groups that increase the affinity of oligonucleotide by a sequence of target nucleic acids, such as poly- (L-lysine). In addition, they can be joined intercalating agents, such as ellipticin, and alkylating agents or metal complexes to sense or antisense oligonucleotides for modify oligonucleotide binding specificities antisense or felt by the target nucleotide sequence.

Sense or antisense oligonucleotides can be introduced into a cell that contains the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4-mediated DNA transfection, electroporation, or by the use of gene transfer vectors, such as the Epstein-Barr virus. In a preferred method, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, in vivo or ex vivo . Suitable retroviral vectors include, but are not limited to, those derived from murine retroviruses M-MuLV, N2 (a retrovirus derived from M-MuLV) or the double copy vectors designated as DCT5A, DCT5B and DCT5C (see WO 90/13641 ).

The sense or antisense oligonucleotides they can also be introduced into a cell that contains the target nucleotide sequence by forming a conjugate with a ligand binding molecule, as described in WO 04/31/753. Suitable ligand binding molecules include, but without limitation, cell surface receptors, growth factors, other cytokines, or other ligands that are bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not interfere substantially with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, nor block the sense or antisense oligonucleotide input or its version conjugated in the cell.

Alternatively, a sense oligonucleotide or antisense can be introduced into a cell that contains the target nucleic acid sequence by forming a oligonucleotide-lipid complex, as described in WO 90/10448. The sense oligonucleotide complex or antisense-lipid dissociates preferably in the inside the cell using an endogenous lipase.

Antisense or sense RNA or DNA molecules they generally have at least about 5 bases of length, about 10 bases in length, about 15 bases in length, approximately 20 bases in length, approximately 25 bases in length, approximately 30 bases in length, about 35 bases in length, about 40 bases in length, approximately 45 bases in length, approximately 50 bases in length, approximately 55 bases in length, approximately 60 bases in length, approximately 65 bases in length, approximately 70 bases in length, approximately 75 bases in length, approximately 80 bases in length, approximately 85 bases in length, approximately 90 bases of length, approximately 95 bases of length, approximately 100 bases in length, or more.

The probes can also be used in PCR techniques to generate a group of sequences for the PRO coding sequence identification closely related.

Nucleotide sequences encoding a PRO can also be used to construct hybridization probes to map the gene encoding the PRO and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided in the present invention can be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, binding analysis against known chromosomal markers, and hybridization screening with libraries.

When PRO coding sequences encode a protein that binds to another protein (for example, when the PRO is a receiver), the PRO can be used in trials to identify the other proteins or molecules involved in the union interaction. Through such procedures, you can identify inhibitors of binding interaction receptor / ligand. The proteins involved in these interactions Binding can also be used to screen inhibitors or peptide agonists or small molecules of the interaction of Union. In addition, the PRO receiver can be used to isolate the ligand or correlative ligand. Screening assays can be design to find candidate compounds that mimic the biological activity of a native PRO or a receptor for PRO. Sayings screening assays will include trials susceptible to screening with a High performance chemical libraries, making them particularly suitable for identifying small molecules as drugs Candidates Among the small molecules contemplated are included synthetic organic or inorganic compounds. Rehearsals are they can perform in a variety of formats, including trials of protein-protein binding, screening assays biochemical, immunoassays and cell-based assays, which are well characterized in the art.

Nucleic acids encoding PRO or its modified forms can also be used to generate animals GM or knock out animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (for example, a mouse or a rat) is an animal which has cells that contain a transgene, which was introduced in the animal or in an ancestor of the animal in prenatal state, by example, an embryonic stage. A transgene is a DNA that is integrated into the genome of a cell from which a transgenic animal In one embodiment, the cDNA encoding PRO is you can use to clone genomic DNA encoding PRO according to the established techniques and genomic sequences used to generate transgenic animals that contain cells that express DNA which encodes PRO. The procedures for generating animals transgenic, particularly animals such as mice or rats, they have become conventional in the art and are described, for example, in United States Patents Nos. 4,736,866 and 4,870,009. Usually, specific cells would be marked for incorporation of the PRO transgene with specific enhancers of tissue. Transgenic animals that include a copy of a transgene encoding PRO introduced in the line germinal of the animal in an embryonic stage to examine the effect  of the highest expression of DNA encoded by PRO. These animals are can be used as test animals for reagents intended to confer protection from, for example, pathological conditions associated with its overexpression. According to this aspect of the invention, the treatment of an animal with the reagent and the reduction of the incidence of the pathological condition, in comparison with untreated animals that carry the transgene, would indicate a potential therapeutic intervention in the condition pathological

Alternatively, non-human PRO homologs can be used to construct a knock out animal with PRO that has a defective or altered gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO and the altered genomic DNA that encodes PRO introduced into an embryonic stem cell of the animal. For example, cDNA encoding PRO can be used to clone genomic DNA encoding PRO according to established techniques. A part of the genomic DNA encoding PRO can be deleted or replaced by another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Usually, several kilobases of unchanged flanking DNA (at the 5 'and 3' ends) are included in the vector [see, for example, Thomas and Capecchi, Cell , 51: 503 (1987) for a description of homologous recombination vectors] . The vector is introduced into an embryonic stem cell line (for example, by electroporation) and the cells in which the introduced DNA has been homologously recombined with the endogenous DNA are selected [see, for example, Li et al ., Cell, 69: 915 (1992)]. The selected cells are then injected into an animal blast (for example, a mouse or a rat) to form aggregation chimeras [see, for example, Bradley, in Teralocarcinomas and Embryonic Stem Cells: A Practical Approach , EJ Robertson, ed. (IRL, Oxford, 1987), pages 113-152]. Next, the chimeric embryo can be implanted in a suitable pseudopregnated female raised animal and the embryo can be produced to create a knock out animal. The progeny carrying homologously recombined DNA in their germ cells can be identified by standard techniques and used to raise animals in which all cells thereof contain homologously recombined DNA. Knock out animals can be characterized, for example, by their ability to defend against certain pathological conditions and by their development of pathological conditions due to the absence of the PRO polypeptide.

The nucleic acid encoding PRO polypeptides can also be used in gene therapy. In gene therapy applications, genes are introduced into cells to achieve synthesis in vivo synthesis of a therapeutically effective genetic product, for example, for the replacement of a defective gene. "Gene therapy" includes both conventional gene therapy in which a lasting effect is achieved by a single treatment, such as the administration of gene therapeutic agents, which involves the once or repetitive administration of a therapeutically effective DNA or RNA. Antisense DNAs and RNAs can be used as therapeutic agents to block the expression of certain genes in vivo . It has also been observed that short antisense oligonucleotides can be imported into cells in which they act as inhibitors, despite their low intracellular concentrations caused by their limited uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 [1986]). The oligonucleotides can be modified to increase their uptake, for example, by replacing their negatively charged phosphodiester groups with uncharged groups.

There are a variety of techniques available to introduce nucleic acids into viable cells. The techniques vary depending on whether the nucleic acid is transferred in cells grown in vitro , or in vivo in the cells of the desired host. Suitable techniques for the transfer of nucleic acid in mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation procedure, etc. Preferred current in vivo gene transfer techniques include transfection with viral vectors (usually retroviral) and liposome-mediated transfection of viral envelope proteins (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]) . In some situations, it is desirable to provide the source of nucleic acids with an agent that recognizes the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor in the cell. target, etc. When liposomes are used, proteins that bind to a cell surface membrane protein associated with endocytosis can be used to direct and / or facilitate uptake, for example, capsid proteins or fragments of the same tropics for a specific cell type, antibodies to proteins that undergo cyclic internalization, proteins that direct intracellular localization and increased intracellular half-life. The technique of receptor-mediated endocytosis is described in, for example, Wu et al., J. Biol. Chem . 262, 4429-4432 (1987); and Wagner et al., Proc. Natl Acad. Sci. USA 87, 3410-3414 (1990). For a review of the gene labeling and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).

PRO polypeptides described herein invention can also be used as weight markers molecular for the purpose of a protein electrophoresis and the isolated nucleic acid sequences can be used to recombinantly express said markers.

The nucleic acid molecules that encode PRO polypeptides or fragments thereof described in the The present invention are useful for the identification of chromosomes. In this regard, there is a current need to identify new chromosome markers, as they are currently available relatively few chromosome marker reagents, based on Sequence data available. Each acid molecule PRO nucleic of the present invention can be used as chromosome marker.

PRO polypeptides and acid molecules nucleic of the present invention can also be used as diagnosis for tissue characterization, in which the PRO polypeptides of the present invention can be expressed differentially in one tissue compared to another, preferably in a diseased tissue compared to a tissue normal of the same type of tissue. The nucleic acid molecules of PRO will be used to generate probes for PCR, Northern analysis, Southern analysis and Western analysis.

The PRO polypeptides described in the present invention can also be used as therapeutic agents. The PRO polypeptides of the present invention can be formulated according to known procedures for preparing pharmaceutically useful compositions, through which the PRO product thereof is combined in a mixture with a pharmaceutically acceptable carrier vehicle. The therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with physiologically acceptable carriers, excipients or optional stabilizers ( Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors at the doses and concentrations used, and include buffers, such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates that include glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN?, PLURONICS? or PEG.

The formulations to be used for in vitro administration must be sterile. This is easily achieved by filtration through sterile filtration membranes, prior to or after lyophilization and reconstitution.

Therapeutic compositions herein invention are generally placed in a container that has a sterile access container, for example, a bag or vial of intravenous solution that has a plug penetrable by a needle hypodermic injection

The administration path is in accordance with known procedures, for example, injection or infusion through intravenous, intraperitoneal, intracerebral routes, intramuscular, intraocular, intraarterial or intralesional, topical administration, or by release systems controlled.

The desired doses and drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the particular intended use. The determination of the appropriate dose or route of administration is within the knowledge of a regular physician. Animal experiments provide a reliable guide for the determination of effective doses for human therapy. Scaling between effective dose species can be carried out following the principles established by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al ., Eds., Pergamon Press, New York, 1989, pages 42-96.

When the in vivo administration of a PRO or agonist or antagonist polypeptide thereof is used, normal dosage amounts may vary from about 10 ng / kg to 100 mg / kg of mammalian body mass or more per day, preferably about 1 \ mug / kg / day up to 10 mg / kg / day, depending on the route of administration. Guidelines for specific doses and release procedures are provided in the literature; see, for example, US Patent Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration that is directed to an organ or tissue, for example, may require release in a different way than that of another organ or tissue.

When administration by controlled release of a PRO polypeptide in a formulation with release characteristics suitable for the treatment of any disease or disorder that requires administration of the PRO polypeptide is desired, microencapsulation of the PRO polypeptide is contemplated. Microencapsulation of recombinant proteins for controlled release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2, and MN rgp 120. Johnson et al., Nat. Med ., 2 : 795-799 (1996); Yasuda, Biomed. Ther ., 27: 1221-1223 (1993); Hora et al., Bio / Technology , 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polyactide Polyglycolide Microsphere Systems", in Vaccine Design: The Subunit and Adjuvant Approach , Powell and Newman, eds, (Plenum Press: New York, 1995), pages 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and US Patent No. 5,654,010.

The controlled release formulations of these proteins were developed using polylactic-coglycolic acid polymer (PLGA) due to their biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids can be rapidly purified inside the human body. In addition, the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide / glycolide polymer", in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pages 1-41.

The present invention comprises procedures of screening compounds to identify those that mimic the PRO polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists). Screening trials for drug candidates agonists are designed to identify compounds that bind or complex with PRO polypeptides encoded by genes identified in the present invention, or interfere with the interaction of encoded polypeptides with other proteins cell phones. Such screening tests will include susceptible trials. of screening high performance chemical libraries, making them particularly suitable for identifying small molecules as Candidate drugs

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The tests can be performed in a variety of formats including binding assays protein-protein, biochemical screening assays, immunoassays and cell-based assays, which are fine characterized in the art.

All trials for antagonists have in common that require contact of the candidate drug with a PRO polypeptide encoded by a nucleic acid identified in the present under conditions and for a sufficient time to allow these two components to interact.

In binding assays, the interaction is the binding and the complex formed can be isolated or detected in the mixture of reaction. In a particular embodiment, the PRO polypeptide encoded by the gene identified in the present invention or the Candidate drug is immobilized on a solid phase, for example, a microtiter plate, by covalent or non-covalent bonds covalent Non-covalent bonding is generally performed by the coating of the solid surface with a solution of PRO polypeptide and drying. Alternatively, a immobilized antibody, for example, a monoclonal antibody, specific for the PRO polypeptide to be immobilized, to anchor it to the solid surface. The test is performed by adding the non-immobilized component, which can be marked by a mark detectable, to the immobilized component, for example, the surface coated containing the anchored component. When the reaction, unreacted components are extracted, for example, by washing, and the complexes anchored to the solid surface When the component originally did not immobilized carries a detectable marker, the detection of the immobilized mark on the surface indicates that the complexation When the originally non-frozen component does not carries a marker, complexation can be detected, by example, using a labeled antibody that binds specifically to the immobilized complex.

If the candidate compound interacts with, but does not bind to a particular PRO polypeptide encoded by a gene identified in the present invention, its interaction with that polypeptide can be analyzed by known methods to detect protein-protein interactions. Such trials include traditional strategies, such as, for example, cross-linking, coinmunoprecipitation and copurification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored using a yeast-based genetic system described by Fields et al. (Fields and Song, Nature (London) , 340: 245-246 (1989); Chien et al., Proc Natl. Acad. Sci. USA , 88: 9578-9582 (1991)). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA binding domain, the other acting as the transcription-activation domain. The yeast expression system described in previous publications (generally referred to as "the two hybrid system") takes advantage of this property and uses two hybrid proteins, one in which the target protein is fused to the DNA binding domain of GAL4, and another, in which the candidate activating proteins are fused to the activation domain. The expression of a GAL1- lacZ reporter gene under the control of a promoter activated by GAL4 depends on the reconstitution of GAL4 activity through protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromatogenic substrate for β-galactosidase. A complete kit (MATCHMAKER ™) to identify protein-protein interactions between two specific proteins is commercially available from Clontech using the two-hybrid technique. This system can also be extended to map protein domains involved in specific protein interactions as well as to specify amino acid residues that are crucial to these interactions.

Compounds that interfere with the interaction of a gene encoding an identified PRO polypeptide in the present invention and other intra- or extracellular components It can be analyzed as follows: usually a reaction mixture is prepared containing the product of the gene and the low intra- or extracellular component conditions and for a time that allows interaction and union of the two products. To analyze the capacity of a Candidate compound to inhibit binding, reaction develops  in the absence and in the presence of the test compound. Also I know you can add a placebo to a third reaction mixture, to use as a positive control The union (complex formation) between the test compound and the intra- or extracellular component present in the mixture it is monitored as described previously in The present invention. The formation of the complex in the reaction or control reactions, but not in the reaction mixture that contains the test compound, indicates that the test compound interferes with the interaction of the test compound and its reaction partner

To analyze antagonists, the PRO polypeptide can be added to a cell together with the compound to be screened for a specific activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is a PRO polypeptide antagonist. Alternatively, the antagonists can be detected by combining the PRO polypeptide and a potential antagonist with the membrane bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO polypeptide can be labeled, for example by radioactivity, so that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the antagonistic potential. The gene encoding the receptor can be identified by numerous methods known to those skilled in the art, for example, "panning" of ligand and separation by FACS. Coligan et al. Current Protocols in Immun ., 1 (2): Chapter 5 (1991). Preferably, expression cloning is used when the polyadenylated RNA is prepared from a PRO polypeptide sensitive cell and a cDNA library created from this RNA is divided into groups and used to transfect COS cells or other cells that They are not sensitive to the PRO polypeptide. Transfected cells that develop on glass specimens are exposed to labeled PRO polypeptides. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site specific protein kinase. After fixation and incubation, the sample holders are subjected to autoradiographic analysis. Positive groups are identified and subgroups are prepared and retransfected using an interactive sub-grouping and receiving process, finally obtaining a single clone that encodes the receptor potential.

As an alternative strategy for receptor identification, the labeled PRO polypeptide can be join by photoafinity with the cell membrane or preparations of Extract expressing the receptor molecule. Crosslinked material It is separated by PAGE and exposed to the X-ray film. marked complex containing the receptor can be cleaved, separate into peptide fragments, and undergo protein microsequencing. Amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene that encodes the potential receiver

In another trial for antagonists, the cells of mammals or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the compound candidate. Then, the capacity of the compound to increase or block this interaction.

More specific examples of antagonists potentials include an oligonucleotide that binds to fusions of immunoglobulin with PRO polypeptide, and, in particular, antibodies that include, without limitation, polyclonal antibodies and monoclonal and antibody fragments, chain antibodies single, anti-idiotypic antibodies and versions chimeric or humanized of said antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist can be a protein. closely related, for example, a mutated form of PRO polypeptide that recognizes the receptor but does not transmit the effect, thus competitively inhibiting the action of the polypeptide PRO.

Another potential PRO polypeptide antagonist is an antisense DNA or RNA construct prepared using antisense technology, when, for example, an antisense DNA or RNA molecule acts to directly block the translation of mRNA by hybridization to labeled mRNA and preventing translation of the protein. Antisense technology can be used to control gene expression through the formation of a triple helix or antisense DNA or RNA, both procedures based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding part of the polynucleotide sequence, which encodes the mature PRO polypeptides of the present invention, is used to design an antisense RNA oligonucleotide approximately 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res ., 6: 3073 (1979); Cooney et al., Science , 241 : 456 (1988); Dervan et al., Science , 251: 1360 (1991)), thus avoiding transcription and production of the PRO polypeptide. The antisense RNA oligonucleotide hybridizes to mRNA in vivo and blocks the translation of the mRNA molecule into the PRO polypeptide (antisense - Okano, Neurochem ., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988) The oligonucleotides described above can also be released to the cells, so that the antisense RNA or DNA can be expressed in vivo to inhibit the production of the PRO polypeptide. When antisense DNA is used, oligodeoxynucleotides are preferred derived from the translation initiation site, for example, between approximately -10 and +10 positions of the nucleotide sequence of the target gene.

Potential antagonists include small molecules that bind to the active site, the binding site to the receptor, or the growth factor or other binding site relevant of the PRO polypeptide, thereby blocking the normal biological activity of the PRO polypeptide. Among the examples Small molecules include, but are not limited to, peptides small or peptide-like molecules, preferably peptides soluble and non-peptide organic or inorganic compounds synthetic

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific division of RNA. Ribozymes act by sequence specific hybridization to complementary target RNA, followed by endonucleolytic division. The ribozyme specific division sites in a potential target RNA can be identified by known techniques. For more details, see, for example, Rossi, Current Biology , 4: 469-471 (1994) and PCT Publication No. WO 97/33551 (published September 18, 1997).

The triple helix-forming nucleic acid molecules used to inhibit transcription should be single chain and composed of deoxynucleotides. The base composition of these oligonucleotides is designed so that it induces triple helix formation through the Hoogsteen base pairing rules, which generally require considerable stretches of purines or pyrimidines in a double-stranded chain. For more details, see, for example, PCT Publication No. WO 97/33551, supra .

These small molecules can be identified by one or more of the screening assays described previously in the present invention and / or by any other Screening technique known to a person skilled in the art.

Diagnostic and therapeutic uses of the molecules described in the present invention can also be based on the successes of positive functional trials disclosed and described below.

F. Anti-PRO antibodies

The present invention further provides anti-PRO antibodies. Among the antibodies of examples include polyclonal, monoclonal antibodies, humanized, bispecific and heteroconjugated.

1. Polyclonal antibodies

Anti-PRO antibodies can Understand polyclonal antibodies. The procedures of Preparation of polyclonal antibodies are known for the skilled. Polyclonal antibodies can be develop in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Usually, the immunizing agent and / or adjuvant will be injected into the mammal by multiple subcutaneous injections or intraperitoneal The immunizing agent may include the PRO polypeptide or a fusion protein thereof. It can be useful conjugate the immunizing agent to a protein known to be immunogenic in the mammal that is immunized. Among the examples of said immunogenic proteins are included, but not limited to, California limpet hemocyanin, serum albumin, bovine thyroglobulin, and soy trypsin inhibitor. Between the Examples of adjuvants that can be used include the Preund complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dichlorinomycolate). The immunization protocol can be selected by an expert in Matter without great experimentation.

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2. Monoclonal antibodies

The anti-PRO antibodies may alternatively be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature , 256 : 495 (1975). In a hybridoma procedure, a mouse, hamster or other appropriate host animal is usually immunized with an immunizing agent to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro .

The immunizing agent will usually include the PRO polypeptide or a fusion protein thereof. Generally, peripheral blood lymphocytes ("PLBs") are used if cells of human origin, or spleen cells, or lymph node cells are used if sources of non-human mammals are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice , Academia Press, (1986), pages 59-103 ]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of non-fused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will usually include hypoxanthine, aminopterin and thymidine ("HAT medium"), whose substances prevent the growth of deficient cells in HGPRT.

Preferred immortalized cell lines are those that efficiently fuse, support a stable high level of antibody expression by the selected antibody producing cells, and are sensitive to a medium, such as HAT medium. The most preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. The human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol ., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pages, 51-63].

Next, the culture medium in which the hybridoma cells are cultured can be analyzed for the presence of monoclonal antibodies directed against the PRO. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay , such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA). Such techniques and tests are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson and Poliard, Anal. Biochem ., 107 : 220 (1980).

After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and can be developed by standard procedures [Goding, supra ]. Suitable culture media for this purpose include, for example, Eagle medium modified by Dulbecco and RPMI-1640 medium. Alternatively, hybridoma cells can be developed in vivo as ascites fluid in a mammal.

Monoclonal antibodies secreted by subclones can be isolated or purified from the culture medium or ascites fluid by purification procedures of conventional immunoglobulins, such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional methods (for example, by the use of oligonucleotide probes that are capable of specifically binding to genes encoding the light and heavy chains of murine antibodies) . The hybridoma cells of the present invention serve as a preferred source of said DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells, such as simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce protein of immunoglobulin, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing the coding sequence for human heavy and light chain constant domains instead of murine homologous sequences [US Patent No. 4,816,567; Morrison et al., Supra ] or covalently binding to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Said non-immunoglobulin polypeptide may be substituted for the constant domains of an antibody of the present invention, or may be substituted for the variable domains of an antigen combination site of an antibody of the invention to create a bivalent chimeric antibody.

The antibodies can be antibodies monovalent The procedures for preparing antibodies Monovalent are known in the art. For example, a procedure involves recombinant expression of the light chain and the modified heavy chain of immunoglobulin. Chain heavy is usually truncated at any point in the Fc region to avoid crosslinking of the heavy chain. Alternatively, the relevant cysteine residues are replaced by another residue of amino acid or are removed to prevent crosslinking.

In vitro procedures are also suitable for preparing monovalent antibodies. The digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be performed using routine techniques known in the sector.

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3. Human and humanized antibodies

The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other sub-binding sequences. antibody antigen) containing a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibody) in which residues of a receptor complementarity determining region (CDR) are replaced by residues of a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit that has the specificity, affinity and capacity desired. In some cases, the Fv framework residues of the human immunoglobulin are replaced by the corresponding non-human residues. Humanized antibodies may also comprise residues that are neither found in the recipient antibody nor in the imported CDR sequences or framework. In general, the humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all regions of FR are those of a consensus sequence of human immunoglobulin. The optimally humanized antibody will also comprise at least a portion of a constant region of immunoglobulin (Fc), usually that of a human immunoglobulin [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-329 (1988); and Presta, Curr. Op. Struct. Biol ., 2 : 593-596 (1992)].

Procedures for humanizing non-human antibodies are known in the art. Generally, a humanized antibody has one or more amino acid residues introduced therein from a non-human origin. These non-human amino acid residues are often indicated as "imported" residues, which are usually acquired from an "imported" variable domain. Humanization can be performed essentially following the procedure 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)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, said "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than one intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of similar sites in rodent antibodies.

Human antibodies can also be produced using several techniques known in the sector, 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 . They are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, page 77 (1985) and Boerner et al., J. Immunol ., 147 (1) : 86- 95 (1991)] Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely deactivated. , the production of human antibodies is observed, which closely resembles those observed in humans in all aspects, including gene readjustment, assembly, and antibody repertoire.This strategy is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., BioTechnology 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).

Antibodies can also be purified by affinity using selection and / or mutagenesis procedures known as described above. Antibodies preferred affinity matured have an affinity that is five times, more preferably 10 times, even more preferably 20 or 30 times more than the starting antibody (usually murine, humanized or human) from which the antibody is prepared matured

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4. Bispecific Antibodies

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

Methods for making bispecific antibodies are known in the art. Usually, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain / light chain partners, in which the two heavy chains have different specificities [Millstein and Neck, Nature , 305 : 537-539 (1983) ]. Due to the random variety of heavy and light immunoglobulin 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 performed by affinity chromatography steps. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J. , 10 : 3655-3659 (1991).

Variable antibody domains with the desired binding specificities (antibody-antigen combination sites) can be fused to sequences of immunoglobulin constant domains. The fusion is preferably with a constant immunoglobulin heavy chain domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) that contains the site necessary for light chain binding present in at least one of the fusions. The DNAs encoding immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and co-transfected into a suitable host organism. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzimology , 121 : 210 (1986).

According to another strategy described in WO 96/27011, you can design the interface between a couple of molecules of antibody to maximize the percentage of heterodimers that are recover from recombinant cell culture. The interface preferred comprises at least a part of the CH3 region of a constant antibody domain. In this procedure, one or more small amino acid side chains of the interface of the first antibody molecule are replaced by side chains older (for example, tyrosine or tryptophan). The "cavities" compensators of similar or identical size to the chain or chains large laterals are created at the interface of the second molecule of antibody by replacing large side chains of amino acids by smaller ones (for example, alanine or threonine). This provides a mechanism to increase the performance of the heterodimer over other unwanted end products, such as homodimers

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (for example, bispecific F (ab ') 2 antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically divided to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize the neighborhood dithiols and prevent the formation of intermolecular disulfide. Next, the Fab 'fragments generated are converted into thionitrobenzoate (TNB) derivatives. Next, one of the Fab'-TNB derivatives is converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Fab 'fragments can be recovered directly from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med . 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody molecule F (ab ') 2. Each Fab 'fragment was secreted separately from E. coli and subjected to a chemical coupling directed in vitro to form the bispecific antibody. Said bispecific antibody formed in this manner was able to bind cells that overexpress the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumors.

Various techniques for manufacturing and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol . 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were bound to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This procedure can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for manufacturing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (V H) connected to a light chain variable domain (V L) by a linker that is too short to allow pairing between the two domains in the same chain. Accordingly, the V_ {H} and V_ {L} domains of one fragment are forced to pair with the complementary V_ {L} and VH {domains of another fragment, thus forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain Fv dimers (sFv) has also been described. See Gruber et al., J. Immunol . 152: 5368 (1994). Antibodies with more than two valences are considered. For example, trypecific antibodies can be prepared. Tun et al., J. Immunol . 147: 60 (1991).

Example bispecific antibodies are can bind to two different epitopes in a PRO polypeptide determined of the present invention. Alternatively, an arm of anti-PRO polypeptide can be combined with one arm that binds to a trigger molecule in a white blood cell, such as a T cell receptor molecule (for example, CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) for centering the cellular defense mechanisms in the cell that expresses the particular PRO polypeptide. Bispecific antibodies are also they can use to confine cytotoxic agents to cells that express a particular PRO polypeptide. These antibodies have a PRO binding arm and an arm that binds to a cytotoxic agent or a chelating radionuclide, such as EOTUBE, DPTA, DOTA or TETA. Other bispecific antibody of interest binds to the PRO polypeptide and It also binds to the tissue factor (TF).

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5. Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently bound antibodies. Such antibodies have been proposed, for example, to direct cells of the immune system to unwanted cells [US Patent No. 4,676,980] and for the treatment of HIV infection [WO 91/00360: WO 92/300373; EP 03089]. It is considered that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those described, for example, in US Patent No. 4,676,980.

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6. Design of the effector function

It may be desired to modify the antibody of the present invention with respect to the effector function in order to increase, for example, the efficacy of the antibody in the treatment of cancer. For example, a cysteine residue or residues can be introduced into the Fc region, thus allowing the formation of disulfide bonds between chains in this region. The homodimeric antibody generated in this manner can improve the ability to internalize and / or increase complement-mediated cytolysis and antibody-dependent 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 increased anti-tumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolf et al. Cancer Research , 53 : 2560-2565 (1993). Alternatively, an antibody can be designed to have dual Fc regions and, thus, can increase complementary lysis and ADCC capabilities. See, Stevenson et al., Anti-Cancer Drug Design , 3: 219-230 (1989).

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7. Immunoconjugates

The present invention also relates to immunoconjugates comprising an antibody conjugated to an agent cytotoxic, such as a chemotherapeutic agent, toxin (by example, an enzymatically active toxin of bacterial origin, fungal, plant or animal, or fragments thereof), or an isotope radioactive (i.e. radioconjugates).

Chemotherapeutic agents useful for the generation of said immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, A chain abrin, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII, and PAPS), momordica charantia inhibitor, curcin, crotine, sapaonaria officinalis inhibitor, gelonin, mitogelin , restrictocin, fenomycin, enomycin, and trichothecenes. There is a set of radionuclides available for the production of radioconjugated antibodies. Some examples are 212 Bi, 131 I, 131 In, 90 Y and 186 Re.

Antibody and cytotoxic agent conjugates are manufactured using a set of bifunctional protein-coupling agents, such as 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), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p- diazonium benzoyl) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science , 238: 1098 (1987). Carbon-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminopentaacetic acid (MX-DTPA) 14 is an example of chelating agent for conjugation of radionuclides to the antibody. See WO94 / 11026.

In another embodiment, the antibody can be conjugate a "receptor" (such as streptavidin) for use in the pre-marking of tumors in which the conjugate antibody-receptor is administered to the patient, followed by removal of unbound conjugate from the circulation using a clarifying agent and then the administration of a "ligand" (eg, avidin) that is conjugates a cytotoxic agent (for example, a radionuclide).

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8. Immunoliposomes

The antibodies described in the present invention can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl Acad. Sci. USA , 82 : 3688 (1985); Hwang et al., Proc. Natl Acad. Sci USA , 77 : 4030 (1980); and US Patent Nos. 4,485,045 and 4,544,545. Liposomes with a longer circulation time are described in US Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation process with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem ., 257 : 286-288 (1982) by a disulfide bond exchange reaction . Optionally, the liposome contains a chemotherapeutic agent (such as Doxorubicin). See Gabizon et al., J. National Cancer Inst ., 81 (19): 1484 (1989).

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9. Pharmaceutical antibody compositions

Antibodies that specifically bind to a PRO polypeptide identified in the present invention, as well as other molecules identified by the screening assays described previously in the present invention, they can be administered to the treatment of various disorders in the form of compositions Pharmaceuticals

If the PRO polypeptide is intracellular and complete antibodies are used as inhibitors, internalization antibodies are preferred. However, lipofections or liposomes can also be used to release the antibody, or an antibody fragment, in the cells. When antibody fragments are used, the smaller inhibitor fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequences of an antibody, peptide molecules can be designed that maintain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl Acad. Sci. USA , 90 : 7889-7893 (1993). The formulation of the present invention may also contain more than one active compound as necessary for the particular disease being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent or a growth inhibiting agent. Said molecules are suitably present combined in amounts that are effective for the desired purpose.

The active substances can also be introduced into prepared microcapsules, for example, by coacervation techniques or by interphase polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems. (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences , supra .

The formulations to be used in in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

Release preparations can be prepared. controlled. Among some examples of release preparations controlled polymer semipermeable matrices are included solid hydrophobic containing the antibody, whose matrices they are in the form of shaped articles, for example, films or microcapsules Among the examples of release matrices controlled include polyesters, hydrogels (for example, poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, ethylene-vinyl acetate copolymer no degradable, lactic acid-glycolic acid copolymers degradable, such as LUPRON DEPOT? (microspheres injectables composed of lactic acid-acid copolymer glycolic and leuprolide acetate), and acid poly-D - (-) - 3-hydroxybutyric.  While polymers, such as ethylene acetate Vinyl and lactic acid-glycolic acid allow the release of molecules for about 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibodies remain in the body for a period over time, they can be denatured or added as a result of exposure to moisture at 37 ° C, resulting in a loss of Biological activity and possible changes in immunogenicity. Be can devise reasonable strategies for stabilization depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is link formation Intermolecular S-S through exchange thio-disulfide, stabilization can be achieved by modifying sulfhydryl residues, lyophilization of acidic solutions, moisture content control, proper use of additives, and the development of compositions of specific polymer matrices.

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G. Antibody uses anti-PRO

The anti-PRO antibodies of the present invention have several utilities. For example, anti-PRO antibodies can be used in diagnostic assays for PRO, for example, by detecting their expression (and in some cases, differential expression) in specific cells, specific tissues or serum. Several diagnostic assay techniques known in the sector can be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays performed in heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques , CRC PRess , Inc. (1987) pages 147-158]. Antibodies used in diagnostic assays can be labeled with a detectable group. The detectable group should be able to produce, directly or indirectly, a detectable signal. For example, the detectable group may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 I, a fluorescent or chemiluminescent compound, such such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art can be used to conjugate the antibody to the detectable group, including those procedures described by Hunter et al., Nature , 144 : 945 (1962); David et al., Biochemistry , 13 : 1014 (1974); Pain et al., J. Immunol. Meth ., 40 : 219 (1981); and Nygren, J. Histochem. and Cytochem ., 30 : 407 (1982).

Anti-PRO antibodies also they are useful for the affinity purification of PRO from a culture of recombinant cells or of natural origin. In this process, the PRO antibodies are immobilized on a suitable support, such as a Sephadex resin or filter paper, using procedures known in the art. Then the immobilized antibody is contacted with a sample that contains the PRO to be purified, and then the support is washed with a suitable solvent that will remove substantially all of the material in the sample except for the PRO, which is attached to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the PRO from the antibody.

The following examples are offered only with illustrative objectives, and are not intended to limit the Scope of the present invention.

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Examples

Reagents commercially available to referenced in the examples were used according to the manufacturer's instructions unless indicated contrary. The origin of the cells identified in the following examples, and throughout the document, by the access numbers of The ATCC belongs to the American Type Culture Collection, Manassas, GOES.

Example 1 Screening for extracellular domain homology to identify new polypeptides and the cDNAs that encode them

Extracellular domain (ECD) sequences (including the secretion signal sequence, if any) of approximately 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. EST databases included public databases (for example, Dayhoff, GenBank), and private databases (for example, LIFESEQ?, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the BLAST or BLAST-2 software (Altschul et al ., Methods in Enzymology 266: 460-480 (1996)) as a comparison of the ECD protein sequences with a 6-frame translation of the sequences of ITS T. Comparisons with a BLAST rating of 70 (or in some cases 90) or higher that did not encode known proteins were grouped and assembled into consensus DNA sequences with the "phrap" program (Phil Green, University of Washington, Seattle, WA ).

Using this screening for homology of extracellular domain, consensus DNA sequences were assembled in relation to the other EST sequences identified using phrap In addition, the consensus DNA sequences obtained are extended often (but not always) using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as much as possible using the sources of EST sequences described above.

Based on the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify a cDNA library containing the sequence of interest by PCR and for use as probes to isolate a clone of coding sequence. full length for a PRO polypeptide. Direct and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to produce a PCR product of approximately 100-1000 bp in length. The sequences of the probes are usually 40-55 bp in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 bp. In order to screen several libraries for a full length clone, DNA from the libraries was screened by PCR amplification, as by Ausubel et al ., Current Protocols in Molecular Biology, with the PCR primer pair.

The cDNA libraries used to isolate cDNA clones were constructed by standard methods using commercially available reagents, such as those from Invitrogen, San Diego, CA. The cDNA was primed with oligo dT containing a NotI site, joined by the blunt end to SalI hemikinase adapters, divided with NotI, appropriately sized by gel electrophoresis, and cloned in a defined orientation in a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes et al ., Science, 253: 1278-1280 (1991)) at the unique XhoI and NotI sites.

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Example 2 Isolation of cDNA clones by amylase screening 1. Preparation of cDNA library primed with oligo dT

MRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2). This RNA was used to generate a library of CDNA primed with oligo dT in the pRK5D vector using reagents and protocols of Life Technologies, Gaithersburg, MD (Super Script Plasmid System). In this procedure, the size was separated by Double stranded cDNA with more than 1000 bp and bound cDNA was cloned by SalI / NotI in the vector divided by XhoI / NotI. pRK5D is a cloning vector that has a start site of the sp6 transcription followed by a restriction enzyme site SfiI that precedes the XhoI / NotI cDNA cloning sites.

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2. Preparation of primed cDNA library randomly

A secondary cDNA library was generated with in order to preferentially represent the 5 'ends of the clones of primary cDNA. The sp6 RNA was generated from the primary library (described above) and this RNA was used to generate a randomly barley cDNA library in the pSST-AMY.0 vector using reagents and Life Technologies protocols (Super Script Plasmid System, referenced above). In this procedure, the cDNA of double chain separated at 500-1000 bp, joined by blunt ends to NotI adapters, split with SfiI and be they cloned into the vector divided by SfiI / NotI. pSST-AMY.0 is a cloning vector that has a yeast alcohol dehydrogenase promoter that precedes cDNA cloning sites and mouse amylase sequence (the mature sequence without the secretion signal) (followed by the yeast alcohol dehydrogenase terminator), after cloning sites Thus, the cDNAs cloned in this vector that fuse in the reading frame with the amylase sequence will lead to the secretion of amylase from colonies of yeast properly transfected.

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3. Transformation and detection

The DNA of the library described in paragraph 2 above it was cooled in ice to which DH10B bacteria was added electrocompetent (Life Technologies, 20 ml). Then the mixture of bacteria and vectors was electroporated as recommended by the manufacturer. Subsequently, SOC medium (Life Technologies, 1 ml) and the mixture was incubated at 37 ° C for 30 minutes Next, the transformants were placed in 20 standard 150 mm LB plates containing ampicillin and were incubated for 16 hours (37 ° C). They scratched off the plates positive colonies and the bacterial residue DNA was isolated using standard protocols, for example, CsCl gradient. TO then the purified DNA followed the following protocols with yeast.

The methods with the yeast were divided into Three categories (1) Yeast transformation with the vector combined plasmid / cDNA: (2) clone detection and isolation of yeast that secrete amylase; and (3) PCR amplification of the directly from yeast colonies and purification of DNA for sequencing and subsequent analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain It has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL +, SUC +, GAL +. Preferably, yeast mutants that present post-translational mechanisms poor Such mutants may have alleles deficient in the translocation in sec71, sec72, sec62, sec71 being the most preferred truncated. Alternatively, they can preferably be used in combination with yeast expressing antagonist amylase (including antisense nucleotides and / or ligands) that interfere with the normal action of these genes, other proteins involved in this post-translational mechanism (for example, SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p or SSA1p-4p) or the formation of complexes of these proteins.

The transformation is performed based on the protocol described by Gietz et al ., Nucl. Acid Res., 20: 1425 (1992). The transformed cells were then inoculated with agar in the broth of the YEPD complex medium (100 ml) and grown overnight at 30 ° C. YEPD broth was prepared as described in Kaiser et al ., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). Next, the overnight culture was diluted to approximately 2 x 10 6 cells / ml (approximately OD 600 = 0.1) in new YEPD broth (500 ml) and redeveloped to 1 x 10 <2>. 7} cells / mol (approximately DO600 = 0.4-0.5).

Next, the cells were collected and prepared for transformation by transferring in bottles with GS3 rotor at 5,000 rpm for 5 minutes, the supernatant and then resuspended in sterile water, and it centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. He discarded the supernatant and cells were subsequently washed with LiAc / TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li 2 OOCCH 3), and were resuspended in LiAc / TE (2.5 ml).

The transformation took place by mixing the prepared cells (100 µl) with salmon testis DNA denatured single chain (Lofstrand Labs, Gaithersburg, MD) and Transforming DNA (1 µg, vol. <10 µm) in tubes microcentrifuge The mixture was mixed briefly by Cetrifugation, then 40% PEG / TE (600 µL, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li2 OOCCH3, pH 7.5). This mixture was gently stirred and incubated at 30 ° C with stirring. for 30 minutes Next, the cells were subjected to thermal shock at 42 ° C for 15 minutes, and the reaction vessel in a microcentrifuge at 12,000 rpm for 5-10 seconds, decanted and resuspended in TE (500 µL, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. Then the cells will diluted in TE (1 ml) and aliquots (200 µl) were extended in the selective medium previously prepared in plates of 150 mm growth (VWR).

Alternatively, instead of multiple small reactions, the transformation was performed using a only large-scale reaction, where the reaction amounts are They climbed accordingly.

The selective medium used was synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared in Kaiser et al ., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208-210 (1994). The transformants were developed at 30 ° C for 2-3 days.

The detection of colonies that secrete amylase was performed by the inclusion of red starch in the selective growth medium. The starch was coupled to red dye (Reactive Red-120, Sigma) as in the procedure described by Biely et al ., Anal. Biochem., 172: 176-179 (1988). The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0.15% (w / v), and buffered with potassium phosphate to a pH of 7.0 (50-100 mM final concentration ).

Positive colonies were collected and they scratched in a new selective medium (on 150 mm plates) with the in order to obtain well-isolated and identifiable individual colonies. Well-isolated individual colonies positive for Amylase secretion by direct starch incorporation red in SCD-Ura buffered agar. The positive colonies for their ability to break starch giving place to a clear halo around the positive colony visualized directly.

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4. Isolation of DNA by PCR amplification

When a positive colony was isolated, it was collected a part of it with a stick and diluted in sterile water (30 µl) in a 96-well plate. At this time, the positive colonies were frozen and saved for later analysis or were amplified immediately. An aliquot was used of cells (5 µl) as a template for the PCR reaction in one volume 25 µl containing: 0.5 µ Klentaq (Clontech, Palo Alto, AC); 4.0 µm 10 mM dNTPs (Perkin Elmer-Cetus); 2.5 µL Kentaq buffer (Clontech); 0.25 µl direct oligo 1; 0.25 µl inverse oligo 2; 12.5 µl distilled water. The direct oligonucleotide sequence 1 was:

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5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3 ' (SEC ID NO: 169)

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The reverse oligonucleotide sequence 2 was:

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5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3 ' (SEC ID NO: 70)

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Then the PCR of the following is performed way:

25

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The underlined regions of oligonucleotides they hybridized to the ADH promoter region and the amylase region, respectively, and a 307 bp region of the vector was amplified pSST-AMY.0 when there was no insert present. Usually, the first 18 nucleotides of the 5 'end of these oligonucleotides contained hybridization sites for primers of sequencing Thus, the total product of the PCR reaction of an empty vector had 343 bp. However, the cDNA fused to the signal sequence gave rise to nucleotide sequences considerably longer.

After PCR, an aliquot of the reaction (5 µl) was examined by gel electrophoresis on a 1% agarose gel using a Tris-Borate-EDTA buffer system (TBE) described by Sambrook et al., Supra . Clones that give rise to a single larger 400 bp strong PCR product were subsequently analyzed by DNA sequencing after purification with a Qiaquick 96 PCR cleaning column (Qiagen Inc., Chatsworth, CA).

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Example 23 Isolation of cDNA clones using algorithm analysis signal

Several acid sequences were identified nucleic encoding polypeptides by applying a private signal sequence search algorithm developed by Genentech, Inc. (South San Francisco, CA) after ESTs, as well as EST fragments grouped and assembled from public databases (for example, GenBank) and / or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, CA). The algorithm of the signal sequences computes an assessment of the secretion signal in based on the character of the DNA nucleotides surrounding the first methionine codon (ATG) and optionally the second codon of methionine at the 5 'end of the sequence or sequence fragment in consideration. Nucleotides after the first ATG must encode at least 35 unambiguous amino acids without any codon Stop If the first ATG has the required amino acids, it is not Examine the second. If none meets the requirement, the sequence Candidate is not valued. In order to determine if the sequence EST contains an authentic signal sequence, the DNA and the corresponding amino acid sequences surrounding the ATG codon are assessed using a set of seven sensors (parameters of evaluation) known to be associated with secretion signals. The use of this algorithm resulted in the identification of numerous nucleic acid sequences encoding the polypeptides.

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Example 4 Isolation of cDNA clones encoding PRO polypeptides humans

Using the techniques described in the Examples 1 to 3 above, numerous clones of Full length cDNA encoding PRO polypeptides as they are describe here. These cDNAs were then deposited under the conditions of the Budapest Treaty with the American Type Culture Collection, 10801 University Blvd., Manassas VA 20110-2209. USA (ATCC), as shown in the Table 7 below:

TABLE 7

26

These deposits were made as stipulated in the Budapest Treaty on Recognition International Deposit of Microorganisms for the purpose of Patent Procedure and Regulation under it (Treaty of Budapest). This ensures the maintenance of a crop viable deposit for 30 years from the date of Deposit. Deposits will be available through the ATCC as the terms of the Budapest Treaty, and are subject to agreement between Genentech, Inc. and ATCC, which ensures availability permanent and unrestricted progeny of deposit culture for public use after the grant of the respective patent American or after opening to public inspection of any US or foreign patent application, whichever first, and ensures the availability of the progeny to the person authorized by the United States Patent Commissioner and Marks in accordance with standard 35 USC \ NAK 122 and the standards of Commissioner as agreed (including 37 CFR \ NAK 1.14 with particular reference to 886 OG 638).

The assignee of this application has agreed that if a crop of the materials in the deposit died or be lost or destroyed when grown under conditions suitable, the materials will be immediately replaced in a notification by other equals. Material availability deposited is not interpreted as a license to perform the invention contravening the rights granted under the authority of any government in accordance with its patent laws.

Example 5 Use of PRO as a hybridization probe

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

The DNA that comprises the coding sequence of the full-length or mature PRO polypeptide as described in the present invention is used as a screening probe Homologous DNAs (such as those encoding natural variants of PRO) in human tissue cDNA libraries or genomic libraries  of human tissue.

Hybridization and washing of filters that contain any library DNA are made according to the following conditions of high astringency. The hybridization of the Probe derived from radiolabelled PRO polypeptide to filters is Performs in a 50% solution of formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x of Denhardt solution, and 10% dextran sulfate at 42 ° C for  20 hours. The washing of the filters is carried out in a solution 0.1x aqueous SSC and 0.1% SDS at 42 ° C.

Next, the DNAs can be identified that have a desired sequence identity with the DNA that encodes a full length native sequence PRO polypeptide using standard techniques known in the sector.

Example 6 Expression of PRO in E. coli

This example illustrates the preparation of a non-glycosylated form of PRO by recombinant expression in E. coli .

The DNA sequence encoding the PRO polypeptide is initially amplified using selected PCR primers. Primers should contain sites for restriction enzymes that correspond to sites for restriction enzymes in the selected expression vector. A variety of expression vectors can be used. An example of a suitable vector is pBR322 (derived from E. coli , see Bolivar et. Al ., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with a restriction enzyme and dephosphorylated. Next, the PCR amplified sequences are joined in the vector. The vector will preferably include sequences encoding an antibiotic resistant gene, a trp promoter, a leader poly-His sequence (including the first six STII codons, poly-His sequence, and dividing site for enterokinase), the coding region of PRO, the lambda transcriptional terminator, and an argU gene.

Next, the binding mixture is used to transform a selected strain of E. coli using the procedures described in Sambrook et. al., supra . Transformants are identified by 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 be developed overnight in a liquid culture medium such as the LB broth supplemented with antibiotics. The overnight crop can be used later to inoculate a larger culture scale. Then the cells develop up to a desired optical density, during which the promoter of the expression is activated.

After culturing the cells for many more hours, cells can be collected by centrifugation. The cell residue obtained by centrifugation can be solubilize using various agents known in the art, and the solubilized PRO protein can then be purified using a metal chelating column in conditions that allow strong protein binding.

The PRO can be expressed in E. coli in a form labeled with poly-His according to the following procedure. DNA encoding PRO is initially amplified using selected PCR primers. The primers contain sites for restriction enzymes that correspond to the restriction enzyme sites in the selected expression vector, and other useful sequences that provide efficient and reliable translation initiation, rapid purification on a metal chelating column and removal proteolytic with enterokinase. The PCR-amplified poly-His tagged sequences are then linked into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (laclq ) The transformants are first developed in LB containing 50 mg / ml carbenicillin at 30 ° C with stirring until an OD of 3-5 to 600 nm is reached. The cultures are then diluted 50-100 times in CRAP medium (prepared by mixing 3 , 57 g of (NH 4) 2 SO 4, 0.71 g of sodium citrate? 2 O, 1.07 g of KCl, 5.36 g of Difco yeast extract, 5.36 g of Sheffield hycase SF in 500 ml of water, as well as 110 mM MPOS, pH 7.3, 0.55% glucose (w / v) and 7 mM MgSO 4) and develop over approximately 20 -30 hours at 30 ° C. with agitation Samples are taken to verify expression by SDS-PAGE analysis, and the entire culture is centrifuged to sediment the cells.The cell pellets are frozen until purification and ren aturalization

The E. coli paste from 0.5-1 l fermentations (6-10 g of sediment) is resuspended in 10 volumes (w / v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Sodium sulphite are added solid and sodium tetrathionate to final concentrations of 0.1 M and 0.02 M, respectively, and the solution is stirred overnight at 4 ° C. This stage results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman ultracentrifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelating column buffer (6M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to purify. The purified extract is loaded into a 5 ml Qiagen Ni 2 + -NTA chelating column equilibrated in the buffer of the metal chelating column. 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. Fractions containing the desired protein are grouped and stored at 4 ° C. The protein concentration is estimated by its absorbance at 280 nm using the extinction coefficient calculated based on its amino acid sequence.

Proteins are renatured by diluting the shows slowly in fresh prepared renaturation 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. The volumes of renaturation are chosen so that the protein concentration final is between 50 and 100 micrograms / ml. The solution of renaturation is gently stirred at 4 ° C for 12-36 hours The renaturation reaction is deactivates by adding TFA to a final concentration 0.4% (pH of approximately 3). Before a purification additional protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to a final concentration of 2-10%. The protein Renatured is chromatographed on a reverse phase column R1 / H pores with a 0.1% TFA mobile buffer with elution with a 10 to 80% acetonitrile gradient. Aliquots of fractions with an absorbance A 280 in gels of polyacrylamide-SDS and the fractions that are grouped contain the homogenous renatured protein. In general, the adequately renatured samples of most proteins elute at the lowest concentrations of acetonitrile, since these samples are the most compact with their hydrophobic interiors protected from interaction with the reverse phase resin. Aggregated samples are eluted normally at higher acetonitrile concentrations. In addition to separating the misfolded forms of proteins in the desired way, the stage Reverse phase also eliminates endotoxin from samples.

Fractions containing the PRO polypeptide desired folding are grouped and acetonitrile is removed with a gentle stream of nitrogen directed to the solution. Proteins were formulated in 20 mm Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using Superfine G25 (Pharmacia) resins balanced in the formulation buffer and sterile filtered.

Many of the PRO polypeptides described herein are expressed satisfactorily as described previously.

Example 7 PRO expression in mammalian cells

This example illustrates the preparation of a form potentially glycosylated from PRO by expression recombinant in mammalian cells.

The vector pRK5 (see EP 307,247 published March 15, 1989) is used as an expression vector. Optionally, PRO DNA binds at pRK5 with restriction enzymes selected to allow insertion of PRO DNA using binding procedures such as those described in Sambrook et. al., supra . The resulting vector is called pRK5-PRO.

In one embodiment, the selected host cells can be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in a medium such as DMEM supplemented with fetal calf serum and optionally, nutritional components and / or antibiotics. Approximately 10 µg of pRK5- [PRO] DNA is mixed with about 1 µg of DNA encoding the VA RNA gene [Thimmappaya et. al., Cell , 31 : 543 (1982)] and is dissolved in 500 µl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 µl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO 4, and a precipitate is allowed to form for 10 minutes at 25 ° C. The precipitate is suspended and added to the 293 cells and allowed to stand for approximately four hours at 37 ° C. The culture medium is aspirated and 2 ml of 20% glycerol in PBS is added for 30 seconds. Next, the 293 cells are washed with serum-free medium, fresh medium is added and the cells are incubated for approximately 5 days.

Approximately 24 hours after transfections, the culture medium is extracted and replaced by culture medium (alone) or culture medium containing 200 µCi / ml of 35 S-cysteine and 200 µCi / ml of 35 S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated in a filter of centrifugation and loaded on a 15% SDS gel. The processed gel can be dried and exposed to a film during a period of specific time to reveal the presence of the PRO polypeptide. The cultures that contain transfected cells may experience an additional incubation (in serum free medium) and the medium is examined in specific bioassays.

In an alternative technique, PRO can be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et. al ., Proc. Natl Acad. Sci., 12: 1575 (1981). 293 cells develop to maximum density in a rotating flask and 700 µg of pRK5- [PRO] DNA is added. First, the cells are concentrated in the rotating flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated in the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and reintroduced into the rotating flask containing the tissue culture medium, 5 µg / ml bovine insulin and 0.1 / mug / bovine transferrin. After about four days, the conditioned medium is centrifuged and filtered to remove cells and cell debris. The sample containing the expressed PRO can then be concentrated and purified by any selected procedure, such as dialysis and / or column chromatography.

In another embodiment, PRO may be expressed in CHO cells. The vector pRK5- [PRO] can be transfected into cells CHO using known reagents, such as CaPO4 or DEAE-dextran. As described previously, cell cultures can be incubated, and the medium can be substituted by culture medium (alone) or medium containing a radiolabel, such as 35 S-methionine. After determining the presence of the PRO polypeptide, the medium of culture can be substituted by serum free medium. Preferably, the cultures are incubated for approximately 6 days and, at Then, the conditioned medium is collected. Then the medium containing the expressed PRO can be concentrated and purified by any selected procedure.

The epitope tagged PRO can be expressed also in host CHO cells. The PRO can be subcloned out of the vector pRK5. The subclone insert can undergo PCR to merge into the frame with a selected epitope tag, such as a poly-His tag in a vector of Baculovirus expression. The PRO insert tagged with poly-His can then be subcloned into a vector directed by SV40 containing a selection marker, such as DHFR, to select stable clones. Finally the cells CHO can be transfected (as described above) with the vector directed by SV40. Marking can be done, such as described above, to verify the expression. He culture medium containing the PRO labeled with expressed poly-His can then concentrate and purified by any selected procedure, such as by chelation affinity chromatography with Ni 2+.

PRO can be expressed in CHO and / or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.

Stable expression in CHO cells is performed. Using the following procedure. Proteins are expressed as a construction of IgG (immunoadhesin), in which the coding sequences for soluble forms (for example, extracellular domains) of the respective proteins are fused to a constant region sequence of IgG1 containing the hinge, CH2 and domains CH2 and / or is a form labeled with poly-His.

After PCR amplification, the respective DNAs are subcloned into a CHO expression vector using standard techniques described in Ausubel et. al., Current Protocols of Molecular Biology , Unit 3.16, John Wiley and Sons (1997). The CHO expression vectors are constructed to have compatible 5 'and 3' restriction sites of the DNA of interest to allow adequate transfer of the cDNA. The vector used for expression in CHO cells is as described in Lucas et. al., Nucl. Acid Res . 24: 9 (1774-1779 (1996), and uses the SV40 early promoter / enhancer to direct expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression allows selection for stable plasmid maintenance after the transfection.

Twelve micrograms of the desired plasmid DNA are introduced into approximately 10 million CHO cells using commercially available Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim) transfection reagents. Cells develop as described in Lucas et. al., supra . Approximately 3 x 10 7 cells are frozen in a blister for further growth and production as described below.

Blisters containing plasmid DNA are defrost by placing in a water bath and mix by agitation. The content is 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 selective medium (PS20 filtered at 0.2 µm with serum 5% fetal bovine diafiltered at 0.2 µm). Then the cells are fractionated on a 100 mL shaker that contains 90 mL of selective medium. After 1-2 days, the cells transferred to a 250 mL shaker filled with 150 mL of medium selective growth and incubate at 37 ° C. After others 2-3 days, 3 x 10 5 cells / mL are seeded in 250 mL, 500 mL and 2000 mL stirrers. The cellular medium is changed by fresh medium by centrifugation and resuspension in the medium of production. Although any CHO media can be used adequate, actually used a means of production described in U.S. Patent No. 5,122,469, issued June 16 1992. In a 3L production stirrer, it is sown at the rate of 1.2 x 10 6 cells / ml. On day 0, the number of cells and pH. On day 1, samples of the agitator are taken and Start bubbling with filtered air. On day 2, they are taken Stirrer samples, the temperature is changed to 33 ° C, and added 30 mL of glucose 500 g / L and 0.6 mL of 10% antifoam (for example 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion). Throughout the production, the pH is adjusted according to the need staying at values around 7.2. After 10 days, or until the viability falls below 70%, the cell culture is collected by centrifugation and filtered to through a 0.22 µm filter. The filtrate is stored at 4 ° C or It is loaded immediately into columns for purification.

For labeled constructions of poly-his, proteins are purified using a Ni2 + column - NTA (Qiagen). Before the purification, imidazole is added to the conditioned medium until a 5 mM concentration. The conditioned medium is pumped into a 6 ml column of Ni2 + - NTA balanced at 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM of imidazole at a flow rate of 4-5 ml / min. to 4 ° C. After loading, the column is washed with an equilibration buffer additional and the protein is eluted with equilibrium buffer that It contains 0.25 M imidazole. The highly purified protein is desalt later in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a column  G25 Superfine (Pharmacia) 25 ml and stored at -80 ° C.

The immunoadhesin constructs (which contains Fc) are purified from the conditioned medium as and as follows. The conditioned medium is pumped into a 5 ml Protein A (Pharmacia) column that had been equilibrated in a 20 mM sodium phosphate buffer, pH 6.8. After if loaded, the column is washed extensively with buffer equilibrium before elution with 100 mM citric acid, pH 3.5. The eluted protein is neutralized immediately by collecting fractions 1 ml in tubes containing 275 µL of 1M Tris buffer, pH 9. The highly purified protein is subsequently desalted in a storage buffer, as described above for proteins labeled with poly-his. The homogeneity is calculated by SDS polyacrylamide gels and by amino acid sequencing N-terminals through Edman degradation.

Many of the PRO polypeptides described herein are expressed satisfactorily as described previously.

Example 8 PRO expression in yeast

The following procedure describes the expression PRO recombinant in yeast.

First, the expression vectors of Yeast are constructed for intracellular production or secretion of PRO from the ADH2 / GAPDH promoter. The DNA encoding PRO and the promoter are inserted into sites for restriction enzymes suitable in the plasmid selected to direct expression intracellular PRO. For secretion, the DNA encoding PRO it can be cloned into the selected plasmid, along with the DNA that encodes the ADH2 / GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a sequence signal / secretory leader of the alpha factor or yeast invertase, and linker sequences (if needed) for the expression of PRO.

Yeast cells, such as the strain AB110 of the yeast, can then be transformed with the expression plasmids described above and grown in fermentation media selected. Yeast supernatants transformed can be analyzed by acid precipitation 10% trichloroacetic acid and a separation by SDS-PAGE, followed by staining the gels with Coomassie blue.

The recombinant PRO can be isolated subsequently and purified by extracting the cells of yeast from the fermentation medium by centrifugation and, to then the concentration of the medium using filters specific cartridge The concentrate containing PRO can be further purified using chromatography resins in Selected column

Many of the PRO polypeptides described herein are expressed satisfactorily as described previously.

Example 9 Expression of PRO in infected insect cells of Baculovirus

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

The sequence encoding PRO is merged into 5 'direction of an epitope tag contained in a vector of baculovirus expression. Such epitope tags include poly-His tags and labels immunoglobulin (such as Fc regions of IgG). One can be used variety of plasmids, including plasmids derived from commercially available plasmids, such as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired part of the PRO coding sequence, such as the sequence encoding the extracellular domain of a transmembrane protein or sequence which encodes the mature protein if the protein is extracellular, it PCR amplifies with primers complementary to the regions 5 'and 3'. The 5 'primer can incorporate sites for enzymes from flanking constraint (selected). The product is digested to then with these restriction enzymes selected and you subclone in the expression vector.

Recombinant baculovirus is generated by co-transfection of the anterior plasmid and BaculoGold ™ virus (Pharmingen) DNA in Spodoptera frugiperda ("Sf9") (ATCC CRL 1711) cells using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses are collected and used for further amplifications. Viral infection and protein expression are performed as described in O'Reilley et. al ., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

The expressed poly-His labeled PROs can then be purified, for example, by Ni2 + -chelate affinity chromatography as indicated below. The extracts are prepared from the Sf9 recombinant cells infected with the virus as described by Rupert et. al., Nature , 362 : 175-179 (1993). Briefly, the Sf9 cells are washed, resuspended in the sonication buffer (25 ml of HEPES, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; NP- 40 to 0.1%; 0.4 M KCl), and are sonicated twice for 20 seconds on ice. The sonicates are purified by centrifugation, and the supernatant is diluted 50 times in the loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0 filter , 45 µm. A Ni2 + -NTA agarose column (commercially available from Qiagen) with a bed volume of 5 ml is prepared, washed with 25 ml of water and equilibrated with 25 ml of the loading buffer. The filtered cell extract is loaded into the column at 0.5 ml per minute. The column is washed to the baseline at A 280 with the loading buffer, at which point the fraction collection begins. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes the specifically unbound protein. After reaching the baseline at A 280 again, the column develops with a gradient of 0 to 500 mM imidazole in the secondary wash buffer. One ml fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2 + -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the PRO labeled with eluted His 10 are grouped and dialyzed against the loading buffer.

Alternatively, the purification of the PRO labeling with IgG (or with Fc) can be done using techniques of known chromatography, including, for example, chromatography on column with Protein A or protein G.

Many of the PRO polypeptides described herein are expressed satisfactorily as described previously.

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Example 10 Preparation of antibodies that bind to PRO

This example illustrates the preparation of monoclonal antibodies that can specifically bind to PRO.

Techniques for producing monoclonal antibodies are known in the sector and are described, for example, in Coding, supra . Immunogens that can be used include purified PRO, fusion proteins containing PRO and cells expressing recombinant PRO on the cell surface. Immunogen selection can be performed according to the person skilled in the art without extensive experimentation.

Mice, such as Balb / c, are immunized with PRO immunogen emulsified in Freund's complete adjuvant and it is injected subcutaneously or intraperitoneally in an amount of 1-100 micrograms. Alternatively, the immunogen it is emulsified in the MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the bases of the hind legs of the animal. Then the mice immunized are reinforced 10 to 12 days later with immunogen additional emulsified in the selected adjuvant. Then, for several weeks, mice can also be reinforced with additional immunization injections. Serum samples they can be obtained periodically from the mice by samples of retro-orbital blood to be analyzed in ELISA assays to detect antibodies anti-PRO.

After detecting an antibody titer suitable, "positive" animals for antibodies are given You can inject a final intravenous injection of PRO. From three to four days later, the mice are sacrificed and the Spleen cells Next, the spleen cells fuse (using 35% polyethylene glycol) to a myeloma cell line selected murine, such as P3X63AgU.1, available from ATCC, No.  CRL 1597. Fusions generate hybridoma cells that can be place next in 96 tissue culture plates wells containing a HAT medium (hypoxanthine, aminopterin and thymidine) to inhibit the proliferation of non-fused cells, myeloma hybrids and spleen cell hybrids.

The hybridoma cells will be screened in a ELISA for reactivity against PRO. Cell determination of "positive" hybridomas that secrete antibodies desired monoclonal against PRO is within the technique.

Positive hybridoma cells can be inject intraperitoneally into Balb / c syngeneic mice to produce ascites fluids containing antibodies monoclonal anti-PRO. Alternatively, the hybridoma cells can develop in flasks or bottles in tissue culture roller. The purification of monoclonal antibodies produced in ascites fluids are can be performed using precipitation with ammonium sulfate, followed by gel exclusion chromatography. Alternatively, you can affinity chromatography based on the binding of the antibody to protein A or protein G.

Example 11 Purification of PRO polypeptides using Antibodies Specific

The native or recombinant PRO polypeptides are they can purify by a variety of standard techniques in protein purification techniques. For example, the pro-PRO polypeptide, mature PRO polypeptide, or the pre-PRO polypeptide by chromatography of immunoaffinity using antibodies specific for the polypeptide PRO of interest. In general, a column of immunoaffinity by covalent antibody couplings PRO anti-polypeptide to a chromatography resin activated

Polyclonal immunoglobulins are prepared to from immune sera by precipitation with sulfate of ammonium or by purification in immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J). Likewise, the monoclonal antibodies are prepared from ascites fluid of mouse by precipitation with ammonium sulfate or Immobilized Protein A chromatography. Immunoglobulin partially purified covalently binds to a resin chromatographic, such as the SEPHAROSE? activated with CnBr (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked and the derived resin is washed according to Manufacturer's instructions.

Said immunoaffinity column is used in the purification of the PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of entire cell or a subcellular fraction obtained by differential centrifugation by adding detergent or by other procedures known in the art. Alternatively, the soluble PRO polypeptide containing a signal sequence could secrete in a useful amount in the medium in which the cells They grow

A preparation containing PRO polypeptide soluble is passed through the immunoaffinity column, and the column is lava under conditions that allow preferential absorbance of PRO polypeptide (eg, high ionic strength buffers in presence of detergent). Then the column is eluted in conditions that disrupt antibody / PRO polypeptide binding (for example, a low pH buffer, such as about a pH of 2-3, or a high concentration of chaotrope, such as urea or thiocyanate ion), and the PRO polypeptide is collected.

Example 12 Drug screening

The present invention is particularly useful. to screen compounds by using PRO polypeptides or binding fragments thereof in any of a set of drug screening techniques. The PRO polypeptide or fragment used in said test may be free in solution, fixed to a solid support, adsorbed on a cell surface or located intracellularly A drug screening procedure uses eukaryotic or prokaryotic host cells, which are transformed stably with recombinant nucleic acids that express the PRO polypeptide or fragment. Drugs are screened against those transformed cells in competitive binding assays. These cells, both viable and fixed, can be used to standard binding assays. You can measure, for example, the formation of complexes between the PRO polypeptide or a fragment and the agent That is being tested. Alternatively, you can examine the decrease in complex formation between the PRO polypeptide and your target cell or target receptors caused by the agent that Its testing.

Thus, the present invention provides screening procedures for drugs or any other agent that may affect a disease or disorder associated with the polypeptide PRO. These procedures comprise contacting said agent. with a PRO polypeptide or fragment thereof and assay (I) of the presence of a complex between the agent and the PRO polypeptide or a fragment, or (II) of the presence of a complex between the PRO polypeptide or fragment and cell, by procedures well known in the art. In said binding tests Competitive, the PRO polypeptide or fragment are normally marked. After a suitable incubation, the PRO polypeptide or a free fragment are separated from the bound form, and the amount of Free or uncomplexed marker is a measure of the ability of the specific agent to bind to the PRO polypeptide or to interfere with the PRO / cell polypeptide complex.

Another technique for drug screening provides high performance screening of compounds that have a suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published September 13, 1984. Briefly, a large number of compounds are synthesized different small test peptides on a solid substrate, such like plastic needles or some other surface. As I know Applies to a PRO polypeptide, the test peptide compounds are They react with PRO polypeptide and wash. The PRO polypeptide bound by methods well known in the technique. The purified PRO polypeptide can also be coated. directly on plates to use in screening techniques drugs mentioned above. In addition, they can be used non-neutralizing antibodies to capture the peptide e immobilize it on the solid support.

The present invention also contemplates the use of competitive drug screening assays in that neutralizing antibodies capable of binding to polypeptides PRO specifically compete with a test compound to join to PRO polypeptide or fragments thereof. In this way, they can antibodies used to detect the presence of any peptide that shares one or more antigenic determinants with the PRO polypeptide.

Example 13 Rational drug design

The objective of rational drug design is to produce structural analogs of biologically active polypeptides of interest (i.e., a PRO polypeptide) or small molecules with which they interact, for example, agonists, antagonists or inhibitors. Any of these examples can be used to create drugs that are more active or stable forms of the PRO polypeptide or that enhance or interfere with the function of the PRO polypeptide in vivo (cf. Hodgson, Bio / Technology , 9 : 19-21 (1991)) .

In one strategy, the three-dimensional structure of the PRO polypeptide, or of a PRO-inhibitor polypeptide complex, is determined by X-ray crystallography, by computer modeling or, more commonly, by a combination of the two strategies. The shape and loads of the PRO polypeptide should be established to elucidate the structure and to determine the active site or sites of the molecule. Less frequently, useful information regarding the structure of the PRO polypeptide could be obtained by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design analog molecules of the PRO polypeptide type or to identify effective inhibitors. Useful examples of rational drug design may include molecules that have improved activity or stability, as shown by Braxton and Wells, Biochemistry , 31 : 7796-7801 (1992) or that act as inhibitors, agonists or antagonists of native peptides as shown by Athauda et. al., J. Biochem ., 113 : 742-746 (1993).

It is also possible to isolate an antibody target specific, selected by a functional assay, such and as described above and then fix your crystal structure This strategy, in principle, produces a initial drug on which the drug design can be based later. It is possible to avoid a crystallography of the whole protein by generating antibodies anti-idiotypic (anti-ids) for a pharmacologically active functional antibody. As picture mirror of a mirror image, the site of attachment of the anti-ids is expected to be a receiver analog original. Then the anti-ids could be used to identify and isolate peptides from peptide banks produced chemically or biologically. Isolated peptides They would then act as the initial drug.

Due to the present invention, they can be manufacture sufficient quantities of the PRO polypeptide to perform analytical studies, such as in crystallography of X-rays. In addition, knowledge of the sequence of PRO polypeptide amino acids provided herein invention will provide a guide for users of techniques of computer modeling instead of or in addition to the X-ray crystallography.

Example 18 Differential expression distribution of normal tissue versus to tumor

Oligonucleotide probes were constructed at from some of the nucleotide sequences encoding the PRO polypeptide shown in the accompanying figures for its use in quantitative PCR amplification reactions. Probes of oligonucleotides were chosen to produce a fragment amplified approximately 200-600 pairs of bases from the 3 'end of its associated template in a reaction of standard PCR. The oligonucleotide probes were used in standard quantitative PCR amplification reactions with cDNA libraries isolated from different human tissue samples normal and human tumor and were analyzed by electrophoresis in agarose gel to obtain a quantitative determination of expression level of the nucleic acid encoding the polypeptide PRO in the various tumor and normal tissues analyzed. Be used β-actin as a control to ensure that equivalent amounts of nucleic acid were used in each reaction. The identification of the differential expression of acid nucleic encoding the PRO polypeptide in one or more tissues tumor compared to one or more normal tissues thereof tissue type makes the molecule diagnostic useful for the determination of the presence or absence of tumor in a subject suspected of owning a tumor, as well as therapeutically as a target for the treatment of a tumor in a subject that possesses said tumor. These trials provided the following results.

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Example 19 Identification of receptor / ligand interactions

In this assay, several polypeptides were analyzed PRO for the ability to join a panel of potential molecules receptors or ligands in order to identify the receptor / ligand interactions. The identification of a ligand for a known receptor, a receptor for a known ligand or a new receptor / ligand pair is useful for a set of indications, for example, addressing of bioactive molecules (bound to the ligand or receptor) to a cell known to express the receptor or ligand, the use of the receptor or ligand as a reagent to detect the presence of the ligand or receptor in a composition suspected of containing them, where the composition may comprise cells suspected of expressing the ligand or receptor,  modulation of growth or other biological activity or immunologic of a cell known to express or respond to ligand receptor, modulation of the immune response of cells or towards cells that express the receptor or ligand, allowing the preparation of agonists, antagonists and / or targeted antibodies against the receptor or ligand that will modulate growth or a biological or immunological activity of a cell that expresses the receptor or ligand, and several indications that will be apparent to The subject matter expert.

The test is performed as indicated by continuation. A PRO polypeptide of the present is expressed invention suspected of being a ligand for a receptor as a fusion protein that contains the Fc domain of a human IgG (an immunoadhesin). The receptor-ligand binding is detects allowing the interaction of the immunoadhesin polypeptide with cells (eg, Cos cells) that express the receptors PRO candidate polypeptide and immunoadhesin visualization bound with fluorescent reagents directed against the domain of Fc fusion and microscope examination. The cells that express Candidate receptors are produced by transfection transient, in parallel, of defined subgroups of a library of cDNA expression vectors encoding PRO polypeptides that They can act as receptor molecules. Then the cells  they are incubated for 1 hour in the presence of the immunoadhesin of PRO polypeptide under analysis for possible receptor binding. TO The cells are then washed and fixed with paraformaldehyde. The cells are then incubated with antibody conjugated to fluorescent directed against the Fc part of the immunoadhesin of the PRO polypeptide (for example, Fc antibody goat anti-human conjugated to FITC). TO then the cells are washed again and examined by microscope. A possible interaction is assessed by presence of fluorescent labeling of cells transfected with cDNA encoding a receptor or group of PRO polypeptide receptors particular and the absence of similar fluorescent labeling of cells similarly prepared that have been transfected with other cDNAs or cDNA groups. If a defined group of cDNA expression vectors by interaction with a PRO polypeptide immunoadhesin, cDNA samples individuals that comprise the group are analyzed individually (the group is "broken") to determine the specific cDNA that encodes a receptor capable of interacting with immunoadhesin of the PRO polypeptide.

In another embodiment of this test, it is left interact with a potential PRO polypeptide ligand tagged with epitope (for example with an 8 histidine tag "His") with a panel of potential receptor PRO polypeptide molecules that have been expressed as fusions with the human IgG Fc domain (immunoadhesins). After 1 hour of co-incubation with the PRO polypeptide tagged with epitope, candidate receptors each one is immunoprecipitated with protein A particles and the particles are washed. The potential interaction with the ligand is determined by Western blot analysis of the immunoprecipitated complexes with antibody directed towards the epitope tag. It is valued that an interaction takes place if observe a band of the anticipated molecular weight of the protein epitope tagged in western blot analysis with a candidate recipient, but it is not observed to occur with the others panel members of potential recipients.

Using these trials, they have been identified Here the following receptor / ligand interactions:

(one)

PRO10272 joins PRO5801.

(2)

PRO20110 binds to the human IL-17 receptor (Yao et al ., Cytokine 9 (11): 794-800 (1997); also designated here as PRO1) and to PRO20040.

(3)

PRO10096 joins PRO20233.

(4)

PRO19670 joins PRO1890.

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The previous written report is considered to be enough to allow a subject matter expert to perform the present invention The present invention is not limited by the deposited construction, since the deposited realization intends be a single illustration of certain aspects of the invention and any construction that is functionally equivalent will is within the scope of the present invention. The deposit of material of the present invention does not constitute an admission that the description contained herein is inadequate to allow embodiment of any aspect of the invention, including the better way of it, nor should it be interpreted as limiting the scope of the claims to the illustrations it represents. In fact, they will be apparent to those skilled in the art from from the above description various modifications of the invention in addition to those shown and described here and will be found in the scope of the appended claims

They are also described here:

1. A procedure to detect a polypeptide designated as A, B, C or D in a sample suspected of containing a polypeptide A, B, C or D, said said procedure contacting said sample with a polypeptide designated here as E, F, G, H or I and determine the formation of a conjugate of polypeptides A / E, B / F, B / G, C / H or D / I in said sample, where the formation of said conjugate is indicative of the presence of a polypeptide A, B, C or D in said sample and where A is a PRO1027 polypeptide, B is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide, F is a PRO1 polypeptide, G is a polypeptide PRO20040, H is a PRO20233 polypeptide and I is a polypeptide PRO1890.

2. Procedure according to paragraph 1, where said sample comprises cells suspected of expressing said polypeptide A, B, C or D.

3. Procedure according to paragraph 1, where said polypeptide E, F, G, H or I is labeled with a marker detectable

4. Procedure according to paragraph 1, where said Polypeptide E, F, G, H or I is attached to a solid support.

5. Procedure for detecting a polypeptide designated as E, F, G, H or I in a sample suspected of containing a polypeptide E, F, G, H or I, said method comprising contacting said sample with a polypeptide designated herein as A, B, C or D and determine the formation of a conjugate of A / E, B / F, B / G, C / H or D / I polypeptides in said sample, where the formation of said conjugate is indicative of the presence of a polypeptide A, B, C or D in said sample and where A is a polypeptide PRO10272, B is a polypeptide PRO20110, C is a polypeptide PRO10096, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide, F is a PRO1 polypeptide, G is a PRO20040 polypeptide, H is a PRO20233 polypeptide and I is a PRO1890 polypeptide.

6. Procedure according to paragraph 5, where said sample comprises cells suspected of expressing said polypeptide E, F, G, H or I.

7. Procedure according to paragraph 5, where said Polypeptide A, B, C or D is labeled with a detectable marker.

8. Procedure according to paragraph 5, where said Polypeptide A, B, C or D is attached to a solid support.

9. Procedure of binding a molecule bioactive to a cell that expresses a polypeptide designated as A, B, C or D, said procedure comprising contacting said cell with a polypeptide designated as E, F, G, H or I that is bound to said bioactive molecule and allow said polypeptides A, B, C or D and said polypeptides E, F, G, H or I bind to each other, thus joining said bioactive molecules with said cell, where A is a PRO10272 polypeptide, B is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide, F is a PRO1 polypeptide, G is a polypeptide PRO20040, H is a PRO20233 polypeptide and I is a polypeptide PRO1890.

10. Procedure according to paragraph 9, where said bioactive molecule is a toxin, a radiolabel or a antibody.

11. Procedure according to paragraph 9, where said bioactive molecule causes the death of said cell.

12. Procedure for binding a molecule bioactive to a cell that expresses a polypeptide designated as E, F, G, H or I, said procedure comprising contacting said cell with a polypeptide designated as A, B, C or D that is bound to said bioactive molecule and allow said polypeptides A, B, C or D and said polypeptides E, F, G, H or I bind to each other, thus joining said bioactive molecules with said cell, where A is a PRO10272 polypeptide, B is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide, F is a PRO1 polypeptide, G is a polypeptide PRO20040, H is a PRO20233 polypeptide and I is a polypeptide PRO1890.

13. Procedure according to paragraph 12, where said bioactive molecule is a toxin, a radiolabel or a antibody.

14. Procedure according to paragraph 12, where said bioactive molecule causes the death of said cell.

15. Modulation procedure of at least a biological activity of a cell that expresses a polypeptide designated as A, B, C or D, said procedure comprising putting said cell in contact with a polypeptide designated as E, F, G, H or I or an anti-A, B, C or polypeptide antibody D, whereby said polypeptide E, F, G, H or I or antibody anti-A, B, C or D polypeptide binds to said polypeptide A, B, C or D, thus modulating at least one activity biological of said cell, where A is a PRO10272 polypeptide, B is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide, F is a PRO1 polypeptide, G is a PRO20040 polypeptide, H is a polypeptide PRO20233 and I is a PRO1890 polypeptide.

16. Procedure according to paragraph 15, where Kill that cell.

17. Modulation procedure of at least a biological activity of a cell that expresses a polypeptide designated as E, F, G, H or I, said procedure comprising contacting said cell with a polypeptide designated as A, B, C or D or an anti-E, F, G polypeptide antibody, H or I, whereby said polypeptide A, B, C or D or antibody anti-E, F, G, H or I polypeptide binds to said polypeptide E, F, G, H or I, thus modulating at least one biological activity of said cell, where A is a PRO10272 polypeptide, B is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide, F is a PRO1 polypeptide, G is a PRO20040 polypeptide, H is a polypeptide PRO20233 and I is a PRO1890 polypeptide.

18. Procedure according to paragraph 17, where Kill that cell.

Indication

The content of this application must not be construed as limited to the claims presented, It includes all the material described in the application.

The application maintains the right to submit modifications and / or divisional requests from it, aimed at any subject matter described herein regardless of whether the matter is in the claims of the present application as presented, and regardless of the final destination of any request for European patent from which this application is divided.

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References cited in the description

This list of references cited by the applicant is intended solely to help the reader and not It is part of the European patent document. Although it has been put maximum care in its realization, errors cannot be excluded or omissions and the EPO declines any responsibility to respect.

Patent documents cited in the description

US 5536637 A [0003]

WO 9104753 A [0132]

EP 404097 A [0070]

WO 9010448 A [0133]

WO 9311161 A [0070]

US 4736866 A [0138]

US 4275149 A [0074]

US 4870009 A [0138]

US 5364934 A [0080]

US 4657760 A [0150]

WO 8705330 A [0092]

US 5206344 A [0150]

US 4640835 A [0094]

US 5225212 A [0150]

US 4496689 A [0094]

WO 9703692 A [0151]

US 4301144 A [0094]

WO 9640072 A [0151]

US 4670417 A [0094]

WO 9607399 A [0151]

US 4791192 A [0094]

US 5654010 A [0151]

US 4179337 A [0094]

WO 9733551 A [0165]

US 5428130 A [0097]

WO 9733551 PCT [0166]

WO 8905859 A [0105]

US 4816567 A [0177] [0181]

US 4399216 A [0105]

US 5545807 A [0182]

DD 266710 [0106]

US 55458o6 A [0182]

US 4946783 A [0106]

US 5569825 A [0182]

EP 139383 A [0107]

US 5625126 A [0182]

US 4943529 A [0107]

US 5533435 A [0182]

EP 402226 A [0107]

US 5661016 A [0182]

EP 183070 A [0107]

WO 9308829 A [0185]

EP 244234 A [0107]

WO 9627011 A [0187]

EP 394538 A [0107]

US 4676980 A [0192]

WO 9100357 A [0107]

WO 9100360 A [0192]

US 5010182 A [0110]

WO 92200373 A [0192]

EP 362179 A [0110]

EP 03089 A [0192]

WO 9013646 A [0110]

WO 9411026 A [01%]

EP 36776 A [0114]

US 4485045 A [0198]

EP 73657 A t01161

US 4544545 A [0198]

GB 2211504 A [0117]

US 5013556 A [0? 98]

EP 11060 A [0120]

US 3773919 A [0204]

EP 117058 A [0120]

EP 307247 A [0248]

WO 9106629 A [0129]

US 5122469 A [025J8]

WO 9010048 A [0130]

WO 8403554 A [0285]

WO 9013641 A [0131]

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Claims (20)

1. Procedure to detect the presence of a tumor in a mammal, said procedure comprising the comparison of the expression level of a gene that encodes a PRO1865 polypeptide in (a) a sample of cell analysis of esophageal tissue or skin obtained from said mammal and (b) in a control sample of known normal tissue cells thereof cell type, in which a higher or lower level of the expression of said gene encoding a PRO1865 polypeptide in said Esophageal or skin tissue analysis sample compared to the control sample is indicative of the presence of tumor of esophagus or melanoma, respectively, in said mammal, in which PRO1865 polypeptide is a polypeptide that has at least one 80% identity in the amino acid sequence with: (a) the amino acid sequence shown in the SEQ ID No. 132; (b) the amino acid sequence encoded by the full length coding sequence of the deposited DNA under the access number ATCC 203543; (c) the amino acid sequence of the polypeptide shown in SEQ ID No. 132, which lacks its signal peptide associated; (d) the amino acid sequence of a domain extracellular polypeptide shown in SEQ ID No. 132, with its associated signal peptide; or (e) the amino acid sequence of a domain extracellular polypeptide shown in SEQ ID No. 132, which lacks its signal peptide, and in which a superior expression is indicative of the presence of an esophageal tumor or melanoma. 2. Method according to claim 1, in The level of identity is at least 85%. 3. Method according to claim 1, in The level of identity is at least 90%. 4. Method according to claim 1, in The level of identity is at least 95%. 5. Method according to claim 1, in The level of identity is at least 99%. 6. Method according to claim 1, in which the polypeptide comprises: (a) the amino acid sequence shown in the SEQ ID No. 132; (b) the amino acid sequence encoded by the full length coding sequence of the deposited DNA under the access number ATCC 203543; (c) the amino acid sequence of the polypeptide shown in SEQ ID No. 132, which lacks its signal peptide associated; (d) the amino acid sequence of a domain extracellular polypeptide shown in SEQ ID No. 132, with its associated signal peptide; or (e) the amino acid sequence of a domain extracellular polypeptide shown in SEQ ID No. 132, which It lacks its signal peptide. 7. Procedure to detect the presence of a tumor in a mammal, said procedure comprising the comparison of the level of expression of a PRO1865 polypeptide in (a) a sample of esophageal or skin tissue cell analysis obtained from said mammal and (b) in a control sample of known normal tissue cells of the same type of cells, in the that a higher or lower level of the expression of said PRO1865 polypeptide in said tissue analysis sample of esophagus or skin compared to the control sample is indicative of the presence of esophageal tumor or melanoma, respectively, in said mammal, in which the polypeptide PRO1865 is as defined in any of the claims 1 to 6 and in which a superior expression is indicative of the presence of an esophageal tumor or melanoma. 8. Method according to claim 7, in wherein said PRO1865 polypeptide is detected using an antibody  which specifically binds to a PRO1865 polypeptide as it is defined in any one of claims 1 to 6. 9. Method according to claim 8, in which the antibody is detectably labeled.

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10. Method according to claim 8, in which the antibody is a monoclonal antibody, an antibody humanized or an antibody fragment. 11. Method according to claim 10, in which the antibody fragment is selected from the group that it consists of the Fv, Fab, Fab 'and F (ab') 2 fragments. 12. Procedure to detect the presence of a tumor in a mammal, said method comprising (a) contacting an antibody that specifically binds to a PRO1865 polypeptide with a sample of cell analysis esophageal tissue or skin obtained from the mammal with a sample of control of known normal tissue cells of the same type of cells, and (b) detect the formation of a complex between said antibody and said polypeptide in the test sample and the control sample, in which a greater or lesser amount of complexes formed in said tissue analysis sample of esophagus or skin compared to the control sample is indicative of the presence of esophageal tumor or melanoma, respectively, in the mammal, in which the PRO1865 polypeptide it is as defined in any one of claims 1 to 6 and in which a greater number of complexes is indicative of the presence of an esophageal tumor or melanoma. 13. Procedure to detect the presence of tumor in a mammal, said procedure comprising the comparison of the expression level of a nucleic acid encoding PRO1865 in (a) a sample of tissue cell analysis of esophagus or skin obtained from said mammal, and (b) in a sample of control of known normal tissue cells of the same type of cells, in which a higher or lower level of expression of expression of said nucleic acid encoding PRO1865 in said Esophageal or skin tissue analysis sample compared to the control sample is indicative of the presence of tumor of esophagus or melanoma, respectively, in said mammal, in which the nucleic acid encoding PRO1865 is a nucleic acid that It has at least 80% identity in the acid sequence nucleic with: (a) a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID No. 132; (b) the nucleotide sequence shown in the SEQ ID No. 131; (c) the length coding sequence complete of the nucleotide sequence shown in SEQ ID No. 131; (d) the length coding sequence complete DNA deposited under the ATCC access number 203543; (e) a nucleotide sequence encoding the polypeptide shown in SEQ ID No. 132, which lacks its peptide associated signal; (f) a nucleotide sequence encoding the extracellular domain of the polypeptide shown in SEQ ID No. 132, with its associated signal peptide; or (g) a nucleotide sequence encoding the extracellular domain of the polypeptide shown in SEQ ID No. 132, which lacks its associated signal peptide, and in which the superior expression is indicative of the presence of an esophageal tumor or melanoma. 14. Method according to claim 13, in The level of identity is at least 85%. 15. Method according to claim 13, in The level of identity is at least 90%. 16. Method according to claim 13, in The level of identity is at least 95%. 17. Method according to claim 13, in The level of identity is at least 99%. 18. Method according to claim 13, in which the nucleic acid comprises: (a) a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID No. 132; (b) the nucleotide sequence shown in the SEQ ID No. 131; (c) the length coding sequence complete of the nucleotide sequence shown in SEQ ID No. 131; (d) the length coding sequence complete DNA deposited under the ATCC access number 203543; (e) a nucleotide sequence encoding the polypeptide shown in SEQ ID No. 132, which lacks its peptide associated signal; (f) a nucleotide sequence encoding the extracellular domain of the polypeptide shown in SEQ ID No. 132, with its associated signal peptide; or (g) a nucleotide sequence encoding the extracellular domain of the polypeptide shown in SEQ ID No. 132, It lacks its associated signal peptide. 19. Procedure according to any of the claims 13 to 18, wherein said nucleic acid which encodes PRO1865 is detected using an acid probe nucleic acids that hybridize specifically with said nucleic acid encoding PRO1865. 20. Method according to claim 19, in which the nucleic acid probe is labeled so detectable