ES2327785T3 - PROCEDURES AND COMPOUNDS TO INHIBIT THE GROWTH OF NEOPLASTIC CELLS. - Google Patents

Abstract

The present invention concerns methods and compositions for inhibiting neoplastic cell growth. In particular, the present invention concerns antitumor compositions and methods for the treatment of tumors. The invention further concerns screening methods for identifying growth inhibitory, e.g., antitumor compounds. In addition, the present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

Procedures and compounds to inhibit the Neoplastic cell growth.

Field of the Invention

The present invention relates to procedures and compounds to inhibit cell growth neoplastic In particular, the present invention relates to antitumor compounds and procedures for the treatment of tumors The invention further relates to screening procedures to identify growth inhibitor compounds, by example antitumor compounds.

Background of the invention

Malignant tumors (cancers) constitute the second leading cause of mortality in the United States, after heart disease (Boring et al., CA Cancel J. Clin ., 43 ; 7 (1993)).

Cancer is characterized by increased number of anomalous, or neoplastic, cells from a tissue normal that proliferate to form a tumor mass, the invasion of contiguous tissues by these neoplastic tumor cells and the generation of malignant cells that eventually spread through from the circulatory system or the lymphatic system to the nodules regional lymphatic and remote sites (metastases). In a cancerous situation, a cell proliferates in conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, which are characterized by a different degree of invasive capacity and aggressiveness.

Despite recent advances in the cancer treatment, it is very necessary to find new agents Therapeutics capable of inhibiting cell growth neoplastic Therefore, it is the objective of the present invention identify compounds capable of inhibiting growth of neoplastic cells, such as cancer cells.

Summary of the Invention A. Accomplishments

The invention relates to means for Tumor treatment, including cancers, such as breast cancer, prostate, colon, lung, ovary, kidney and system cancers central nervous (CNS), leukemia, melanoma, etc., in mammals, preferably human.

In one aspect, the present invention relates to to compositions of matter for use in the inhibition of neoplastic cell growth comprising an effective amount of a PRO224 polypeptide as defined herein, mixed with a pharmaceutically acceptable carrier. As determined in the claims, in a preferred embodiment, the composition of matter comprises a growth inhibitory amount of a PRO224 polypeptide. In another preferred embodiment, the composition It comprises a cytotoxic amount of a PRO224 polypeptide. Optionally, the subject compositions may contain one or more additional and / or cytotoxic growth inhibiting agents and / or other chemotherapeutic agents.

In another aspect, the invention relates to subject compositions useful for the treatment of a tumor in a mammal comprising a therapeutically effective amount of a PRO224 polypeptide as defined in the claims. He Tumor is preferably a cancer.

In another aspect, the invention relates to an in vitro method for inhibiting the growth of a tumor cell comprising exposing a cell to an effective amount of a PRO224 polypeptide as defined herein, or a medical use of said polypeptide. in such an in vivo procedure, as defined in the claims. Here we describe an article of manufacture comprising:

to)

a container;

b)

a composition comprising an active agent contained within the container, in which the composition is effective to inhibit neoplastic cell growth, e.g. eg the tumor cell growth, and the active agent in the Composition is a PRO224 polypeptide as defined herein, or an agonist thereof; Y

C)

a label attached to said container, or a leaflet included in said container referring to the use  of said PRO224 polypeptide or agonist thereof, for the inhibition of neoplastic cell growth, in which the agonist can be an antibody that binds to the PRO224 polypeptide. The agonist can be an anti-PRO224, an antibody agonist, or a small molecule that emulates the biological activity of a PRO224 polypeptide. Articles of similar manufacturing comprising a PRO224 polypeptide as defined herein, or an agonist thereof in an amount that It is therapeutically effective for the treatment of the tumor. Too articles of manufacture comprising a polypeptide are described PRO224 as defined herein, or an agonist thereof, and other growth inhibitor, cytotoxic agent or agent Chemotherapeutic

B. Additional Embodiments

In embodiments of the present invention, the invention refers to an isolated nucleic acid molecule that comprises a nucleotide sequence encoding a polypeptide PRO224.

In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% amino acid sequence identity, preferably at least about 81% sequence identity, more preferably at least approximately a sequence identity of 82%, still more preferably at least approximately a sequence identity of 83%, still more preferably at least approximately a sequence identity of 84%, still more preferably at least approximately a sequence identity of 85%, still more preferably at least about a sequence identity of 86%, still more preferably at least about a sequence identity of 87%, still more preferably at least about a sequence identity of 88%, still more preferably at least about one identity d and sequence of 89%, still more preferably at least about a sequence identity of 90%, still more preferably at least about a sequence identity of 91%, still more preferably at least about a sequence identity of 92% , still more preferably at least about a sequence identity of 93%, still more preferably at least about a sequence identity of 94%, still more preferably at least about a sequence identity of 95%, still more preferably by at least about a sequence identity of 96%, still more preferably at least about a sequence identity of 97%, still more preferably at least about a sequence identity of 98% even more preferably at least about a identity of 99% sequence to (a) a DNA molecule that encodes a PRO224 polypeptide with a full length amino acid sequence as described herein, an amino acid sequence that is missing the signal peptide, an extracellular domain of a transmembrane protein, with or without the signal peptide, as herein describe or any other specifically defined fragment of the full length amino acid sequence as described herein, or (b) the complement of the molecule
of DNA from (a).

In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence with at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about one identity of sequence of 82%, even more preferably at least about a sequence identity of 83%, even more preferably at least about a sequence identity of 84%, even more preferably at least about a sequence identity of 85%, even more preferably at least about a sequence identity of 86%, even more preferably at least about a sequence identity of 87%, even more preferably at least about a sequence identity of 88%, even more preferably at least minus about 89% sequence identity, even more preferably at least about a sequence identity of 90%, even more preferably at least about a sequence identity of 91%, even more preferably at least about a sequence identity of 92%, even more preferably at least about a sequence identity of 93%, even more preferably at least about a sequence identity of 94%, even more preferably at least about a sequence identity of 95%, even more preferably at least about a sequence identity of 96%, even more preferably at least about a sequence identity of 97%, even more preferably at least about a sequence identity of 98%, even more preferably at least about a sequence identity of 99% at ( a) a DNA molecule comprising the coding sequence of a full length PRO224 DNA polypeptide as described herein, the coding sequence of a PRO224 polypeptide lacking signal peptides as described herein, the coding sequence of an extracellular domain of a transmembrane PRO224 polypeptide, with or without the peptide signal, as described herein or the coding sequence of any other specifically defined fragment of the amino acid sequence throughout its length as described herein or (b) the complement of the molecule of
DNA of (a).

In another aspect, the invention relates to a isolated nucleic acid molecule comprising a sequence of nucleotides with at least about one identity of 80% sequence, preferably at least about one 81% sequence identity, more preferably at least approximately 82% sequence identity, even more preferably at least about one identity of 83% sequence, even more preferably at least approximately 84% sequence identity, even more preferably at least about one identity of 85% sequence, even more preferably at least approximately a sequence identity of 86%, even more preferably at least about one identity of 87% sequence, even more preferably at least approximately a sequence identity of 88%, even more preferably at least about one identity of 89% sequence, even more preferably at least approximately a sequence identity of 90%, even more preferably at least about one identity of 91% sequence, even more preferably at least approximately a sequence identity of 92%, even more preferably at least about one identity of 93% sequence, even more preferably at least approximately 94% sequence identity, even more preferably at least about one identity of 95% sequence, even more preferably at least approximately 96% sequence identity, even more preferably at least about one identity of 97% sequence, even more preferably at least approximately 98% sequence identity, even more preferably at least about one identity of 99% sequence to (a) a DNA molecule that encodes the same mature polypeptide encoded by any human protein cDNA deposited with the ATCC as described herein, or (b) the complement of the DNA molecule of (a).

Another aspect of the invention relates to a isolated nucleic acid molecule comprising a sequence of nucleotides encoding a PRO224 polypeptide that is either transmembrane domain deleted or domain deleted, or is complementary to such a coding nucleotide sequence, in which the transmembrane domain (s) of such polypeptide are describe (n) herein. Therefore, the soluble extracellular domains of PRO224 polypeptides here described.

It is described that fragments of a PRO224 polypeptide, of a coding sequence, or complement thereof, can find a use, such as hybridization probes, to encode fragments of a PRO224 polypeptide that can optionally encode a polypeptide comprising a site. binding for a PRO224 antibody or as antisense oligonucleotide probes. Such nucleic acid fragments normally have a length of at least 20 nucleotides, preferably a length of at least 30 nucleotides, more preferably a length of at least 40 nucleotides, even more preferably a length of at least 50 nucleotides, including more preferably a length of at least 60 nucleotides, even more preferably a length of at least 70 nucleotides, even more preferably a length of at least 80 nucleotides, even more preferably a length of at least 90 nucleotides, even more preferably a length of at least 100 nucleotides, even more preferably a length of at least 110 nucleotides, even more preferably a length of at least 120 nucleotides, even more preferably a length of at least 130 nucleotides, even more preferably a length of at least 140 nucleotides, even more pr preferably a length of at least 150 nucleotides, even more preferably a length of at least 160 nucleotides, even more preferably a length of at least 170 nucleotides, even more preferably a length of at least 180 nucleotides, even more preferably a length of at least 190 nucleotides, even more preferably a length of at least 200 nucleotides, even more preferably a length of at least 250 nucleotides, even more preferably a length of at least 300 nucleotides, even more preferably a length of at least 350 nucleotides, even more preferably a length of at least 400 nucleotides, even more preferably a length of at least 450 nucleotides, even more preferably a length of at least 500 nucleotides, even more preferably a length of at least minus 600 nucleotides, even more preferably e a length of at least 700 nucleotides, even more preferably a length of at least 800 nucleotides, even more preferably a length of at least 900 nucleotides, even more preferably a length of at least 1000 nucleotides, in which in in this context the term "approximately" indicates the length of the nucleotide sequence referenced plus or minus 10% of said referenced length. It is indicated that new fragments of the nucleotide sequence of a PRO224 polypeptide encoding aligning the nucleotide sequence of the PRO224 polypeptide encoding other known nucleotide sequences can be determined routinely using any of a number of known sequence alignment programs and determine which nucleotide sequence fragment (s) of the PRO224 polypeptide it encodes is new. All such nucleotide sequences of the PRO224 polypeptide that it encodes are contemplated herein. Also contemplated are PRO224 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO224 polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO224 polypeptide fragments comprising a binding site for a
anti-PRO224 antibody.

In another embodiment, the invention relates to an isolated PRO224 polypeptide encoded by one of the sequences of nucleic acid identified below.

In a certain aspect, the invention relates to an isolated PRO224 polypeptide, which comprises an amino acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably by at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about one identity of sequence of 85%, even more preferably at least about a sequence identity of 86%, even more preferably at least about a sequence identity of 87%, even more preferably at least about a sequence identity of 88% , even more preferably at least about one identity d and sequence of 89%, even more preferably at least about a sequence identity of 90%, even more preferably at least about a sequence identity of 91%, even more preferably at least about a sequence identity of 92% , even more preferably at least about a sequence identity of 93%, even more preferably at least about a sequence identity of 94%, even more preferably at least about a sequence identity of 95%, even more preferably by at least about a sequence identity of 96%, even more preferably at least about a sequence identity of 97%, even more preferably at least about a sequence identity of 98%, even more preferably at least about one identity 99% sequence to a PRO224 polypeptide with a sec A complete amino acid sequence as described herein, an amino acid sequence 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 specifically defined fragment of the complete amino acid sequence as described
at the moment.

In another aspect, the invention relates to an isolated PRO224 polypeptide, comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably by at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about one identity of sequence of 85%, even more preferably at least about a sequence identity of 86%, even more preferably at least about a sequence identity of 87%, even more preferably at least about a sequence identity of 88% , even more preferably at least about one identity of sequence of 89%, even more preferably at least about a sequence identity of 90%, even more preferably at least about a sequence identity of 91%, even more preferably at least about a sequence identity of 92%, even more preferably at least about a sequence identity of 93%, even more preferably at least about a sequence identity of 94%, even more preferably at least about a sequence identity of 95%, even more preferably at least less about a sequence identity of 96%, even more preferably at least about a sequence identity of 97%, even more preferably at least about a sequence identity of 98%, even more preferably at least about one identity of 99% sequence to an encoded amino acid sequence by any of the human protein cDNA deposited with the ATCC as described in
the present.

In another aspect, the invention relates to a PRO224 polypeptide comprising an amino acid sequence that score at least about 81% positive, preferably at least about 81% positive, more preferably at least about 82% of positive, even more preferably at least about 82% positive, even more preferably at least approximately 83% positive, even more preferably at least about 84% positive, even more preferably at least about 85% positive, even more preferably at least about 86% of positive, even more preferably at least about 87% positive, even more preferably at least approximately 88% positive, even more preferably at least about 89% positive, even more preferably at least about 90% positive, even more preferably at least about 91% of positive, even more preferably at least about 92% positive, even more preferably at least approximately 93% positive, even more preferably by at least about 94% positive, even more preferably at least about 95% positive, even more preferably at least about 96% of positive, even more preferably at least about 97% positive, even more preferably at least approximately 98% positive, even more preferably at least about 99% positive when compared with the amino acid sequence of a PRO224 polypeptide with a complete amino acid sequence as described herein, an amino acid sequence that is missing the signal peptide as it describes herein, an extracellular domain of a protein transmembrane, with or without the signal peptide, as described in the present or any other specifically defined fragment of the complete amino acid sequence as described in the Present.

In a specific aspect, the invention is refers to a PRO224 polypeptide isolated without the signal sequence N-terminal and / or methionine inhibitor and is encoded by a nucleotide sequence encoding such a sequence of amino acids as described above. Are also described processes to produce it, in which these processes comprise culturing a host cell comprising a vector that comprises the appropriate nucleic acid molecule that encodes under suitable conditions for the expression of the PRO224 polypeptide and recover PRO224 polypeptide from cell culture.

Another aspect of the invention relates to a PRO224 polypeptide isolated which is either transmembrane domain deleted or inactivated transmembrane domain. The processes for produce the same are also described herein, in which those processes include cultivating a host cell that comprises a vector comprising the appropriate acid molecule nucleic encoding under conditions suitable for expression of the PRO224 polypeptide and recover the PR0024 polypeptide from the culture mobile.

Agonists of a native PR0024 polypeptide as defined herein. He agonist can be an anti-PR0024, an antibody agonist or a small molecule.

A method for identifying agonists to a PR0024 polypeptide comprising contacting the PR0024 polypeptide with a candidate molecule and controlling a biological activity mediated by said PR0024 polypeptide is also described herein. Preferably, the PR0024 polypeptide is a PR0024 polypeptide.
native.

In yet another embodiment, the invention is refers to a composition of matter comprising a polypeptide PR0024 as described herein, together with a carrier for use as defined in the claims. Optionally, the Carrier is a pharmaceutically acceptable carrier.

Another embodiment of the present invention is aimed at the use of a PR0024 polypeptide for preparation of a medicine useful in the treatment of a condition that is sensitive to PR0024 polypeptide, as defined in The claims.

Vectors comprising DNA encoding any of the described polypeptides are also described herein. Host cells comprising any such vector are also described. By way of example, the host cells may be CHO, E. Coli ., Yeast or insect cells infected by baculovirus. A process to reduce any of the polypeptides described herein is also 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 described comprising any of the polypeptides described herein fused to a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules comprise any of the polypeptides described herein fused to a sequence of tag epitopes or an Fc zone of an immunoglobulin.

An antibody that binds is also described. specifically to any of the polypeptides described above or later. Optionally, the antibody is an antibody monoclonal, humanized antibody, antibody fragment or single chain antibody.

Also described herein are probes of oligonucleotides useful for isolating nucleotide sequences from CDNA and genomic or as antisense probes, in which such tests can be derived from any of the nucleotide sequences described above or later.

Brief description of the drawings

Figure 11 shows the nucleotide sequence (SEQ ID NO: 24) of a cDNA containing a nucleotide sequence encoding a natural sequence PRO224, in which the sequence of Nucleotides (SEQ ID NO: 24) is a clone designated herein. as DNA33221-1133. It is also presented in bold and underlined the positions of the start and end codons.

Figure 12 shows the amino acid sequence (SEQ ID NO: 25) of a natural PRO224 polypeptide sequence that It is derived from the coding sequence of SEQ ID Nº: 24 shown in figure 11.

Detailed description of the invention

When the term polypeptide is used or "PRO224" protein sequence PRO224 polypeptides are included natural and variants of PRO224 (which are also determined in the Present). The PRO224 polypeptide can be isolated from a variety of sources, such as types of human tissues or from from another source, or prepared by procedures recombination and / or synthesis.

A "natural sequence PRO224" comprises a polypeptide having the same amino acid sequence as the naturally occurring PRO224 polypeptide. Such a natural sequence PRO224 polypeptide can be isolated from nature or can be produced by recombination and / or synthesis means. The term "natural sequence PRO224" specifically includes truncated or secreted forms of natural occurrence (eg, an extracellular domain sequence), variant forms of natural occurrence (eg, spliced forms alternately) and allelic variants of natural occurrence of the PRO224 polypeptide . In one embodiment of the invention, the natural sequence PRO224 polypeptide is a mature or full length natural sequence PRO224 polypeptide as shown in Figure 12 (SEQ ID NO: 25). Also, although the PRO224 polypeptide referred to in Figure 12 (SEQ ID NO: 25) begins with the methionine residue designated therein as amino acid position 1, it is conceivable and possible to use another well located methionine residue. above or below position 1 of the amino acid of Figure 12 (SEQ ID NO: 25) as amino acid residue of the polypeptide initiator
PRO224.

The "extracellular domain" or "ECD" of a polypeptide referred to herein refers to a form of the polypeptide that essentially lacks the transmembrane and cytoplasmic domains. Generally, an ECD of a polypeptide will have less than about 1% of such transmembrane and / or cytoplasmic domains and, preferably, will have less than about 0.5% of such domains. It will be understood that any domain, or domains, transmembrane identified for the polypeptides of the present invention is identified according to the criteria used systematically in the art to identify that type of hydrophobic domain. The exact limits of a transmembrane domain may vary but most likely they will not do so in more than about 5 amino acids at each end of the domain, as initially identified and as shown in the accompanying figures. As such, in one embodiment of the present invention, the extracellular domain of a polypeptide of the present invention comprises 1 to X amino acids of the mature amino acid sequence, wherein X is any amino acid of the 5 amino acids on each side of the boundary. extracellular domain / domain
transmembrane

The approximate location of the "signal peptide" of the various PRO polypeptides referred to herein are shown in the accompanying figures. However, it is noted that the limit of the C-terminal end of a signal peptide may vary, but most likely it does not do so by more than about 5 amino acids on each side of the C-terminal limit of the signal peptide, as initially identified herein, in which the limit of the C-terminal end of the signal peptide can be identified according to the criteria used systematically in the art to identify that type of amino acid sequence element (eg, Nielsen and others, Prot. Eng ., 10 : 1-6 (1997) and von Heinje et al., Nucl. Acids. Res ., 14 : 4,683-4,690 (1986)). In addition, it has also been observed that, in some cases, the cleavage of a signal sequence from a secreted polypeptide is not completely uniform, whereby more than one secreted species is produced. These mature polypeptides, in which the signal peptide is cleaved into no more than about 5 amino acids on each side of the C-terminal end of the signal peptide, as identified herein, and the polynucleotides encoding them, are contemplated. by the present invention.

The "PRO224 variant polypeptide" makes reference to an active PRO224 polypeptide (other than a PRO224 natural sequence polypeptide) as defined subsequently, which has at least about one 80% amino acid sequence identity with the sequence of amino acids of: (a) of 1 or about 31 to 282 residues of the PRO224 polypeptide shown in Figure 12 (SEQ ID NO: 25), (b) of X to 282 residues of the PRO224 polypeptide shown in Figure 2 (SEQ ID NO: 25), (c) 1 or about 31 to X of Figure 12 (SEQ ID NO: 25), in which X is any amino acid from amino acid 226 to amino acid 235 of Figure 12 (SEQ ID NO: 25), or (d) another fragment specifically from the sequence of amino acids shown in figure 12 (SEQ ID NO: 25).

Such PRO224 variants include, for example, PRO224 polypeptides in which one or more amino acid residues are added, or removed, at the N or C terminal ends, so as within one or more internal domains of the sequence natural.

Generally, a PRO224 variant will have at least about an amino acid sequence identity of 80%, more preferably an amino acid sequence identity of at least 81%, more preferably an amino acid sequence identity of at least at least 82%, more preferably an amino acid sequence identity of at least 83%, more preferably an amino acid sequence identity of at least 84%, more preferably an amino acid sequence identity of at at least 85%, more preferably an amino acid sequence identity of at least 86%, more preferably an amino acid sequence identity of at least 87%, more preferably an amino acid sequence identity of at least 88%, more preferably an amino acid sequence identity of at least 89%, more preferably a sequence identity of a minoacids of at least 90%, more preferably an amino acid sequence identity of at least 91%, more preferably an amino acid sequence identity of at least 92%, more preferably a sequence identity of amino acids of at least 93%, more preferably an amino acid sequence identity of at least 94%, more preferably an amino acid sequence identity of at least 95%, more preferably an identity of the amino acid sequence of at least 96%, more preferably an amino acid sequence identity of at least 97%, more preferably an amino acid sequence identity of at least 98% and even more preferably an identity of the amino acid sequence of at least 99% with: (a) residues 1 or about 31 to 282 of the PRO224 polypeptide shown in Figure 12 (SEQ ID NO: 25), (b) X to 282 residues of the PRO224 polypeptide shown in Figure 12 (SEQ ID NO: 25), (c) 1 or about 31 to X of FIGURE 12 (SEQ ID NO: 25), wherein X is any amino acid of amno acid 226 to amino acid 235 of Figure 12 (SEQ ID NO: 25), or (d) another fragment specifically from the amino acid sequence shown in Figure 12
(SEQ ID NO: 25).

PRO224 variant polypeptides do not include the natural sequence of the PRO224 polypeptide. Usually, PRO224 variant polypeptides are at least one length of 10 amino acids, often at least 20 amino acids in length, more often at least 30 amino acids in length, more often at least about 40 amino acids in length, more often at least 50 amino acids in length, more often at least 60 amino acids in length, more often at least 70 amino acids in length, more often at least 80 amino acids in length, more often at least 90 amino acids in length, more often at least about 100 amino acids in length, more often at least 150 amino acids in length, more often at least 200 amino acids in length, more often at least 250 amino acids in length, more often at least 300 amino acids in length, or more.

As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison software. This source code can be systematically collected for use in a UNIX operating system that provides the sequence comparison software.
ALIGN-2

In addition, tables 2A-2D show hypothetical examples to use the procedure described below to determine the identity of the amino acid sequence in% (tables 2A-2B) and the identity of the nucleic acid sequence in% (tables 2C-2D) using the ALIGN-2 sequence comparison software, in which "PRO" represents the amino acid sequence of a hypothetical PRO224 polypeptide of interest; "comparison protein" represents the amino acid sequence of a polypeptide with which the "PRO" polypeptide of interest is compared; "PRO-DNA" represents a hypothetical PRO224 that encodes a nucleic acid sequence of interest; "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule with which the "PRO-DNA" nucleic acid molecule of interest is compared; "X", "Y" and "Z" each represent different hypothetical amino acid residues; and "N", "L" and "V" each represent different nucleotides
hypothetical

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TABLE 1

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one

2

3

4

5

6

7

8

9

10

eleven

12

13

14

fifteen

16

17

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TABLE 2A

100

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TABLE 2B

101

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TABLE 2C

102

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2D TABLE

104

The "percentage (%) of identity of the amino acid sequence "in terms of the sequences of the PRO224 polypeptide identified herein is defined as the percentage of amino acid residues of a sequence of choice which are identical to the amino acid residues of a sequence PRO224, after aligning the sequences and entering spaces, if it is necessary, to achieve the maximum percentage of identity of the sequences, and without considering any conservative substitution as part of the sequence identity. Alignment in order to determine the percent identity of the amino acid sequence it can be achieved in several ways that are part of the state of the technique, for example, using a computer program available such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). The experts in the matter can determine the appropriate parameters to determine alignment, including any algorithm necessary to achieve maximum alignment along the entire length of the sequences that are compared. For the purposes of this, no However, the values in% of the sequence identity of amino acids are obtained as explained below using the sequence comparison software ALIGN-2, in which the complete source code for the ALIGN-2 program is provided in table 1. The sequence comparison software ALIGN-2 is owned by Genentech, Inc., and the code source represented in table 1 has been presented together with the User documentation in the U.S. Copyright Office, Washington D.C., 20559, where he has registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is available at Genentech, Inc., South San Francisco, California, or can be compiled from the source code provided in the Table 1. The ALIGN-2 program should be compiled to its use in a UNIX operating system, preferably a system UNIX V4.0D digital. All comparison parameters of sequences are set by the ALIGN-2 program And they don't vary.

For the purposes of this, the% of amino acid sequence identity of a sequence determined from amino acids A with, or against, a sequence of certain amino acids B (which can be expressed alternatively as a given amino acid sequence that has or comprises a certain% identity of the amino acid sequence with, or against, a given amino acid sequence B) is calculated as follows:

100 times the fraction X / Y

where X is the number of residues of amino acids considered identical pairings by the program of alignment of ALIGN-2 sequences in that alignment of A and B of the program, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of the amino acid sequence B, the% identity between the sequences of amino acids of A and B will not be equal to% identity between amino acid sequences of B and A. As examples of% calculations of amino acid sequence identity, tables 2A-2B show how to calculate% identity of the amino acid sequence between the amino acid sequence designated "comparison protein" and the sequence of designated amino acids "PRO".

Unless specifically stated otherwise, all% identity values of the amino acid sequence used herein have been obtained as described above using the ALIGN-2 sequence comparison software. However, the% identity of the amino acid sequence can also be determined using the NCBI-BLAST2 sequence comparison program (Altschul et al., Nucleic Acids Res ., 25 : 3,389-3,402 (1997)). The NCBI-BLAST2 sequence comparison program can also be downloaded at http: //www.ncbi. nlm.nih.gov. The NCSI-BLAST2 uses various search parameters, in which all search parameters have been set as default values, including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity lenght = 15/5, multi-passe-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.

In situations where the NCBI-BLAST2 to compare amino acid sequences, % identity of the amino acid sequence of a sequence of determined amino acids A with, or against, a sequence of determined B amino acids (which can be expressed alternatively as a given amino acid sequence that has or comprises a certain% identity of the amino acid sequence with, or against, a certain sequence of amino acids B) is calculated as follows:

100 times the fraction X / Y

where X the number of residues of amino acids considered with identical matching by the program NCBI-BLAST2 sequence alignment in that alignment of A and B of the program, and where Y is the total number of amino acid residues in B. It will be appreciated that where the amino acid sequence length A is not equal to the length of the amino acid sequence B, the% identity between amino acid sequences of A and B will not be equal to% identity between the amino acid sequences of B and TO.

In addition, the% identity of the amino acid sequence can also be determined using the WU-BLAST-2 software (Altschul et al., Methods in Enzymology , 266 : 460-480 (1996)). Most of the WU-BLAST-2 search parameters have been set as default values. Those that have not been set as default values, that is, as adjustable parameters, have been set with the following values:

overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11 and scoring matrix = BLOSUM62. For the purposes of this, a value of% identity of the sequence of Amino acids are determined by dividing: (a) the number of residues of amino acids with identical matching between the sequence of PRO polypeptide amino acids of interest from the natural PRO polypeptide and amino acid sequence of interest of comparison (i.e. the sequence against which it has been compared the PRO polypeptide of interest, which may be a variant of the PRO polypeptide), as determined in WU-BLAST-2, for (b) the total number of amino acid residues of the PRO polypeptide of interest. By example, in the statement: "a polypeptide comprising a amino acid sequence A that has at least 80% of amino acid sequence identity with the sequence of amino acids B ", the amino acid sequence A is the sequence of comparison amino acids of interest and amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.

The "PRO224 variant polypeptide" or "nucleic acid sequence of the PRO224 variant" makes reference to a nucleic acid molecule that encodes a PRO224 polypeptide as defined below and which has so less about a nucleic acid sequence identity 80% with either (a) a nucleic acid sequence that encodes residues 1 or about 31 to 282 of the PRO224 polypeptide shown in Figure 12 (SEQ ID No. 25), (b) acid sequence nucleic encoding amino acids X to 282 of the PRO224 polypeptide shown in Figure 12 (SEQ ID No. 25), in which X is any amino acid residue 26 to 35 of Figure 12 (SEQ ID No. 25), (c) 1 or about 31 to X of Figure 12 (SEQ ID No. 25), in the that X is any amino acid from amino acid 226 to amino acid 235 of Figure 12 (SEQ ID No. 25), or (d) an acid sequence nucleic encoding another fragment specifically derived from the amino acid sequence shown in Figure 12 (SEQ ID No. 25). Generally, a PRO224 variant polypeptide will therefore have less about an acid sequence identity 80% nucleic, more preferably a sequence identity of nucleic acid of at least 81%, more preferably a nucleic acid sequence identity of at least 82%, more preferably a nucleic acid sequence identity of at least 83%, more preferably an identity of the nucleic acid sequence of at least 84%, plus preferably an identity of the nucleic acid sequence of at least 85%, more preferably an identity of the nucleic acid sequence of at least 86%, plus preferably an identity of the nucleic acid sequence of at least 87%, more preferably an identity of the nucleic acid sequence of at least 88%, plus preferably an identity of the nucleic acid sequence of at least 89%, more preferably an identity of the nucleic acid sequence of at least 90%, plus preferably an identity of the nucleic acid sequence of at least 91%, more preferably an identity of the nucleic acid sequence of at least 92%, plus preferably an identity of the nucleic acid sequence of at least 93%, more preferably an identity of the nucleic acid sequence of at least 94%, plus preferably an identity of the nucleic acid sequence of at least 95%, more preferably an identity of the nucleic acid sequence of at least 96%, plus preferably an identity of the nucleic acid sequence of at least 97%, more preferably an identity of the nucleic acid sequence of at least 98% and even more preferably an identity of the nucleic acid sequence of at least 99% with (a) a nucleic acid sequence that encodes residues 1 or about 31 to 282 of the polypeptide PRO224 shown in Figure 12 (SEQ ID No. 25), (b) a sequence of nucleic acid encoding amino acids X to 282 of the polypeptide PRO224 shown in Figure 12 (SEQ ID No. 25), in which X is any amino acid residue from 26 to 35 of Figure 12 (SEQ ID No.: 25), (c) 1 or about 31 of X of Figure 12 (SEQ ID No. 25), or (d) a nucleic acid sequence encoding another fragment specifically derived from the amino acid sequence shown in Figure 12 (SEQ ID No.: 25). The variants of PRO224 polypeptide does not include the nucleotide sequence PRO224.

Generally, the polypeptides of the variant PRO224 are at least 30 amino acids in length, often at least about 60 amino acids in length, more often than by at least about 90 amino acids in length, more often than minus about 120 amino acids in length, more often at least about 150 amino acids in length, more often at least about 180 amino acids in length, more often at least 210 amino acids in length, more often at least 240 amino acids in length, more often at least about 270 amino acids in length, more often at least 300 amino acids in length, more often at least 450 amino acids in length, more often at least 600 amino acids in length, more often at least 900 amino acids in length, or more.

The "percentage (%) of identity of the nucleic acid sequence "in terms of acid sequences  PRO224 polypeptide nucleic encoding identified in the present is defined as the percentage of nucleotides in a sequence of choice that are identical to the nucleotides of a PRO224 polypeptide nucleic acid sequence encoding, after aligning the sequences and entering spaces, if it is necessary, to achieve the maximum percentage of identity of the sequences The alignment in order to determine the percentage of nucleic acid sequence identity can be achieved from several ways that are part of the state of the art, by example, using an available computer program such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters to determine alignment, including any algorithm necessary to achieve maximum alignment to along the entire length of the sequences that are compared. For the purposes of this, however, the values in% of the identity of the nucleic acid sequence are obtained as explained later using the computer program of ALIGN-2 sequence comparison, in which the Full source code for the ALIGN-2 program provided in table 1. The comparison software for ALIGN-2 sequences is owned by Genentech, Inc., and the source code represented in table 1 has been presented along with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where he has registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is is available at Genentech, Inc., South San Francisco, California, or it can be compiled from the source code provided in table 1. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably a UNIX V4.0D digital system. All parameters Sequence comparison are set by the program ALIGN-2 and do not vary.

For the purposes of this, the% of nucleic acid sequence identity of a sequence determined from nucleic acid C with, or against, a sequence of certain amino acids D (which can be expressed alternatively as a given C nucleic acid sequence that has or comprises a certain% identity of the amino acid sequence with, or against, a given amino acid sequence D) is calculated as follows:

100 times the fraction W / z

where W is the number of nucleotides considered identical pairings by the alignment program of ALIGN-2 sequences in that alignment of C and D of the program, and where Z is the total number of nucleotides in B. As examples of calculations of the% identity of the sequence of nucleic acid, tables 2C-2D show how calculate% identity of nucleic acid sequence between the nucleic acid sequence designated "comparison DNA" and the designated amino acid sequence "PRO-DNA"

Unless specifically stated otherwise, all% identity values of the nucleic acid sequence used herein have been obtained as described above using the ALIGN-2 sequence comparison software. However, the% identity of the nucleic acid sequence can also be determined using the NCBI-BLAST2 sequence comparison program (Altschul et al., Nucleic Acids Res ., 25 : 3,389-3,402 (1997)). The NCBI-BLAST2 sequence comparison program can also be downloaded at http: //www.ncbi. nlm.nih.gov. The NCSI-BLAST2 uses various search parameters, in which all search parameters have been set as default values, including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity lenght = 15/5, multi-passe-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.

In situations where the NCBI-BLAST2 to compare sequences,% of nucleic acid sequence identity of a sequence of certain amino acids C with, or against, an acid sequence determined D nucleic (which can alternatively be expressed as a given C nucleic acid sequence that has or comprises certain% identity of the nucleic acid sequence with, or versus, a certain sequence of nucleic acid B) is calculated as follows:

100 times the fraction W / z

where W the number of nucleotides considered as identical pairing by the program of NCBI-BLAST2 sequence alignment in that alignment of C and D of the program, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, the% identity between C and D nucleic acid sequences will not be equal to% of identity between the nucleic acid sequences of D and C.

In addition, the% of the identity values of the nucleic acid sequence can also be determined using the WU-BLAST-2 software (Altschul et al., Methods in Enzymology , 266 : 460-480 (1996)). Most of the WU-BLAST-2 search parameters have been set as default values. Those that have not been set as default values, that is, as adjustable parameters, have been set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11 and scoring matrix = BLOSUM62. For the purposes of the present, a% identity value of the nucleic acid sequence is determined by dividing: (a) the number of nucleotides with identical matching between the nucleic acid sequence of the nucleic acid molecule of interest of the PRO polypeptide which encodes with a sequence derived from the new PRO polypeptide nucleic acid sequence encoding and the nucleic acid sequence of comparative interest (i.e., the sequence against which the PRO polypeptide nucleic acid molecule encoding has been compared , which may be a variant of the PRO polynucleotide), as determined in WU-BLAST-2, by (b) the total number of nucleotides of the nucleic acid molecule of interest of the PRO polypeptide encoding. For example, in the statement "an isolated nucleic acid molecule comprising an amino acid sequence A having at least 80% identity of the nucleic acid sequence with the nucleic acid sequence B", the nucleic acid sequence A is the nucleic acid sequence of comparative interest and the nucleic acid sequence B is the nucleic acid sequence of the nucleic acid molecule of interest of the PRO polypeptide encoding.

In other embodiments, polynucleotides of the PRO224 variant are nucleic acid molecules that encode a PRO224 polypeptide active, respectively, and which are capable of hybridize, preferably under stringent conditions of hybridization and washing, to nucleotide sequences encoding the full length nucleotide sequences that encode the full length PRO224 polypeptide shown in Figure 12 (SEQ  ID No. 25), polypeptides of the PRO224 variant may be those encoded by a polynucleotide of the PRO224 variant.

The term "positive", in the context of amino acid sequence identity comparisons made as described above, includes residues of amino acid in the compared sequences that are not only identical, but those that also have similar properties. Residues of amino acid that get a positive value to an amino acid residue of interest are those that are identical to the amino acid residue of interest or are a preferred substitution (as defined in the table 3 below) of the amino acid residue of interest.

For the purposes of this, the percentage of the positive value of an amino acid sequence determined A, a, with, or against an amino acid sequence determined B (which can be expressed alternatively as a determined amino acid sequence A that has or comprises a certain percentage of positives to, with, or against a sequence of determined amino acid B) is calculated as follows:

100 times the fraction of X / Y

where X is the number of residues of amino acids that achieve a positive value as defined previously using the sequence alignment program ALIGN-2 of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A is not equal to the length of the amino acid sequence B, the% positive from A to B will not be the same % positive from B to TO.

"Isolated", when used to describe the various polypeptides referred to herein, means that the polypeptide has been identified and separated and / or recovered from a component of its natural environment. Preferably, the isolated polypeptide lacks association with all the components with which it is naturally associated. The contaminating components of their natural environment are materials that would usually interfere with the diagnosis or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (I) to a degree sufficient to obtain at least 15 residues of an internal amino acid sequence or of the N-terminus by using a rotary cup sequencer, or (2) until homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. The isolated polypeptide includes a polypeptide in situ within recombinant cells, since at least one component of the natural environment of PRO224 will be missing. Generally, however, the preparation of the isolated polypeptide will require at least one purification step.

An "isolated" nucleic acid molecule encoding a PRO224 polypeptide or a nucleic acid molecule "isolated" encoding an antibody anti-PRO224 is a nucleic acid molecule that identifies and separates from at least one acid molecule contaminating nucleic with which it is generally associated in the natural source of the PRO224 nucleic acid encoding or a anti-PRO224 nucleic acid molecule that encodes Preferably, the isolated nucleic acid lacks association with all the components with which it is naturally associated. An isolated nucleic acid molecule of PRO224 encoding or a nucleic acid molecule of anti-PRO224 that encodes is different in terms of shape or fit in nature. So, isolated nucleic acid molecules are distinguished from PRO224 nucleic acid molecule encoding or of the anti-PRO224 nucleic acid molecules that encodes, as it exists in natural cells. However, a isolated nucleic acid molecule encoding a polypeptide PRO224 or an isolated nucleic acid molecule that encodes a anti-PRO224 antibody includes acid molecules PRO224 nucleic or nucleic acid molecules of anti-PRO224 contained in cells that express generally PRO224 polypeptides or antibodies anti-PRO224 in which, for example, the molecule of nucleic acid is in a different chromosomal location than the of natural cells.

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

The nucleic acid is "operably linked" when in a functional relationship with another acid sequence nucleic. For example, DNA for a secretory leader or Presence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide; a promoter or enhancer is operatively at a coding sequence if it affects the transcription of the sequence; or a ribosome binding site operatively binds to a coding sequence if positioned to facilitate the translation. Generally, "operably linked" means that the DNA sequences that bind are contiguous and, in the case of a secretory leader, continuous and in reading phase. However, the Enhancers do not have to be contiguous. The union is achieved ligand in convenient restriction sites. If such sites do not there are synthetic oligonucleotide adapters or linkers They are used according to conventional practice.

The term "antibody" is used in its broader sense and specifically covers for example single anti-PRO224 monoclonal antibodies (including agonist antibodies), antibody compounds anti-PRO224 with polyepitopic specificity, of single chain, anti-PRO224 antibodies, and fragments of anti-PRO224 antibodies (see below). The term "monoclonal antibody" as used in the present refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, all  population antibodies are identical except for possible naturally occurring mutations that may occur in smaller amounts

The "restriction" of hybridization reactions is easily determined by someone with a general knowledge of the art, and is generally an empirical calculation that depends on the length of the probe, the washing temperature and the salt concentration. In general, longer probes require higher temperatures for proper hybridization, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to rehybridize when complementary strains occur in an environment 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, higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less. For additional details and explanation of the stringency of hybridization reactions, see Ausubel et al. , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Rigorous conditions" or "conditions very rigorous, "as used herein, may be identified by those that: (1) use an ionic force low and a high temperature for washing, for example 0.015 M of sodium chloride / 0.0015 M sodium cotrate / 0.1% dodecyl sulfate sodium at 50 ° C; (2) use an agent during hybridization denaturing, such as formamide, for example, 50% (v / v) of formamide with 0.1% bovine albumin / 0.1% Flcol / 0.1% Polyvinylpyrrolidone / 50 nM sodium phosphate buffer with a pH of 6.5 with 750 mM sodium chloride, 15 mN citrate sodium at 42 ° C; or (3) use 50% formamide, 5 X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhard's solution, DNA of ultrasound salmon sperm (50 µg / ml), 0.1% SDS, and 10% sulfate dextract at 42 ° C, with washes at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a high consistency wash of 0.1 x SSC with EDTA at 55 ° C.

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

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The term " tagged epitope," when used herein, refers to a chimeric polypeptide comprising a PRO224 polypeptide fused to a " tag polypeptide." The tag polypeptide has enough residues to provide an epitope against which an antibody can be created, although it is short enough so that it does not interfere with the activity of the polypeptide to which it is fused. Preferably the tag polypeptide is also completely unique, such that the antibody does not exhibit substantial cross-reaction with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous protein (a "adhesin") with the effector functions of the domains immunoglobulin constants. Structurally, the immunoadhesins comprise a fusion between a sequence of amino acids with the desired binding specificity, which is different of the site of identification and binding of the antigen of a antibody (ie, it is "heterologous"), and a sequence of constant domains of immunoglobulin. The adhesin part of a immunoadhesin molecule is usually a sequence of contiguous amino acids comprising at least the binding site of a receptor or a ligand. In immunoadhesin, the sequence of constant domains of immunoglobulin can be obtained from of any immunoglobulin, such as subtypes of IgG-14, IgG-2, IgG-3, or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM

For the purposes of this, "active" or "activity" refers to a form or forms of PRO224 that conserve biological and / or immunological activity of PRO224 natural or natural appearance, in which activity "biological" refers to a biological function (well inhibitory or stimulator) caused by a natural or appearing PRO224 natural different from the ability to induce the production of a antibody against an antigenic epitope possessed by a PRO224 natural or naturally occurring and "immunological" activity is refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a natural PRO224 or from natural appearance

"Biological activity" in the context of a antibody or other agonist that can be identified by the screening analyzes described herein (for example, a organic or inorganic peptide of small molecule, etc.) is used to refer to the ability of such molecules to induce one or more of the effects listed herein together with the definition of a "therapeutically effective amount." In a specific embodiment, "biological activity" is the ability of inhibiting cell growth or proliferation neoplastic A preferred biological activity is inhibition, which includes the slowdown or complete arrest of the growth of a target tumor cell (for example, cancer). Other activity Preferred biological is the cytotoxic activity that causes death of the target tumor cell (for example, cancer). And yet another Preferred biological activity is the induction of apoptosis of a target tumor cell (for example, cancer).

The expression "immunological activity" means immunological cross reactivity with at least one epitope of a PRO224 polypeptide.

As used herein, "immunological cross reactivity" means that the polypeptide  of choice is able to competitively inhibit the activity qualitative biological of a PRO224 polypeptide that has this activity with polyclonal antisera cultivated against the known PRO224 active polypeptide. Such antisera are prepared in a manner conventional injecting goats or rabbits subcutaneously, by example, the known active analog in Freund's complete adjuvant, followed by an intraperitoneal or subcutaneous booster injection In incomplete Freund. The immunological cross reactivity is preferably "specific", which means that affinity of binding of the molecule with immunological cross reactivity identified (eg, antibody) with the PRO224 polypeptide corresponding is considerably higher (preferably by at least twice, more preferably at least four times, even more preferably at least six times, more preferably at least eight times higher) than the binding affinity of that molecule with any other known natural polypeptide.

As used herein, "tumor" refers to all cell growth or proliferation neoplastic, regardless of whether they are malignant or benign, and all precancerous and cancerous cells and tissues.

The terms "cancer" and "cancerous" are refer to, or describe, the physiological disease in mammals that It is usually characterized by unregulated cell growth. Examples of cancer include, but not exclusively, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers are breast cancer, prostate cancer, colon cancer, cell cancer squamous, small cell lung carcinoma, non-carcinoma microcytic lung, ovarian cancer, neck cancer uterine, gastrointestinal cancer, pancreatic cancer, the glioblastoma, liver cancer, urinary bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, cancer vulvar, thyroid cancer, liver carcinoma and various types of head and neck cancer.

"Treatment" is an intervention that is carried out with the intention of preventing the appearance of a disorder or alter its pathology. Therefore, the "treatment" refers to both therapeutic treatment and to prophylactic or preventive measures. Among those who require treatment includes those who already suffer from the disorder as well like those in whom the disorder has to be prevented. At treatment of a tumor (for example, cancer), an agent therapeutic can directly decrease cell pathology tumor, or make tumor cells more susceptible to treatment with other therapeutic agents, for example, radiation and / or chemotherapy.

The "pathology" of a cancer includes all the phenomena that affect the patient's well-being. This includes, without any limitation, abnormal cell growth or uncontrolled, metastasis, interference with the normal functioning of neighboring cells, the release of cytokines or other secretion products at abnormal concentrations, the suppression or aggravation of the inflammatory response or immunological, etc.

An "effective amount" of the polypeptide described herein or an agonist thereof, as to the growth inhibition of neoplastic cells, is an amount capable of inhibiting, to a greater or lesser extent, cell growth Diana. The term includes an amount capable of inducing an effect inhibitor, cytostatic and / or cytotoxic on growth and / or apoptosis of the target cells. It can be determined empirically and systematically an "effective amount" of a polypeptide PRO224 or an agonist thereof in order to inhibit the Neoplastic cell growth.

A "therapeutically effective amount", in as for the treatment of a tumor, it refers to a quantity capable of inducing one or more of the following effects: (1) inhibition, in lesser or greater degree of tumor growth, which includes, slowdown and complete growth arrest; (2) reduction of the number of tumor cells; (3) reduction of the size of the tumor; (4) inhibition (ie reduction, slowing down or complete arrest) of tumor cell infiltration into peripheral organs; (5) inhibition (i.e. reduction, slowdown or complete arrest) of metastasis; (6) stimulation of the antitumor immune response, which may, but not necessarily, lead to regression or rejection of tumor; and / or (7) relief, to a greater or lesser degree, of one or more of the symptoms associated with the disorder Can be determined empirically and systematically a "quantity therapeutically effective "of a PRO224 polypeptide or of a agonist thereof in order to inhibit the growth of neoplastic cells

A "growth inhibitory amount" of a PRO224 polypeptide or an agonist thereof is an amount capable of inhibiting the growth of a cell, especially a tumor, for example, cancer cells, either in vitro or in vivo . A "growth inhibitory amount" of a PRO224 polypeptide or an agonist thereof can be determined empirically and systematically in order to inhibit the growth of neoplastic cells.

A "cytotoxic amount" of a PRO224 polypeptide or an agonist thereof is an amount capable of causing the destruction of a cell, especially a tumor, for example, cancer cells, either in vitro or in vivo . A "cytotoxic amount" of a PRO224 polypeptide or an agonist thereof can be determined empirically and systematically in order to inhibit the growth of neoplastic cells.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (for example, 131 I, 125 I, 90 Y and 126 Re), agents chemotherapeutics, and toxins such as enzymatically toxins active of bacterial, fungal, plant or animal origin, or fragments thereof.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of a tumor, by example cancer. Examples of chemotherapeutic agents include include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, the cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, for example, paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorrelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, speramycin (see US Patent 4,675,187), the Melphalan and other related nitrogen mustards. The agents hormones that act to regulate or inhibit hormonal action on tumors, such as tamoxifen and onapristone.

When used herein, a "growth inhibitory agent" refers to a compound that inhibits cell growth, especially of a tumor, for example, of cancer cells, either in vitro or in vivo . Thus, the growth inhibiting agent considerably reduces the percentage of target cells in the S phase. Examples of growth inhibiting agents include agents that block cell cycle progression (at a time other than the S phase), such as Agents that induce G1 arrest and phase M arrest. Classic M-phase blockers include vincas (vincristine and vinblastine), taxol and topo II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin These agents that stop G1 also cause the arrest of the S phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. More information can be found in The Molecular Basis of Cancer , Mendelsohn and Israel, eds., Chapter I, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. . 13.

The term "cytokine" is a term generic for proteins released by a population of cells that they act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocines and hormones traditional polypeptides. Cytokines include growth hormones such as growth hormone human, the growth hormone N-methionyl human and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; the relaxin; the prorrelaxin; glycoprotein hormones, such as the hormone follicle stimulator (FSH), the hormone stimulating thyroid (TSH), and luteinizing hormone (LH); the factor of liver growth, fibroblast growth factor: the prolactin; the placental lactogen; necrosis factors tumor α and β; the mullerian inhibitor substance; he peptide associated with murine gonadotropin; the inhibin: the activin; vascular endothelial growth factor; the integrin; thrombopoietin (TPO); growth factors of nerves, such as NGF-?; factor of platelet growth; transforming growth factors (TGF), such as TGF-? And TGF-?; insulin growth factors I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon?,? and?; factors colony stimulants (CSF), such as macrophage CSF (M-CSF); CSF of granulocytic macrophages (GM-CSF); and CSF granulocytes (G-CSF); interleukins (IL), such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-? Or TNF-?; Y other polypeptide factors that include LIF and ligand kit (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture  and biologically active equivalents of sequence cytokines natural.

As used in this patent application, the term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that, compared to the original drug, is less cytotoxic for tumor cells and is capable of being enzymatically activated. or become the most active original form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions , 14, p. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery , Borchardt et al., (Eds.), P. 247-267. Humana Press (1985). The prodrugs of this invention include, but not exclusively, prodrugs containing phosphates, prodrugs containing thiophosphates, glycosylated prodrugs or prodrugs containing phenylacetamide which may optionally be substituted, 5-fluorocytosine prodrugs and other 5-fluorouridine prodrugs, which may derive In a prodrug form for use in the present invention, they include, but not exclusively, those chemotherapeutic agents described above.

The term "agonist" includes specifically agonist antibodies or antibody fragments, fragments or variants of the amino acid sequence of natural PRO224 polypeptides, peptides, organic molecules small, etc. The procedures for the identification of agonists of a PRO224 polypeptide may comprise establishing contact between a tumor cell and a possible agonist molecule, and Determine the inhibition of tumor cell growth.

The "chronic" administration refers to the continued administration of the agent or agents as opposed to a single dose, so that the therapeutic effect is maintained initial (activity) for a prolonged period of time. The "intermittent" administration is a treatment that is not performs successively without interruption; it is rather of cyclic nature

For the purposes of a treatment, "mammal" refers to any animal classified as a mammal, including humans, pets and of farm, as well as those of the zoo, those used in sports, or pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

The administration "in combination with" one or more therapeutic agents includes simultaneous administration (concurrent) and successive in any order.

As used herein, "carriers" includes pharmaceutically acceptable carriers, excipients or stabilizers, which are non-toxic to the cell or the mammal exposed to them at doses and concentrations employed Often, the physiologically acceptable carrier is a pH buffered aqueous solution. Among the examples of carriers Physiologically acceptable buffers such as phosphate are included, citrate and other organic acids; antioxidants like acid ascorbic; low molecular weight polypeptides (less than about 10 waste); 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 such as glucose, mannose, or dextrins; agents chelators such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; I nonionic surfactant such as TWEEN?, polyethylene glycol (PEG) and PLURONICS ™.

The "natural antibodies" and "natural immunoglobulins" are normally glycoproteins heterotetramerics of approximately 150,000 daltons, composed by two identical light chains (L) and two heavy chains (H) identical. Each light chain is attached to a heavy chain by a covalent disulfide bond, although the number of bonds disulfide varies between heavy chains of different isotypes of immunoglobulin Each of the heavy and light chains also it presents regularly spaced intracatenary disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by numerous constant domains. Each light chain presents a variable domain at one end (VL) and a domain constant at its other end; the constant domain of the chain lightweight is aligned with the first constant domain of the chain heavy, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that there is waste of specific amino acids that form an interface between domains Light and heavy chain variables.

The term "variable" refers to the fact that certain parts of the variable domains differ greatly in the sequence between the different antibodies and are used in the binding and specificity of each particular antibody in for its particular antigen. However, the variability is not evenly distributed across all the variable domains of the antibodies. It focuses on three segments called complementarity determining regions (CDR), or hypervariable regions, both in the variable domains of the light chain and in those of the heavy chain. The most highly conserved parts of the variable domains are called framework regions ( framework , FR). The variable domains of the natural heavy and light chains each comprise four FR regions, which broadly adopt a β-sheet configuration, connected by three CDRs, which form loops that connect the β-sheet structure and, in some cases, form part of it. The CDRs of each chain are held together very close to the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibodies (see, Kabat et al., NIH Publ, No. 91 -3242, Vol. 1 , pp. 647-669 (1991)). Constant domains do not directly intervene in the binding of an antibody to an antigen, but show several effector functions, such as the participation of the antibody in the antibody-dependent cellular toxicity.

When used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that is responsible for antigen binding. The hypervariable region comprises amino acid residues of a "complementarity determining region" or "CDR" (ie residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain of the light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD. [1991]) and / or those residues of a "hypervariable loop" (ie residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Clothia and Lesk, J. Mol. Biol., 196: 901-917 [1987]). Lattice residues ( "framework" or "FR") are those residues of variable domains other than residues of the hypervariable region as defined herein.

"Antibody fragments" comprises a portion of an intact antibody, preferably the variable or antigen-binding region of the intact antibody. Examples of antibody fragments are Fab, Fab ', F (ab') 2, and Fv fragments; "diabodies"; linear antibodies (Zapata et al., Protein Eng., 8 (10 ): 1,057-1,062); single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papainic antibody digestion produces two identical antigen binding fragments, called fragments "Fab", each with a single antigen binding site, and a residual fragment "Fc", designation that reflects the capacity to crystallize easily. Pepsin treatment produces a F (ab ') 2 fragment having two binding sites at antigen and is still able to form transverse bonds with the antigen.

"Fv" is the minimum antibody fragment which contains a complete identification and binding site to antigen. This region consists of a dimer of a variable domain of a heavy chain and a light chain in close association, not covalent It is in this configuration that the three CDRs of each domain variable interact with each other to determine a binding site to dimer surface antigen V_ {H} -V_ {L}. Together, the six CDRs confer antigen binding specificity to the antibody. Do not However, even a single variable domain (or half of an Fv which comprises only three specific CDRs for an antigen) has the ability to identify the antigen and bind to it, although with a lower affinity than at 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 at the end carboxylic domain CH1 of the heavy chain that includes one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for a Fab 'in which the cysteine residue (s) of the domains constants carry a free thiol group. Antibody fragments F (ab ') 2 were originally produced as pairs of Fab 'fragments joined by hinge cysteines. They also know each other other chemical couplings of antibody fragments.

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

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins They can be assigned to different classes. There are five classes main immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be subdivided into subclasses (isotypes), for example, IgG1. IBG2, IgG3, IgG4, IgA and IgA2.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, all antibodies in the population are identical except for the presence, in smaller amounts, of possible naturally occurring mutations. Monoclonal antibodies are very specific, being directed against a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations that usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinant. In addition to their specificity, one of the advantages of monoclonal antibodies is that they are synthesized by hybridoma culture, which is not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody that has been obtained from a substantially homogeneous population of antibodies, and its construction does not require antibody production by any particular procedure. For example, the monoclonal antibodies to be used according to the present invention can be synthesized by the first hybridoma procedure described by Kohler et al., Nature , 256 : 495 [1975], or can be synthesized by recombinant DNA procedures (see, for example , U.S. Patent 4,816,567). "Monoclonal antibodies" can also be isolated from bacteriophage antibody libraries using, for example, the techniques described.
in Clackson et al., Nature , 352 : 624-628 [1991] and Marks et al., J. Mol. Biol ., 222 : 581-597 (1991).

The monoclonal antibodies of the present invention specifically include "chimeric" antibodies (immunoglobulins) in which a part of the heavy and / or light chain is identical or homologous to corresponding sequences in the antibodies from a particular species or belonging to a class or particular subclass of antibody, while the rest of the chain or chains is identical or homologous to corresponding sequences in antibodies from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided they show the Desired biological activity (US Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA . 81 : 6,851-6,855 [1984]).

The "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-sequences of antibodies that bind to antigens) that contain a minimal sequence from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues from CDRs of the recipient are replaced by residues from CDRs of a non-human species (donor antibody) such as a mouse, rat or rabbit that has the specificity, affinity and capacity desired. In some cases, the Fv FR residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, humanized antibodies may comprise residues that are neither found in the recipient antibody nor in the imported CDR or framework sequences. These modifications are also made to improve and maximize antibody activity. In general, the humanized antibody will comprise substantially all of at least one, and usually two, of the variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all regions. FR are those of a sequence of a human immunoglobulin. Optimally, the humanized antibody will also comprise at least a part of a constant region of immunoglobulin (Fc), usually that of a human immunoglobulin. For more details, ice, Jones et al., Nature , 321 : 322-323 (1986); Reichmann et al., Nature , 332 : 323-329 [1988]; and Presta, Curr. Op. Struct. Biol 2 : 593-596 (1992). The humanized antibody includes a PRIMATIZED? Antibody in which the antigen binding region of the antibody is derived from an antibody produced by immunization of macaque monkeys with the antigen of interest.

Fv chain fragments single "or" Fv_ {s} "comprise antibody domains V_ {H} and V_ {L}, in which these domains are in a single polypeptide chain. Preferably, the Fv polypeptide it also comprises a polypeptide connector between the domains V_ {H} and V_ {L} that allows Fv_ {s} to form the structure desired for antigen binding. For an analysis of Fv_ {s}, see Pluckthun in: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, p. 269-315 (1994).

The term "dianticibodies" refers to small antibody fragments with two antigen binding sites, whose fragments comprise a heavy chain variable domain (V H) connected to a light chain variable domain (V L) ) in the same polypeptide chain (VH-VL). Using a connector that is too short to allow coupling between the two domains in the same chain, the domains have to be coupled with the complementary domains of another chain and create two antigen binding sites. Dianticibodies are described in greater detail in, for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl Acad. Sci. USA , 90 : 644-6448 (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 their natural environment are materials that could interfere with the diagnosis or therapeutic uses of the antibody, and among them may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified: (I) to more than 95% by weight of the antibody as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of an internal or N-terminal amino acid sequence by means of a rotating cup sequencer, or (3) up to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining . The isolated antibody includes the antibody in situ within recombinant cells since at least one component of the natural environment of the antibody will be missing. Generally, however, the preparation of the isolated antibody will require at least one purification step.

As used herein, the word "marker" refers to a detectable compound that conjugates directly or indirectly with the antibody to generate a "labeled" antibody. The marker can be detectable by itself same (for example, radioisotope markers or markers fluorescent) or, in the case of an enzyme marker, can catalyze the chemical alteration of a substrate compound that is detectable The marker can also be an undetectable entity such as a toxin.

"Solid phase" means a non-matrix aqueous to which the antibody of the present can adhere invention. Among the examples of solid phases included in the This includes those formed partly or completely by glass (e.g. controlled pore glass), by polysaccharides (for example, agarose), polyacrylamides, polystyrene, alcohol Poly and silicones. In some embodiments, depending on the context, the solid phase may comprise the well of a plate of analysis; in others it is a purification column (for example, an affinity chromatography column). That term it also includes a discontinuous solid phase of isolated particles, such as those described in US Pat. US 4,275,149.

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A "liposome" is a small vesicle composed of several types of lipids, phospholipids and / or surfactants  useful in administering a drug (such as a PRO224 polypeptide or an antibody thereof) to a mammal. The Liposome components are usually arranged in a bilayer, similar to the lipid arrangement of biological membranes.

A "small molecule" is defined in the present as the one with a molecular weight below approximately 500 daltons.

II. Compounds and methods of the invention A. PRO224 polypeptide throughout its length

The present invention provides sequences of newly identified and isolated nucleotides encoding polypeptides to which the present application has been made reference as PRO224. In particular, it has been identified and isolated PRO224 polypeptide encoding cDNAs, as described in more detail in the following examples.

As described in the following examples, the PRO224 polypeptide encoding cDNA clones has been presented together with the ATCC. The current nucleotide sequences of the clones can be easily determined by the expert in the matter sequencing the clones presented by systematic procedures in the state of the art. The Predicted amino acid sequences can be determined from nucleotide sequences using the usual techniques. In the case of the PRO224 polypeptide and the coding nucleic acid described herein, applicants have identified what is believed to represent the best identifiable reading frame with Sequence information available for now.

B. PRO224 variants

In addition to the sequence PRO224 polypeptide full natural described herein, the Preparation of PRO224 variants. PRO224 variants can be prepared by introducing appropriate modifications in nucleotides of PRO224 DNA, and / or by synthesis of the PRO224 polypeptide wanted. Those skilled in the art will appreciate that the amino acid modifications can alter processes post-translational PRO224 polypeptide, such as modifying the number or position of glycosylation sites or alter membrane anchor characteristics.

Variations can be made throughout the length of the natural sequence PRO224 or in several domains of the PRO224 described herein, for example, using any of conservative mutations techniques and instructions and not conservatives established above, for example, in the patent from the USA US 5,364,934. Variations can be a substitution, deletion or insertion of one or more codons encoding the PRO224 leading to a modification in the amino acid sequence of the PRO224 compared to the natural sequence PRO224. The variation can be carried out by replacing at least one amino acid by any other amino acid in one or more of the domains of PRO224. It can be determined which amino acid residue can be inserted, replaced or removed without influencing adverse in the desired activity comparing the sequence of PRO0320 with that of known homologous protein molecules and minimizing the number of modifications in the sequence of amino acids performed in regions of high homology. The amino acid substitutions may be the result of substituting an amino acid by another amino acid that has properties similar structural and / or chemical, such as the replacement of a leucine for a serine, that is, amino acid substitutions conservative Insertions or deletions can be carried out in the range of about 1 to 5 amino acids. The variation allowed can be determined by systematically performing insertions, deletions or substitutions of amino acids in the sequence and checking the resulting variants based on the activity manifested by the mature natural sequence or in all its length.

Fragments of the PRO224 polypeptide. Such fragments may be truncated in the terminal end N or the terminal end C, or may lack internal waste, for example, when compared to a protein Natural throughout its length. Some fragments lack waste of amino acids that are not essential for biological activity desired of the PRO224 polypeptide.

PRO224 fragments can be prepared by any of numerous conventional techniques. The fragments of Desired peptides can be chemically synthesized. A technique alternative involves the generation of PRO224 fragments by enzymatic digestion, for example, by treating the protein with a enzyme known to cleave proteins at sites determined by residues of particular amino acids, or by DNA digestion with appropriate restriction enzymes and isolating the fragment wanted. Yet another appropriate technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). The oligonucleotides that determine the ends of the DNA fragment they are used in the 5 'and 3' primers in the CPR. Preferably, PRO224 polypeptide fragments share at least one Biological and / or immunological activity with the PRO224 polypeptide natural shown in Figure 12 (SEQ ID NO: 25).

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In particular embodiments, they are shown conservative substitutions of interest in table 3 under the heading of preferred substitutions. If such substitutions produce a change in biological activity, then it introduce more substantial changes, called substitutions exemplars in table 3, or as also described subsequently in reference to amino acid classes, and are screened the products.

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TABLE 3

18

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Substantial modifications are made to the function or immunological identity of the PRO224 polypeptide selecting substitutions that differ markedly in their effect on the maintenance of: (a) the structure of the skeleton of the polypeptide in the area of substitution, for example, as conformation in sheet or propeller, (b) the load or hydrophobicity of the molecule at the target site, or (c) chain bulge side. Naturally occurring residues are divided into groups. depending on the properties of the usual side chain:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acids: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence the orientation of the chain: gly, pro; Y

(6) aromatic: trp, tyr, phe.

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Non-conservative substitutions will involve exchanging an element of one of these classes for another class. Such substituted residues can also be introduced at the sites. conservative replacement or, more preferably, at sites (not preserved) remaining.

Variations can be made using methods known in the state of the art, such as oligonucleotide-mediated (directed) mutagenesis, alanine scavenging and mutagenesis by PCR [Carter et al., Nucl. Acids Res . 13 : 4,331 (1986); Zoller and others, Nucl. Acids Res ., 10: 6,487 (1987)], cassette insertion mutagenesis [Wells et al., Gene , 34 : 315 (1995)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London Sera , 317 : 415 (1986)] or other known techniques can be carried out on the cloned DNA to produce the DNA of the PRO224 variant.

Scanning amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is usually a preferred scanning amino acid among this group because it eliminates the side chain beyond beta carbon and is less likely to alter the conformation of the variant's main chain [Cunningham and Wells, Science , 244 : 1.081-1.085 (1989 )]. Alanine is also usually preferred because it is the most frequent amino acid. In addition, he is often in both a hidden and exposed position [Creighton, The Proteins , (WH Freeman & Co., NY); Chothia, J. Mol. Biol ., 150 : 1 (1976)]. If the alanine substitution does not produce sufficient amounts of variant, an isostatic amino acid can be used.

C. Modifications of PRO224

The covalent modifications of PRO224 are included within the scope of this invention. A kind of covalent modification includes the reaction of residues of Targeted amino acids of a PRO224 polypeptide with an agent of organic bypass that is capable of reacting with side chains selected or with the residues of the terminal ends N or C of PRO224. Derivatization with bifunctional agents is useful, for example, for the formation of transversal links. PRO224 in a support matrix or water insoluble surface for use in the procedure to purify antibodies anti-PRO224, and vice versa. Between agents commonly used for the formation of cross links They include, for example: 1,1-bis (diazoacetyl) -2-phenylethane,  glutaraldehyde, N-hydroxysuccinimide esters, by example, esters with 4-salicylic acid, imidoesters homobifunctional, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), maleimides bifunctional such as bis-Nmaleimido-1,8-octane and agents such as methyl-3 - [(p-acidophenyl) dithio] Proprioimidate

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

Another type of covalent modification of PRO224 polypeptide included within the scope of this invention comprises the alteration of the natural glycosylation pattern of the polypeptide. For the purposes of this, by "alteration of the natural glycosylation pattern "means to eliminate one or more parts of carbohydrates found in the sequence PRO224 natural (either by removal of the underlying glycosylation site, or by elimination of glycosylation by chemical means and / or enzymatic), and / or adding one or more glycosylation sites that they do not exist in the natural sequence PRO224. In addition, the expression includes qualitative changes in protein glycosylation natural, which imply a change in nature and proportions of the various parts of carbohydrates present.

The addition of glycosylation sites in the PRO224 polypeptide can be carried out by altering The amino acid sequence. The alteration can be made, by example, by adding, or replacing with, one or more serine or threonine residues in the natural sequence PRO224 (for glycosylation sites linked to O). The sequence of PRO224 amino acids can optionally be altered through changes in the DNA, in particular by mutating the DNA that encodes the PRO224 polypeptide in preselected bases such that they are generated codons that will be transformed into the desired amino acids.

Other ways to increase the number of carbohydrate parts in the PRO224 polypeptide are by chemical or enzymatic coupling of glycosides to the polypeptide. Such procedures are described in the prior art, for example, in WO 87/05330 published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem . P. 259-306 (1981).

The removal of the carbohydrate parts present in the PRO224 polypeptide can be carried out chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as targets in glycosylation. Chemical deglycosylation techniques are known in the state of the art and have been described, for example, by Hakimuddin et al., Arch. Biochem. Biophys ., 259 : 52 (1987) and by Edge et al., Anal. Biochem ., 118: 131 (1981). Enzymatic cleavage of carbohydrate parts in polypeptides can be achieved by utilizing a variety of endo and exoglucosidases as described in Thotakura et al., Meth. Enzymol ., 138 : 350 (1987).

Another type of covalent modification of PRO224 comprises linking the PRO224 polypeptide with one of a variety of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes, in the established manner previously in US Pat. US 4,640,835; 4,496,689; 4,301,144; 4,610,411; 4,791,192 or 4,179,337.

The PRO224 polypeptide of the present invention It can also be modified so that it forms a molecule chimeric comprising the PRO224 fused with another polypeptide heterologous or with an amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of the PRO224 polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The tag epitope is generally located at the amino or carboxylic ends of the PRO224 polypeptide. The presence of such labeled forms (tagged) PRO224 polypeptide can be detected using an antibody against the tag polypeptide. Also, the provision of the tag epitope allows the PRO224 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 well known in the state of the art. Examples include the tags poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly); the influenza HA tag polypeptide and its 12CA5 antibody [Field et al., Mol. Cell Biol ., 8 ; 2,159-2,165 (1988)]; the c-myc tag and its antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 [Evan et al., Molecular and Cellular Biology , 5 : 3,610-3,616 (1985)]; and the glycoprotein D (gD) tag of the herpes simplex virus and its antibody [Paborsky et al., Protein Engineering , 3 (6): 547-553 (1990)]. Other tag polypeptides are the Flag [Hopp et al., BioTechnology , 6 : 1,204-1,210 (1988)] peptide; the KT3 epitope peptide (Martin et al., Science , 255 : 192-194 (1992)]; a tubulin-α epitope peptide [Skinner et al., J. Biol. Chem . 266 : 15.163-15.166 (1991)] and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA , 87 : 6,393-6,397 (1990)].

In an alternative embodiment, the molecule chimeric may comprise a fusion of the PRO224 polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (to which also referred to as "immunoadhesin"), such fusion could be the Fc region of an IgG molecule. Ig fusions include preferably substitution of a soluble form (domain transmembrane removed or inactivated) of a PRO224 polypeptide in instead of at least one variable region within a molecule of Ig. In a particularly preferred embodiment, the fusion of the immunoglobulins include the hinge, CH2 and CH3, or the hinge, the CH1, CH2 and CH3 regions of an IgG1 molecule. For production of immunoglobulin fusions see also US Pat. US 5,428,130 published June 27, 1995.

D. Preparation of PRO224

The description that follows refers mainly to the production of PRO224 by culturing cells transformed or transfected with a vector containing PRO224 nucleic acid. Obviously, the use of alternative procedures, well known in the state of the art, in the preparation of PRO224 is also contemplated. For example, the sequence of the PRO224 polypeptide, or parts thereof, can be produced by direct 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 : 2,149-2,154 (1963)]. Protein synthesis can be performed in vitro using manual techniques or by automation. Automated synthesis can be performed, for example, using a peptide synthesizer (Applied Biosystems Peptide Synthesizer; Foster City, CA) and following the manufacturer's instructions. Several parts of the PRO224 polypeptide can be chemically synthesized separately and then combined by chemical or enzymatic methods to produce the full length PRO224 polypeptide.

1. DNA isolation encoding PRO224

The DNA encoding PRO224 can be obtained at from a cDNA library prepared from a tissue that it is believed to contain the mRNA of PRO224 and to express it at a level detectable Therefore, DNA from human PRO224 can be obtained from a cDNA library prepared from tissue human, as described in the examples. The gene that encodes the PRO224 can also be obtained from a library genomic or by known synthetic procedures (for example, automated synthesis of nucleic acids).

Library screening can be performed with probes (such as PRO224 antibodies or oligonucleotides of at least about 20-80 bases) designed for the identity of the gene of interest or the protein encoded by it. Screening of cDNA or a genomic library with the selected probe can be performed using standard interventions, such as those 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 PRO224 is to use the CPR methodology [Sambrook et al., Supra ; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The examples offered below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be sufficiently long and not ambiguous in such a way that the appearance of false positives is minimized. The oligonucleotide is preferably labeled so that it can be detected in the hybridization of DNA in the library where it will be screened. Labeling procedures are well known in the art, and include the use of radiolabels as biotinylation of 32 P-labeled ATP or enzyme labeling. Hybridization conditions, including moderate restriction and high restriction, are provided in Sambrook et al., Supra .

The sequences identified in such Library screening procedures can be compared and aligned  with other known sequences presented and available in bases of public data such as GenBank or in other databases of private sequences The sequence identity (at the level of amino acids or nucleotides) within certain regions of the molecule or along the entire length of the sequence can determined using known procedures in the state of the technique and as described herein.

Nucleic acid with a sequence that encodes proteins can be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence referred to herein for the first time, and, if necessary, using methods of amplification of conventional primers as described in Sambrook et al., supra , to detect precursors and process mRNA intermediates that may not have undergone reverse transcription of cDNA.

2. Selection and transformation of host cells

Host cells are transfected or transformed with expression or cloning vectors described herein for the production of PRO224 and cultured in conventional nutrient media appropriately modified to induce promoters, select transformers, or amplify the genes encoding the desired sequences. Culture conditions, such as medium, temperature, pH and the like, can be selected by any person skilled in the art. In general, the principles, protocols and practical techniques to maximize the productivity of cell cultures can be found in: Mammalian Cell Biotechnology: a Practical Approach , M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra .

The methods of transfection of eukaryotic cells and transformation of prokaryotic cells are known to those skilled in the art, for example, CaCl 2, CaPO 4, mediated by liposomes and electroporation. Depending on the host cell used, the transformation is carried out using standard techniques appropriate for such cells. Calcium treatment that uses 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 some plant cells, as described in Shaw et al., Gene , 23 : 315 (1983) and in WO 89/05859 published on June 29, 1989. In In the case of mammalian cells lacking such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology , 52 : 456-457 (1978) can be used. General aspects of the transfections of the mammalian host cell system have been described in US Pat. US 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 : 3,829 (1979). However, other methods can also be used to introduce DNA to cells, such as nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, for example, polybrene, polynithine. For various techniques on the transformation of mammalian cells, see, Keown et al., Methods in Enzymology , 185 : 527-537 (1990) and Mansour et al., Nature , 336 : 348-352 (1988).

Suitable host cells for cloning or expression of DNA in the vectors of the present invention include prokaryotes, yeasts, or higher eukaryotic cells. Suitable prokaryotes include, but not exclusively, eubacteria, such as gram-negative or gram-positive organisms, for example, enterobacteria such as E. coli . Various strains of E. coli are available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include enterobacteria 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 41 P described in DD 266,710 published April 12, 1989). Pseudomonas , such as P. aeruginosa , and Streptomyces . These examples are illustrative but not exclusive. Strain W3110 is a particularly preferred host or parent host because it is a frequent host strain for fermentation of recombinant DNA products. Preferably the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to effect a genetic mutation in the genes encoding endogenous host proteins, with examples of such hosts including E. coli strain W3110, which has the complete genotype tonA; E. coli W3110 strain 9E4, which presents the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which presents the complete genotype tonA ptr3 phoA E15 (argF-1 ac) 1 69 degP ompT kon '; E. coli W3110 strain 37D6, which presents the complete genotype tonA ptr3 phoA E15 (argF-l ac) 1 69 degP ompT rbs7 ilvG kon '; E. coli W3110 strain 40B4, which is a 37D6 strain with a degP deletion mutation not resistant to kanamycin; and a strain of E. coli presenting a mutant periplasmic protease referred to in US Pat. US 4,946,783 published August 7, 1990. Alternatively, in vitro cloning procedures are suitable, for example, CPR or other nucleic acid polymerase reactions.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are appropriate hosts for cloning or expression of hosts for vectors encoding PRO224. Saccharomyces cerevisiae is a frequently used lower eukaryotic host microorganism. Among others are Schizasaccharomyces pombe (Beach and Nurse, Nature , 290 : 140 [1981]; EP 139,383 published May 2, 1985); Kluyveromyces hosts (US Patent 4,943,529; Fleer et al., Bio / Technology , 9 : 968-975 (1991)) such as, for example, K. lactis (MW98-8C, CBS683, CBS4574; 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. marxiamus : yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol ., 28 : 265-278 [1988]); Candida ; Trichoderma reesia (EP 244,234): Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA , 76 : 5,259-5,263 [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 on 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]). Methylotrophic yeasts are suitable herein and include, but not exclusively, yeasts capable of growing on methanol selected from the genera Hansenula , Candida , Kloeckera , Pichia , Saccharomyces , Torulopsis and Rhodotorula . A list of specific species that are exemplars of this class of yeasts can be found in: C. Anthony, The Biochemistry of Methylotrophs , 269 (1982).

Suitable host cells for the expression of glycosylated PRO224 come from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Useful examples of mammalian host cell lines include Chinese hamster ovarian cells (CHO) and COS cells. More specific examples include a monkey renal CVI line transformed by SV40 (COS-7. ATCC CRL 1651); a human embryonic renal line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol ., 36 : 59 (1977)); Chinese hamster ovarian cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA , 77 : 4216 (1980)); murine sertoli cells (TM4, Mather, Biol. Reprod ., 23 : 243-251 (1990)); human lung cells (W138, ATCC CCL 75): human liver cells (Hep G2, HB 8065); and murine mammary tumor (MMT060562, ATCC CCL51). The selection of the appropriate host cell is considered to be part of the skills in the state of the art.

3. Selection and use of a replicable vector

The nucleic acid (for example, cDNA or DNA genomic) encoding PRO224 can be inserted into a vector replicable for cloning (DNA amplification) or for the expression. Several vectors are available. The vector can be, for example, a plasmid, a cosmid, a viral particle, or a phage. The appropriate nucleic acid sequence may be inserted into the vector through a variety of interventions. In In general, the DNA is inserted into an appropriate site, or sites, of the restriction endonuclease using techniques known in the state of the art The components of the vector generally include, but not exclusively, one or more of a signal sequence, an origin of replication, one or more marker genes, an element enhancer, a promoter and a termination sequence of the transcription. The construction of suitable vectors containing one or more of these components uses standard ligation techniques which are known to the person skilled in the art.

PRO224 can be produced recombinantly 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-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be part of the DNA encoding the PRO224 that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the alkaline phosphatase group, penicillinase, Ipp, or heat stable enterotoxin II leaders. For the secretion of yeast the signal sequence may be, for example, the leader of the yeast invertase, leader alpha factor (including the leaders of the α-factor of Saccharomyces and Kluyveromyces , the last described in US Pat. 5,010,182), or acid phosphatase leader, glucoamylase leader of C. albicans (EP 362,179 published April 4, 1990), or the signal described in WO 90/13646 published November 15, 1990. In the expression of mammalian cells, mammalian signal sequences can be used to direct the secretion of proteins, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretion leaders.

Both expression vectors and those of cloning contain a nucleic acid sequence that allows to the vector replicate in one or more selected host cells. Such sequences are well known for a variety of Bacteria, yeasts and viruses. The origin of plasmid replication pBR322 is suitable for most gram-negative bacteria, the origin of plasmid 2P is suitable for yeast, and they are several viral origins (SV40, polyoma, adenovirus, VSV or BPV) useful in cloning vectors in cells mammals

Expression and cloning vectors usually contain a selection gene, also called selectable marker. The usual selection genes encode proteins that: (a) confer resistance to antibiotics or others toxins, for example, ampicillin, neomycin, methotrexate, or tetracycline; (b) complement auxotrophic deficiencies, or (c) provide important nutrients that are not available in complex media, for example, the gene that encodes the D-Alanine racemase for bacilli.

An example of selectable markers suitable for mammalian cells are those that allow the identification of competent cells to use the nucleic acid encoding PRO224, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and disseminated as described by Urlaub et al., Proc. Natl Acad. Sci. USA , 77 : 4,216 (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 cannot grow on tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics , 85 : 12 (1977)].

Expression and cloning vectors normally contain a promoter operably linked to the nucleic acid sequence encoding PRO224 to direct mRNA synthesis. Promoters identified by a variety of possible host cells are well known. Promoters suitable for the use of prokaryotic hosts include the beta-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 : 4,057 (1980); EP 36,776], and hybrid promoters such as the tac [deBoer et al., Proc. Natl Acad. Sci. USA, 80 : 21-25 (1983)]. Promoters that are used in bacterial systems will also contain a Shine-Dalgamo (SD) sequence operatively linked to the DNA encoding PRO224.

Examples of promoter sequences suitable for use with yeast hosts include 3-phosphoglycerate kinase promoters [Hitzeman et al., J. Biol. Chem ., 255 : 2,073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg ., 7 : 149 (1968); Holland, Biochemistry , 17 : 4,900 (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 glucokinase.

Other yeast promoters, which are promoters inducible that have the additional advantage of transcription controlled by growth conditions, are the regions promoters for alcohol dehydrogenase 2, isocytochrome C, the acid phosphatase, degrading 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 the Yeast expression is described in EP 73,657.

Transcription of PRO224 from vectors in mammalian host cells it is controlled, for example, by promoters obtained from virus genomes such as the polyoma virus, avian smallpox virus (Kingdom patent Kingdom UK 2,211,504, published July 5, 1989), adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma, cytomegalovirus, a retrovirus, hepatitis B and simian virus 40 (SV40), from promoters of heterologous mammals, for example, the actin promoter or an immunoglobulin promoter, and from shock promoters of heat, provided that said promoters are compatible with the host cell systems.

The transcription of a DNA encoding the PRO224 by higher eukaryotes can be increased by inserting an enhancer sequence in the vector. The enhancers are cis-acting DNA elements, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Several enhancer sequences are currently known from of mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Habitually, however, an enhancer from a virus will be used eukaryotic cell. Examples include the enhancer SV40 in the last part of the end of the replication source (100-270 bp), the enhancer of the early promoter of cytomegalovirus, the polyoma enhancer in the last part of the end of origin of replication, and enhancers of adenovirus The enhancer can be spliced on the vector in the position 5 'or 3' of the sequence encoding the '- - PRO224, but preferably it is located in the 5 'position of the promoter.

Expression vectors used in cells eukaryotic host (yeasts, fungi, insects, vegetables, animals, humans, or nucleated cells from others multicellular organisms) will also contain necessary sequences for the completion of transcription and for stabilization mRNA Such sequences are normally available at from untranslated regions 5 ', and occasionally 3', of DNA or Eukaryotic or viral cDNA. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the part untranslated from the mRNA encoding the PRO224.

Still other methods, vectors and host cells suitable for adaptation of PRO224 synthesis in recombinant vertebrate cell culture are described in: Gething et al., Nature , 293 : 620-625 (1981); Mantei et al., Nature , 281 : 40-46 (1979); EP 117,060 and EP 117,058.

4. Detection of gene amplification / expression

Gene amplification and / or expression can be determined in a sample directly, for example, by conventional Southern and Northern blotting to quantify mRNA transcription [Thomas, Proc. Natl Acad. Sci. USA , 77 : 5,201-5.205 (1980)], point transfer (DNA analysis), or by in situ hybridization, using an appropriately labeled probe, depending on the sequences provided herein. Alternatively, antibodies capable of identifying specific duplications may be employed, including DNA duplications, RNA duplications and hybrid DNA-RNA duplications or DNA-protein duplications. The antibodies can in turn be labeled and the analysis can be performed at the sites where the duplicity binds to the surface, so that on the formation of duplicity on the surface the presence of antibody bound to the duplicity can be detected.

Alternatively, gene expression can determined by immunological procedures, such as immunohistochemical staining of cells or tissue parts and analysis of cell culture or body fluids, to quantify directly the expression of gene product. Antibodies utility for immunohistochemical staining and / or analysis of Sample liquids can be either monoclonal or polyclonal, and can be prepared in any mammal. Conveniently, antibodies can be prepared against a PRO224 polypeptide of natural sequence or against a synthetic peptide depending on the DNA sequences provided herein or against a exogenous sequence fused with a DNA of PRO224 and coding for a specific antibody epitope.

5. Purification of polypeptides

The forms of PRO224 can be recovered from a culture medium or host cell lysates. If they are attached to the membrane can be released from the membrane using a suitable detergent solution (for example, Triton-X 100) or by enzymatic cleavage. The cells used in the expression of PRO224 can be altered by various physical or chemical means, such as cycles of freezing-liquefaction, sonication, alteration mechanics, or agents that lyse cells.

It may be desirable to purify the PRO224 from cellular proteins or recombinant polypeptides. The following interventions are exemplary of suitable purification interventions: by fractionation in an ion exchange column; ethanol precipitation; Reverse phase HPLC; chromatography on silica or on a cation exchange resin, such as DEAE; chromatofocalization; SDS-PAGE; precipitation of ammonia sulfate; gel filtration using, for example, Sephadex G-75; Sepharose protein A columns to remove contaminants such as IgG; and metal chelating columns to bind labeled forms (tagged) PRO224 epitope. Various protein purification procedures can be used and such procedures are known in the state of the art and have been described for example in: Deutscher, Methods in Enzymology , 182 (1990); Scopes, Protein Purification: Principles and Practice . Springer-Verlag, New York (1982). The stage or stages of purification selected will depend, for example, on the nature of the production process used and on the particular PRO179, PRO224 produced.

E. Antibodies

Some of the possible drugs that may be used in the compounds and processes herein invention are antibodies and antibody fragments that emulate the biological activity of a PRO224 polypeptide.

1. Polyclonal antibodies

The procedures for the preparation of polyclonal antibodies are known to the expert in matter. Polyclonal antibodies can be cultured in a mammal, for example, by one or more injections of an agent immunizing and, if desired, an adjuvant. Usually the agent immunizing and / or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The agent immunizing may include the PRO224 polypeptide or a protein of fusion of it. It may be useful to conjugate the immunizing agent with a protein that is known to be immunogenic in the mammal that is immunized Examples of such immunogenic proteins include include but not exclusively barnacle hemocyanin, albumin serum, bovine thyroglobulin and trypsin inhibitor of soy. Examples of adjuvants that can be used are include complete Freund's adjuvant and adjuvant MPL-TDM (monophosphoryl lipid A, dicorinomicolate synthetic trehalose). The immunization protocol can be selected by any subject matter expert.

2. Monoclonal antibodies

The 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, a 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 PRO224 polypeptide of a fusion protein thereof. Generally, peripheral blood lymphocytes ("PBL") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if human mammalian sources are not desired. 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 , Academic Press, (1986) p. 59-103]. Immortalized cell lines are normally transformed into mammalian cells, particularly myeloma cells originating in rodents, cattle and humans. Normally, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the non-fused immortalized cells. For example, if the progenitor cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will usually include hypoxanthine, aminopterin and thymidine ("HAT medium"), substances that prevent the growth of deficient cells for HGPRT.

Preferred immortalized cell lines are those that fuse efficiently, sustain a stable high expression of the antibody by the cells that produce the selected antibody and are sensitive to a medium such as the 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 murine-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol ., 133 : 3,001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) p. 51-63].

The culture medium in which the hybridoma cells are cultured can be analyzed for the presence of monoclonal antibodies directed against PRO224. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding analysis, such as radioimmunoassay (RIA) or adsorption enzyme immunoassay (ELISA). Such techniques and analysis are known in the state of the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem ., 107 : 220 (1980).

Once the desired hybridoma cells have been identified, the clones can be subcloned by limiting dilution procedures and cultured by standard procedures [Coding, supra ]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be cultured in vivo as ascites in a mammal.

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

Monoclonal antibodies can also be synthesized by recombinant DNA methods, such as those described in US Pat. US 4,816,567. DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention act as a preferred source of such DNA. Once rotated, the DNA can be placed in expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovarian cells (CHO), or myeloma cells that otherwise do not produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting homologous murine sequences with the coding sequence of the constant domains of human heavy and light chains [US Pat. US 4,816,567; Morrison et al., Supra ] or by covalent binding of the entire immunoglobulin coding sequence or part of the coding sequence of a polypeptide other than immunoglobulin. Said polypeptide other than immunoglobulin may be substituted in the constant domains of an antibody of the invention, or it may be substituted in the variable domains of an antigen binding site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies can be antibodies monovalent The procedures for the preparation of antibodies Monovalent are well known in the state of the art. By example, a procedure involves the recombinant expression of the light chain and modified heavy chain of immunoglobulin. The heavy chain is usually truncated at any point of the Fc region to avoid the formation of cross links in the heavy chain Alternatively, the relevant residues of cysteine are substituted by other amino acid residues or are removed to avoid the formation of cross links.

In vitro procedures are also suitable for the preparation of monovalent antibodies. The digestion of antibodies to produce fragments thereof, in particular, Fab fragments, can be carried out using systematic techniques known in the state of the art.

3. Human and humanized antibodies

The antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human antibodies (e.g. murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antibody sub-sequences of antigen binding) that contain a minimal sequence from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibody) in which residues of a receptor complementarity determining region (CDR) are replaced by residues from a CDR of a non-human species (donor antibody), such as a mouse , a rat or a rabbit with the specificity, affinity and capacity desired. In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are neither found in the receptor antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and usually two, of the variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions. they are those of a consensus sequence of human immunoglobulin. The humanized antibody will also optimally comprise at least a part 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)].

The procedures for obtaining non-humanized humanized antibodies are well known in the state of the art. Generally, a humanized antibody has one or more amino acid residues from a source that is not human. These non-human amino acid residues are often referred to as "import" residues, which are usually obtained from a variable "import" domain. Humanization can basically be done following the procedure of Winter and his collaborators [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-327 (1988); Verhocyen et al., Science , 239 : 1,534-1,536 (1988)], by substituting rodent CDR or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent 4,816,567), in which substantially less of an 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 from similar sites in rodent antibodies.

Human antibodies can also be produced using several techniques known in the state of the art, including bacteriophage libraries [Hoogenboom and Winter, J. Mol. Biol ., 227 : 381 (1991); Marks et al., J. Mol. Biol ., 222 : 581 (1991)]. The techniques of Cole et al., And Boerner et al., Are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss. P. 77 (1985) and Boerner et al., J. Immunol ., 147 (1) : 86-95 (1991)] Similarly, human antibodies can be synthesized by introducing human immunoglobulin loci to transgenic animals, for example, mice in which they have been inactivated partially or completely the genes of the endogenous immunoglobulin After exposure, human antibody production is observed, which reminds exactly in all aspects the production observed in humans, including the redisposition of genes, the assembly and the repertoire of antibodies. This procedure is described, for example, in US Patents US 5,545,807; US 5,545,806; US 5,569,825; US 5,625,126; US 5,633,425; US 5,661,016 and in the following publications scientists: Mar ks et al., Bio / Technology , 10 : 779-783 (1992); Lonberg et al., Nature , 368 : 856-859 (1994); Morrison, Nature , 368 : 912-13 (1994); Fishwild et al., Nature Biotechnology , 14 : 845-51 (1996); Neuberger, Nature Biothechnology , 14 : 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol ., 13 : 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that present binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO224, the other is for any other antigen, and preferably for a protein or a receptor or a receptor subunit of the cell surface

Methods for synthesizing bispecific antibodies are known in the state of the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain / light chain pairs, in which the two heavy chains have different specificities [Milstein and Neck, Nature , 305 : 537-539 (1983 )]. Given the random assortment of heavy and light immunoglobulin chains, these hybridomas (quadromas) produce a potential mixture often of different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out by affinity chromatography phases. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J. , 10 : 3,655-3,659 (1991).

The variable domains of antibodies that exhibit the desired binding specificities (antibody-antigen binding sites) can be fused with sequences of immunoglobulin constant domains. The fusion preferably takes place with a constant domain of the heavy chain of an immunoglobulin, which comprises at least part of the hinge, CH2 and CH3 regions. It is preferable that the first constant region of the heavy chain (CHI) contains the site necessary for the union of the light chain present in at least one of the fusions. The DNAs that encode the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and are co-transfected into a suitable host organism. For more details on the generation of bispecific antibodies see, for example, Suresh et al., Methods in Enzymology , 121 : 210 (1986).

According to another procedure described in the WO 96/27011, the interface between a pair of molecules of antibodies can be manipulated to maximize the percentage of heterodimers that have been recovered from a cell culture recombinant The preferred interface comprises at least one part of the CH3 region of a constant antibody domain. In this procedure, one or more small side chains of amino acids of the interface of the first antibody molecule are substituted by larger side chains (for example, tyrosine or tryptophan). Size compensatory "cavities" are created identical or similar to that of the chain, or chains, large side over the interface of the second antibody molecule replacing the large side chains of amino acids by smaller ones (for example, alanine or threonine). This provides a mechanism for increase the production of the heterodimer above that of others unwanted end products, such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or as antibody fragments (for example, bispecific F (ab ') 2 antibodies). Techniques for the generation of bispecific antibodies from antibody fragments have been described in the literature on the subject. 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 cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol ligand, to stabilize neighboring dithiols and prevent the formation of intermolecular disulfides. The generated Fab 'fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the TNB-Fab 'derivatives is then converted to thiol Fab' by reduction with mercaptoethylamine and mixed with an equimolar amount of the other TNB-Fab '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 F (ab ') antibody molecule. Each Fab 'fragment was independently secreted from E. coli and subjected to in vitro directed chemical coupling to form the bispecific antibody. The bispecific antibody formed in this way was capable of binding to cells that overexpressed the ErbB2 receptor and normal human T cells, as well as triggering the lytic activity of human cytotoxic lymphocytes against human breast tumors.

Several techniques for synthesizing and isolating bispecific antibody fragments directly from the recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol ., 148 (5): 1,547-1,553 (1992). Leucine zipper peptides from Fos and Jun proteins were bound to the Fab 'portions of the two distinct antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This procedure can also be used for the production of antibody homodimers. The technology related to "dianticibodies" described by Hollinger et al., Proc. Natl Acad. Sci. USA , 90 : 6,444-6,448 (1993) has provided an alternative mechanism for synthesizing bispecific antibody fragments. The fragments comprise a variable domain (V H) in the heavy chain connected to a variable domain (V L) in the light chain by means of a connector that is too short to allow coupling between the two domains of the same chain Accordingly, the V_ {H} and V_ {{}} domains of one fragment are required to couple with the complementary V_ {L} and VH {domains of another fragment, whereby two antigen binding sites are formed. Another strategy for synthesizing bispecific antibody fragments through the use of single chain Fv dimers (sFv) has also been described. See, Gruber et al., J. Immunol ., 152 : 5,368 (1994).

Antibodies with more than two valences are contemplated. For example, trypecific antibodies can be prepared. Tun et al., J. Immunol ., 147 : 60 (1991).

Exemplary bispecific antibodies they can bind to two different epitopes in a PRO224 polypeptide given here. Alternatively, an arm of anti-PRO224 polypeptide with an arm that binds to a trigger molecule in a white blood cell such as a molecule T cell receptor (for example, CD2, CD3, CD28, or B7), or Fc IgG receptors (FcgR), such as FcgRI (CD64), FcgRII (CD32) and FcgRIII (CDJ6) to focus defense mechanisms cell of the cell expressing the PRO224 polypeptide in particular. Bispecific antibodies can also be used. to locate cytotoxic agents for cells expressing a particular PRO224 polypeptide. These antibodies have an arm of binding to PRO224 and an arm that binds to a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the PRO224 polypeptide and also binds to the tissue factor (TF).

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 against unwanted cells [US Pat. US 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. The possibility of preparing antibodies in vitro using methods known in protein synthesis chemistry, including those involving cross-linking agents, is contemplated. For example, immunotoxins can be constructed using a disulfide exchange reaction or forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those described, for example, in US Pat. US 4,676,980.

6. Manipulation of the effector function

It may be desirable to modify the antibody of the invention in terms of its effector function, to increase, for example, the efficacy of the antibody in the treatment of cancer. For example, one or more cysteine residues can be introduced into the Fc region, which allows the formation of intercatenary disulfide bonds in this region. The homodimeric antibody generated in this way may have better internalization capacity and / or greater capacity for complement-mediated cell elimination and antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al., J. Exp. Med ., 176 : 1,191-1,195 (1992) and Shopes, J. Immunol ., 148 : 2,918-2,922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research , 53 : 2,560-2,565 (1993). Alternatively, an antibody that has double Fc regions can be manipulated and therefore complement lysis and ADCC capabilities can be enhanced. See, Stevenson et al., Anti-Cancer Drug Design . 3 : 219-230 (1989).

7. Immunoconjugates

The invention is also related to immunoconjugates comprising an antibody conjugate with a cytotoxic agent, such as a chemotherapeutic agent, a toxin (for example, an enzymatically active toxin of origin bacterial, fungal, plant, or animal, or fragments thereof), or a radioactive isotope (i.e. a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Among the toxins, and the enzymatically active fragments thereof that can be used include: diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), the A chain of ricin, the A chain of abrin, the A chain of modec and atpha-sancin. Aleurites fordii proteins, diantin proteins, Phytolaco americana proteins (PAPI, PAPII and PAPS), Momordica charantia inhibitor, curcin, crotine, Saponaria officinalis inhibitor, gelonin, mitogilin, restrictedtocin, phenycin, Enomycin and trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 112 Bi, 131 I, 131 In, 90 Y and 130 Re.

Antibody and cytotoxic agent conjugates are synthesized using a variety of bifunctional agents that are coupled to proteins such as N-succinimidyl-3- (1-pyridyldithiol) propionate (SPDP), iminothiolane (11), bifunctional derivatives of imidoesters (such such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-acidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) ) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluoride compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science , 238 : 1,098 (1987). Carbon-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) 14 is an exemplary chelating agent of radionuclotide conjugation with the antibody. See, document WO 94/11026.

In another embodiment, the antibody can conjugate with a "receptor" (such as streptavidin) for its use in premarking the tumor in which the conjugate antibody-receptor is administered to the patient, followed by elimination of unbound conjugate circulation using a cleaning agent and then administering a "ligand" (eg, avidin) that is conjugated with an agent cytotoxic (for example, a radionuclotide).

8. Immunoliposomes

The antibodies described herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the state of the art, such as those described in Epstein et al., Proc. Natl Acad. Sci. USA , 82 : 3,688 (1985); Hwang and others, Proc. Natl Acad. Sci. USA , 77 : 4,030 (1980); and U.S. patents US 4,485,045 and US 4,544,545. Liposomes with longer circulation time are described in US Pat. US 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation process with a lipid compound comprising phosphatidylcholine, cholesterol and phosphatidylethanolamine derived from PEG (PEG-PE). Liposomes are removed through filters with a determined 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 disulfide exchange reaction. The liposome may optionally contain a chemotherapeutic agent (such as doxorubicin). See, Gabizon et al., J. National Cancer Inst. , 81 (19): 1,484 (1989).

F. Identification of proteins capable of inhibiting growth or proliferation of neoplastic cells

The proteins described in the present patent application have been analyzed in a table of 60 tumor cell lines currently used in disease-oriented screening of in vitro drugs of the National Cancer Institute (NCI). The objective of this screening is to identify molecules that have cytotoxic and / or cytostatic activity against different types of tumors. The NCI screens more than 10,000 new molecules each year (Monks et al., J. Natl. Cancer Inst ., 83 : 757-766 (1991); Boyd, Cancer; Princ. Pract. Oncol. Update , 3 (10) : 1 -12 ([1989]) The tumor cell lines used in this study have been described in Monks et al., Supra Cell lines whose growth has been considerably inhibited by the proteins of the present patent application are specified in the examples.

The results have shown that proteins tested show cytostatic activity and, in some cases and according to concentrations, cytotoxic activity in a variety of lines cancer cells and, therefore, are candidates for treatment of a tumor

Other analyzes can also be used from cells and animal models for tumors (eg, cancers) to verify the results of the NCI oncology study and to further understand the relationship between the protein identified herein and the development and pathogenesis of the neoplastic cell growth. For example, primary cultures obtained from tumors in transgenic animals (as described below) may be used in analyzes from cells herein, although stable cell lines are preferred. Techniques for obtaining constant cell lines from transgenic animals are well known in the state of the art (see, for example, Small et al., Mol. Cell. Biol ., 5 : 642-648).

G. Animal models

A variety of well-known animal models can be used to further understand the role of the molecules identified herein in the development and pathogenesis of tumors, and to test the efficacy of possible therapeutic agents, including antibodies, and other polypeptide agonists. natural, including small molecule agonists. The in vivo nature of such models makes them particularly useful for predicting responses in human patients. Animal models of tumors and cancers (eg, breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents, for example, murine models. Such models can be generated by introducing tumor cells to syngeneic mice using standard techniques, for example, subcutaneous injection, tail vein injection, spleen implant, intraperitoneal implant, implant under the renal capsule, or orthopine implant, for example, Colon cancer cells implanted in colonic tissue (see, for example, PCT Publication No. WO 97/33551, published September 18, 1997).

Probably the most frequently used animal species in cancer studies are immunodeficient mice and, in particular, nude mice. The observation that the naked mouse with hypoaplasia could be used successfully as a host in human tumor xenotransplants has led to its wide use for this purpose. The autosomal recessive gene nu has been introduced into a large number of congenital strains other than nude mice, including, for example, ASW, A / He, AKR, BALB / c, B10.LP, C17, C3H, C57BL, C57, CBA , DBA, DDD, I / st, NC, NFR, NFS, NFS / N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with hereditary immune abnormalities other than the nude mouse have been raised and used as tumor xenotransplant receptors. For more details see, for example, The Nude Mouse in Oncology Research , E. Boven and B. Winograd, eds., CRC Press, Inc .. 1991.

Cells introduced into such animals can be obtained from known tumor / cancer cell lines, such as, any of the tumor cell lines listed above, and, for example, the B104-1-1 cell line (NIH-3T3 stable cell line transfected with the new protooncogene); NIH-3T3 cells transfected with ras; Caco-2 (ATCC HTB-37); a moderately well differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or from tumors and cancers. Tumor or cancer cell samples can be obtained from patients undergoing surgery, using standard conditions, which involve freezing and storage in liquid nitrogen (Karmali et al., Br. J. Cancer , 48 : 689-696 [1983 ]).

Tumor cells can be introduced into animals, such as the naked mouse, by a variety of procedures. The subcutaneous space in mice is very suitable for tumor transplantation. Tumors can be transplanted subcutaneously as solid blocks, as puncture biopsies using a trocar needle, or as cell suspensions. For solid block or trocar needle transplantation, fragments of tumor tissue of appropriate size are introduced into the subcutaneous space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells can also be injected as subdermal transplants. At this site, the inoculum is deposited between the lower part of the dermal connective tissue and the subcutaneous tissue. Boven and Winograd (1991), supra . Animal models of breast cancer can be generated, for example, by transplantation of rat neuroblastoma cells (from which the new oncogene was initially isolated), or newly transformed NIH-3T3 cells into nude mice, essentially as described by Drebin and others, Proc. Natl Acad. Sci. USA , 83 : 9,129-9,133 (1986).

Likewise, animal models of colon cancer can be generated by the introduction of colon cancer cells in animals, for example, nude mice, which causes the appearance of tumors in these animals. An orthotopic transplant model of human colon cancer in nude mice has been described, for example, in Wang et al., Cancer Research , 54 : 4,726-4,728 (1994) and Too et al., Cancer Research , 55 : 681-684 ( nineteen ninety five). This model is based on the so-called "METAMOUSE" sold by AntiCancer, Inc. (San Diego, California).

Tumors that appear in animals can be removed and cultured in vitro . In vitro culture cells can then be introduced into animals. These tumors can serve as targets for other tests or drug screening. Alternatively, the tumors resulting from this introduction can be isolated and the RNA of the cells can be analyzed previously and the cells isolated after one or more rounds of introduction for differential expression of genes of interest. Such introduction techniques can be carried out with any known tumor or carcinogenic cell line.

For example, Meth A, CMS4, CMSS. CMS21. and WEHI-164 are fibrosarcomas of chemically induced BALB / c female mice (DeLeo et al., J. Exp. Med ., 146 : 720 [1977]), which provide a highly controllable model system to study the antitumor activities of various agents ( Palladino et al., J. Immunol ., 138 : 4,023-4,032). In summary, tumor cells propagate in vitro in cell culture. Before injecting them into the animals, the cell lines are cleaned and suspended in a buffer, at a cell density of about 10x10 6 to 10x10 7 cells / ml. The animals are then infected subcutaneously with 10 to 100 µl of the cell suspension, allowing the appearance of a tumor in a period of one to three weeks.

In addition, Lewis lung carcinoma (3LL) of mice, which is one of the most comprehensively studied experimental tumors, can be used as an experimental tumor model. The efficacy of this tumor model has been correlated with beneficial effects in the treatment of human patients with a diagnosis of small cell lung carcinoma (SCCL). This tumor can be introduced into normal mice by injection of tumor fragments from an affected mouse or from cells maintained in culture (Zupi et al., Br. J. Cancer , 41 , Suppl. 4: 309 [1980]), and the evidence indicates that tumors can be initiated from the injection of even a single cell and that a very high proportion of infected tumor cells survive. For more information on this tumor model see, Zacharski, Haemostasis , 16 : 300-320 [1986]).

One way to evaluate the efficacy of a test compound in an animal model with an implanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of transplanted tumors has been measured with a two or three dimensional gauge. The measure limited to two dimensions does not accurately reflect the size of the tumor, therefore, it is usually converted to the corresponding volume using a mathematical formula. However, the measurement of tumor size is not very precise. The therapeutic effects of a possible drug are best described as growth retardation and specific growth retardation induced by the treatment. Another important variable in the description of the growth of a tumor is the doubling time of the tumor volume. Computer programs are available for the calculation and description of tumor growth, such as the Rygaard and Spang-Thomsen, Proc program. 6th Int. Workshop on Immune-Deficient Animals , Wu and Sheng eds., Basel, 1989, 301. However, it is observed that necrosis and the inflammatory response after treatment can actually lead to an increase in tumor size, at least initially. Therefore, it is necessary to carefully monitor the changes, combining a morphometric procedure and the flow cytometric analysis.

Recombinant (transgenic) animal models can be manipulated by introducing the coding part of the genes identified herein into the genome of animals of interest, using standard techniques for the production of transgenic animals. Animals that can act as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs and nonhuman primates, for example, baboons, chimpanzees and apes. Techniques known in the state of the art for introducing a transgene to such animals include pronucleic microinjection (Hoppe and Wanger, US Pat. No. 4,873,191); gene transfer, mediated by retroviruses, to germ lines (eg, Van der Putten et al., Proc. Natl. Acad. Sci. USA , 82 : 6,148-615 [1985]); gene labeling in embryonic stem cells (Thompson et al., Cell , 56 : 313-321 [1989]); embryo electroporation (Lo, Mol. Cell. Biol ., 3 : 1,803-1,814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell , 57 : 717-73). For a review, see, for example, US Pat. US 4,736,866.

For the purposes of the present invention, transgenic animals include those that only carry the transgene in part of their cells ("mosaic animals"). The transgene can be integrated either as a single transgene, or in concatamers, for example, head-to-head or head-to-tail tandems. It is also possible to selectively introduce a transgene into a particular cell type following, for example, the technique of Lasko et al., Proc. Natl Acad. Sci. USA , 89 : 6,232-6,236 (1992).

The expression of the transgene in transgenic animals can be controlled by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify transgene integration. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, CPR or immunocytochemistry. In addition, animals are examined for signs of tumor or cancer development.

The efficacy of antibodies that bind specifically to the polypeptides identified herein and of other possible drugs can also be tested in treatment of spontaneous animal tumors. A suitable goal for such Studies is feline oral squamous cell carcinoma (SCC). Feline oral SCC is a very invasive malignant tumor that constitutes the most frequent oral malignant tumor of cats, realizing more than 60% of oral tumors observed in this species. It rarely metastasizes to remote sites, although it is low incidence of metastasis may simply be a reflection of the Short survival of cats with this tumor. Normally these tumors cannot be treated by surgery, mainly because of the anatomy of the oral cavity of felines. Not yet There is no effective treatment for this tumor. Before starting the study, each cat undergoes an exploration complete clinic, a biopsy and a CT scan (TC). Cats with a diagnosis of squamous cell tumor Oral sublingual are excluded from the study. The tongue can become paralyzed as a result of such a tumor, and even if the treatment removes the tumor, animals may not be able to feed Each cat is treated repeatedly, during an extended period of time. During the treatment period, and in each of the subsequent checks, it will be necessary to take Daily photographs of the tumors. After the treatment, it subject each cat to another CT. Next, CT scans and radiograms Thoracic are evaluated every 8 weeks. The data is evaluated in looking for differences in survival, response and toxicity in comparison with control groups. The positive response can require tumor regression tests, preferably with improvement of the quality of life and / or increase of the life time.

In addition, other tumors can also be tested spontaneous animals, such as fibrosarcoma, adenocarcinoma, lymphoma, chondroma, leiomyosarcoma of dogs, cats and baboons. Of these, mammary adenocarcinoma of dogs and cats is a model preferred since its appearance and behavior are very similar to those of humans. However, the use of this model is limited by the rare occurrence of this type of tumor in animals.

H. Screening analysis of possible drugs

Screening analyzes of possible drugs have been designed to identify compounds that bind competitively, or they form complexes, with the receptor or receptors of the polypeptides identified herein, or any other signal through such receiver or receivers. These analyzes of screening will include analyzes that allow very productive screening of chemical libraries, which makes them particularly suitable for identifying possible small molecule drugs. Small molecules contemplated include compounds synthetic organic or inorganic, including peptides, preferably soluble peptides, fusions of (poly) peptide-immunoglobulin, and, in in particular, antibodies that include, without limitation some, monoclonal and polyclonal antibodies and fragments of antibodies, single chain antibodies, antibodies antidiotypics, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and fragments of antibodies. The analyzes can be carried out in a variety of formats, such as binding analysis protein-protein, biochemical screening analysis, immunoassay and analysis from cells, which are fine characterized in the state of the art.

In union analyzes, the interaction is the binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, a receiver of a polypeptide encoded by the gene identified herein or the possible drug is immobilized in a solid phase, for example, in a microtiter plate, by covalent or non-covalent bonds covalent A non-covalent bond is usually obtained coating the solid surface with a solution of the polypeptide and drying. Alternatively, an antibody can be used. immobilized, for example, a monoclonal antibody, specific for the polypeptide that will be immobilized to anchor it to the surface solid. The analysis is carried out by adding component no immobilized, which may be marked with a detectable marker, when immobilized component, for example, the coated surface that Contains the anchored component. When the reaction is over, it remove unreacted components, for example, by washing, and the complexes anchored in the solid surface When the non-immobilized component originally carries a detectable marker, the detection of marker immobilized on the surface indicates that the complex formation. When the component originally did not immobilized does not carry a marker, the formation of complexes, for example, using a labeled antibody that binds specifically to the immobilized complex.

If the possible compound interacts with a particular receptor but does not bind to it, its interaction with that polypeptide can be analyzed by well-known methods to detect protein-protein interactions. Such analyzes include traditional procedures, such as cross-linking, coinmunoprecipitation and copurification through gradients or chromatographic columns. In addition, protein-protein interactions can be controlled using a genetic system from yeasts described by Fields and his collaborators [Fields and Song, Nature (London), 340 : 245-246 (1989); Chien and others, Proc. Natl Acad. Sci. USA , 88 : 9,578-9,582 (1991)] as described by Chevray and Nathans [ Proc. Natl Acad. Sci. USA , 89 : 5,789-5,793 (1991)]. Several transcriptional activators, such as GAL4 yeast, consist of two physically separated modular domains, one that acts as the DNA binding domain, while the other functions as a transcription activation domain. The yeast expression system described in the 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 with the DNA binding domain of GAL4, and another, in which the possible activation proteins are fused with the activation domain. The expression of a GAL1-lacZ reporter gene under the control of a GAL4 activated promoter depends on the reconstitution of GAL4 activity by protein-protein interaction. Colonies containing interaction polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER ™) for the identification of protein-protein interactions between two specific proteins using the two hybrid technique is commercially available from Clontech. This system can also be extended to the mapping of protein domains involved in specific protein interactions, as well as to identify amino acid residues that are crucial for these
interactions

I. Pharmaceutical compounds

The polypeptides of the present invention, proteins that specifically bind to agonist antibodies identified herein, as well as other molecules identified by screening analysis described herein, they can be administered for the treatment of tumors, including cancers, in  form of pharmaceutical compounds.

When antibody fragments are used, the smaller inhibitor fragment that specifically binds to the binding domain of the target protein is preferred. For example, from variable region sequences of an antibody, peptide molecules can be designed that retain the ability to bind to the target protein sequence. Such peptides are chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci, USA , 90 : 7,889-7,893 [1993]).

The formulation of the present may also contain more than one active compound, if necessary, for that matter particular treated, preferably those with activities Complementary that do not have adverse effects on each other. Alternatively, or in addition, the compound may comprise an agent. that enhances its function, such as, for example, an agent cytotoxic, cytokine, a chemotherapeutic agent, or an agent growth inhibitor Such molecules exist properly combined in amounts that are effective for the purposes pretended.

The therapeutic formulations of the polypeptides identified herein, or agonists thereof, are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences , 16th edition, Osol. A. ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors at the doses and concentrations used, and include buffers such as: phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonia chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight 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, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; counterions that form salts such as sodium; metal complexes (for example, protein-Zn complexes); and / or non-ionic surfactants such as TWEEN?, PLURONICS? or polyethylene glycol (PEG).

The formulation of the present may also contain more than one active compound if necessary, for that matter particular treated, preferably those with activities Complementary that do not have adverse effects on each other. Alternatively, or in addition, the compound may comprise an agent. cytotoxic, cytokine, or a growth inhibitory agent. Such molecules exist properly combined in amounts that are effective for the intended purposes.

The active ingredients can also be found within microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (by for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences , 16th edition, Osol, A. ed. (1980).

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

The therapeutic compounds of the present they are usually placed in a container that has a port of sterile access, for example, a bag or solution vial IV that has a pierceable cap using a needle hypodermic injection

Release preparations can be prepared prolonged Suitable examples of release preparations are prolonged semipermeable matrices of hydrophobic polymers solids containing the antibody, which matrices are presented as shaped articles, for example, movies or microcapsules Among the examples of release matrices prolonged include polyesters, hydrogels (for example, poly (2-hydroxyethyl methacrylate),  or poly (vinyl alcohol), polylactides (US Pat. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, ethylene-vinyl non-degradable acetate, copolymers of degradable lactic acid-glycolic acid such as LUPRON DEPOT ™ (injectable microspheres composed of the copolymer lactic acid-glycolic acid and leuprólido acetate), and acid poly-D - (-) - 3-hydroxybutyric. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. When the encapsulated antibodies remain in the body for a while prolonged, they can become denatured or form aggregates as result of exposure to moisture at 37 ° C, which results in loss of biological activity and possible changes in the immunogenicity Depending on the mechanism involved, they can Plan reasonable strategies for stabilization. By example, if it is discovered that the aggregation mechanism is the formation of an S-S intermolecular link through from the thio-disulfide exchange, the stabilization by modifying sulfhydryl residues, lyophilizing at from acid solutions, controlling moisture content, using appropriate additives and manufacturing matrix compounds specific polymeric.

J. Treatment procedures

It is planned that the polypeptides of the present invention and its agonists, including antibodies, peptides and small molecule agonists, can be used to treat several tumors, for example, cancers. Among the examples of diseases or disorders to be treated include tumors benign or malignant (for example, renal, liver, carcinomas of kidney, urinary bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, pulmonary, vulvar, thyroid, liver; sarcomas; glioblastomas, and several tumors of head and neck); leukemia and lymphocytic neoplasms; others disorders such as neuronal disorder, glial, astrocitol, hypothalamic and other glandular, macrophageal disorders, epithelial, stromal and blastocellic; and inflammatory disorders, angiogenic and immunological. The antitumor agents of the present invention (including polypeptides described in the present and agonists that emulate their activity, for example, antibodies, peptides and small organic molecules) are administered to a mammal, preferably a human, according to known procedures, such as intravenous administration as a bolus or by continuous infusion over a period of time, or intramuscularly, intraperitoneally, intracerebroespinally, intraocular, intraarterial, intralesive, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or by inhalation.

Other therapeutic regimens can be combined with the administration of the anticancer agents of the present invention. For example, the patient to be treated with such anticancer agents may also receive radiotherapy. Alternatively, or in addition, a chemotherapeutic agent can be administered to the patient. The guidelines for the preparation and administration of such chemotherapeutic agents can be used according to the manufacturers instructions or as determined by the physician skilled in the art. The guidelines for the preparation and administration of such chemotherapy are also described in Chemotherapy Service , ed., MC Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede, or follow, the administration of the antitumor agent of the present invention, or it may be administered simultaneously with it. The anticancer agents of the present invention may be combined with an antiestrogenic compound such as tamoxifen or with an antiprogesterone such as onapristone (see EP 616812) at known doses for such molecules.

It may also be desirable to administer antibodies against tumor associated antigens, such as antibodies that bind ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies that bind to the same antigens or to two or more different antigens associated with cancer can be co-administered to the patient. Sometimes, it may be beneficial to also administer one or more cytokines to the patient. In a preferred embodiment, the anticancer agents herein are co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by the administration of an anticancer agent of the present invention. However, the possibility of simultaneous administration or administration of the anticancer agent of the present invention in the first place is also contemplated. Suitable doses of growth inhibiting agent are those that are currently used and can be reduced given the combined action (synergy) of the growth inhibiting agent and the antibody of the
Present.

For the prevention or treatment of a disease, the appropriate dose of an antitumor agent herein will depend on the type of disease to be treated, as previously determined, on the severity and course of the disease, on whether the agent is administers for preventive or therapeutic purposes the previous treatment, the patient's medical history and the response to the agent, and the criteria of the attending physician. The agent is properly administered to the patient at once or throughout a series of treatments. Animal experiments provide reliable guidance for the determination of effective doses for treatment in humans. The staging of effective doses between 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, p. 42-96.

For example, depending on the type and severity of the disease, a possible dose can be administered to the patient initial of about 1 µg / kg to 15 mg / kg (for example, 0.1-20 mg / kg) of an antitumor agent, therefore, by for example, by one or more independent administrations, or by continuous infusion. A usual daily dose could range from about 1 µg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or for more time, depending on the disease, the treatment is maintained until the desired elimination of the symptoms of the disease. However, other dose regimens may be useful. The evolution of this treatment is easily controlled by Conventional techniques and analysis. Guidance is provided on particular doses and administration procedures in literature on the subject; see, for example, US patents. US 4,657,760; US 5,206,344; or US 5,225,212. It is anticipated that different formulations will be effective for therapeutic compounds different and distinct disorders, that the administration directed to an organ or tissue, for example, may require a mode of administration other than that of another organ or tissue.

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K. Manufacturing items

A manufacturing article is provided that Contains materials useful for the diagnosis or treatment of disorders described above. The article of manufacture It comprises a container and a label. Between the containers Suitable include, for example, bottles, vials, syringes and test tubes. The containers can be made of a variety of materials such as glass or plastic. The recipient contains a compound that is effective for diagnosis or treatment of the disease and may have an access port sterile (for example, the container can be a bag or a vial of intravenous solution that has a pierceable plug by a hypodermic injection needle). The active agent of the compound is an antitumor agent of the present invention. The label of container, or associated therewith, indicates that the compound is used for the diagnosis or treatment of disease choice. The article of manufacture may further comprise a second container comprising a pharmaceutically buffer acceptable, such as phosphate buffered saline, solution of Ringer and dextrose solution. It can also include other commercially desirable materials and the user, including other buffers, thinners, filters, needles, syringes and package leaflets with instructions for use.

The following examples are only for illustrative, and are not intended to limit the scope in any way of the present invention.

Examples

Commercially available reagents at referenced in the examples were used according to the manufacturer's instructions unless otherwise indicated. The source of the cells identified in the following examples, and throughout the entire memory, through accession numbers ATCC is the American Type Culture Collection, Manassas, VA.

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Example 1 Extracellular domain homology screening 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 for 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 carried out using the BLAST or BLAST-2 software program (Altschul et al., Methods in Enzymology , 266 : 460-480 (1996)) by comparing the ECD protein sequences with a 6-frame translation of the EST sequences. These comparisons with a BLAST score of 70 (or in some cases 90) or higher that did not encode known proteins were grouped and assembled in consensus DNA sequences with the "phrap" program (Phil Green, University of Washington, Seattle, Washington).

Using this domain homology screening extracellular, the consensus DNA sequences were assembled with the other EST sequences identified using phrap. In addition, to often (but not always), consensus DNA sequences were extended obtained using repeated BLAST cycles or BLAST-2 and phrap to maximize the sequence consensus using the sources of commented EST sequences previously.

From the consensus sequences obtained as described above, oligonucleotides were synthesized and used to identify by PCR a cDNA library containing the sequence of interest and to use them as probes to isolate a clone of the coding sequence throughout length of a PRO polypeptide. The primers of direct CPR and reverse CPR generally have between 20 and 30 nucleotides and are often designed to provide a CPR product with an approximate length of 100-1,000 bp. The sequences of the probe usually have a length of 40-55 bp. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 pkb. To screen various libraries for a full length clone, the DNA of the libraries was analyzed by PCR amplification, as did Ausubel et al., Current Protocols in Molecular Biology , with the CPR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the oligonucleotides of the probe and one of the primer pairs.

The cDNA libraries used to isolate cDNA clones were constructed by standard procedures using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was prepared with oligo-dT containing a Notl site, directly bound to Sall hemikinase adapters, cleaved with Notl, given the appropriate size by gel electrophoresis and cloned in a given orientation in a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science , 253 : 1,278-1,280 (1991)) at the original Xhol and Notl sites.

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Example 7 Isolation of cDNA clones encoding human PRO224

A consensus DNA sequence was assembled from relative form to other EST sequences using "phrap" as described above in example 1. this consensus sequence It is referred to herein as ADN30845. Based on the sequence consensus DNA 30845, oligonucleotides were synthesized: 1) to identify by PCR a cDNA library containing the sequence of interest, and 2) to use as probes to isolate a clone of the coding sequence in its entire length for PRO224.

A pair of CPR primers (direct e reverse) were synthesized:

CPR primer direct: 5'-AAGTTCCAGTGCCGCACCAGTGGC-3 ' (I KNOW THAT ID NO: 26) primer CPR reverse: 5'-TTGGTTCCACAGCCGAGCTCGTCG-3 ' (I KNOW THAT ID NO: 27)

Additionally, a probe of synthetic oligonucleotide hybridization from the sequence DNA 30845 consensus that had the following sequence of nucleotides:

hybridization probe :

5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAATGCCCACC-3 ' (I KNOW THAT ID NO: 28)

To screen several libraries for a source of a full length clone, DNA from libraries was screened by PCR amplification with the pair of CPR primers identified previously. Therefore, a positive library was used to isolate clones encoding the PRO224 gene using the probe oligonucleotide and one of the PCR primers. RNA was isolated for the construction of cDNA libraries from liver tissue human fetal

DNA sequencing of isolated clones as described above provided the sequence of Full length DNA for DNA 33221-1133 [Figure 11, SEQ ID NO: 24]; and the derived protein sequence for the PRO224.

The complete DNA coding sequence 33221-1133 is included in Figure 11 (SEQ ID NO: 24). DNA clone 33221-1133 contains a single open reading frame with an apparent site of initiation of the translation at positions 33-35 of the nucleotide, and an apparent termination codon in positions 879-881 nucleotide. The precursor of the polypeptide Predicted has a length of 282 amino acids. The analysis of the sequence of the PRO224 along its entire length represented in the figure 12 (SEQ ID NO: 25) evidences the presence of a variety of important domains of the polypeptide, in which the locations given for these important domains of the polypeptide are approximate as described above. The analysis of full length PRO224 polypeptide depicted in Figure 2 evidences the presence of the following: a signal peptide of about 1 amino acid to about 30 amino acids; a extracellular domain of approximately 231 amino acids at approximately 248 amino acids; sites of N-glycosylation of approximately 126 amino acids to about 130 amino acids, from about 195 amino acids at about 199 amino acids, and about 213 amino acids to approximately 217 amino acids; sites of N-myristoylation of approximately 3 amino acids at about 9 amino acids, from about 10 amino acids to approximately 16 amino acids, from approximately 26 amino acids to about 32 amino acids, from about 30 amino acids to approximately 36 amino acids, from approximately 112 amino acids to approximately 118 amino acids, approximately 166 amino acids to about 172 amino acids, from about 212 amino acids at about 218 amino acids, about 224 amino acids to approximately 230 amino acids, of about 230 amino acids to about 236 amino acids, and from about 263 amino acids to about 269 amino acids; membrane lipoprotein lipid binding sites prokaryotic from about 44 amino acids to about 55 amino acids; Leucine zippers patterns of approximately 17 amino acids to approximately 39 amino acids. DNA clone 33221-1133 was presented along with the ATCC on 16 September 1997 and ATCC assigned deposit number 209263. The full length PRO224 protein depicted in Figure 12 it has an estimated molecular weight of approximately 28,991 daltons and a pI of about 4.62.

An analysis of the sequence of PRO224 in all its Length shown in Figure 12 (SEQ ID NO: 25) suggests that it has homology with very low density lipoprotein receptors. Apolipoprotein E receptor and P95 chicken oocyte receptor. Based on a BLAST and FastA sequence alignment analysis of the sequence in its entire length, PRO224 has an identity of amino acid sequence up portions of these proteins in a proportion of 28% to 45%, and a total identity with these proteins in a range of 33% to 39%.

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Example 15 In situ hybridization

In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify gene expression sites, analyze the distribution of transcription tissue, identify and locate viral infection, follow changes in specific mRNA synthesis, and assist in chromosomal mapping.

In situ hybridization was performed following an optimized version of the Lu and Gillert protocol, Cell Vision , I : 169-176 (1994), using 33-P-labeled ribosonde generated by CPR. In summary, human tissues embedded in paraffin, fixed with formalin were sectioned, deparaffinized, deprotected in proteinase K (20 g / ml) for 15 minutes at 37 ° C, and also processed for in situ hybridization as described in Lu and Gillert, supra An antisense ribosonde labeled UTP (33 -P) was generated from a CPR product and hybridized at 55 ° C overnight. The samples were immersed in Kodak NTB2? Nuclear emulsion and exposed for 4 weeks.

Synthesis of ribosonda 33 P

6.0 µL (125 mCi) of 33 P-UTP (Amersham BF 1002, SA <2000 Ci / mmol) They were dried at empty speed. For each tube it contains 33 P-UTP dried, the following are added ingredients:

2.0 µl 5x transcription buffer

1.0 µT DTT (100 mM)

2.0 µl NTP mix (2.5 mM: 10 µl every 10 mM GTP, CTP & ATP + 10 µL H 2 O)

1.0 µL UTP (50 µM)

1.0 µL RNA without

1.0 µL DNA template (1 \ mug)

1.0 µL H 2 O

1.0 µL RNA polymerase (for CPR products T3 = AS, T7 = S, normally).

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These tubes covered up at 37 ° C for a hour. A total of 1.0 µL RQI Dnasa was added, followed by a incubation at 37 ° C for 15 minutes. A total of 90 µL TE 10 mM Tris pH 7.6 / 1 mM EDTA pH 8.0) was added, and the mixture was pipetted into a paper DE81. The remaining solution was loaded in a unit of MICROCON-50? ultrafiltration, and centrifuged using program 10 (6 minutes). The filtration unit is Invested on a second tube and centrifuged using the Program 2 (3 minutes). After the final recovery spin, a total of 100 µL TE was added and then 1 µl of product final was pipetted into a DE81 paper and counted in 6 ml of BIOFLUOR ™.

The probe was run on a TBE / urea gel. A total 1-3 µl of the probe or 5 µl of ARM Mrk III It was added to 3 µl of loading buffer. After heating in a heat block at 95 ° C for three minutes, the gel was placed immediately on the ice. The gel samples were emptied, and the Sample was charged and exposed to 180-250 volts for 45 minutes The gel was wrapped in a plastic wrap (SARAN? brand) and was exposed to the XAR film with a screen intensifier in a freezer at 70ºC one hour until all the night.

Hybridization - 33 P A. Pretreatment of frozen sections

The samples were removed from the freezer, placed in aluminum trays and thawed at temperature ambient for 5 minutes. The trays were placed in a incubator at 55 ° C for five minutes to reduce the condensation. Samples were fixed for 10 minutes in 4% of polyacetal on ice in the extractor hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H2O). After deproteinization in 0.5 µg / ml of Proteinase K for 10 minutes at 37 ° C (12.5 ml of 10 mg / ml stock in 250 ml preheated RNase-free buffer RNase), the sections were washed in 0.5 x SSC for 10 minutes at room temperature. Sections were dehydrated at 70%, 95% and 100% ethanol, 2 minutes each.

B. Pretreatment of embedded sections in paraffin

The samples were deparaffinized, placed in SQ H2O, and washed twice in 2 x SSC at temperature atmosphere, for 5 minutes each time. The sections are deproteinized in 20 µl / ml proteinase K (500 µl of 10 mg / ml in 250 ml of RNase free RNase buffer; 37 ° C, 15 minutes) for human embryonic tissue, or 8 x proteinase K (100 µl in 250 ml of RNase buffer, 37 ° C, 30 minutes) for formalin tissues. He subsequent rinse at 0.5 x SSC and dehydration were performed as described above.

C. Prehybridization

The samples were placed in a box of Plastic lined with Box buffer (a x SSC, 50% formamide) -paper saturated filter The tissue was covered with 50 µl of buffer hybridization (3.75 g dextran sulfate + 6 ml of SQ H2O), it was mixed and heated in the microwave for 2 minutes with the loose cap. After cooling on ice, 18.75 ml of formamide, 3.75 20 x SSC ml, and 9 ml of SQ H2O were added, and the tissue was mixed and incubated at 42 ° C for 1-4 hours.

D. Hybridization

1.0 x 10 4 of cpm probe and 1.0 were heated µl tRNA (50 mg / ml stock) per sample at 95 ° C for 3 minutes. The samples were cooled with ice, and 48 µl of sample hybridization buffer. After mixing, 50 were added [mu] 33 P mix at 50 [mu] of prehybridization in the sample. The samples were incubated overnight at 55 ° C.

E. Washes

Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25 M EDTA, V f = 4L) followed by RNase treatment at 37 ° C for 30 minutes (500 µL of 10 mg / ml in 250 ml of RNase buffer = 20 \ mug / ml. The samples were washed 2 x 10 minutes with 2 x SSC, EDTA at room temperature. The restrictions of the conditions of washing were as follows: 2 hours at 55 ° C, 0.1 x SSC, EDTA (20 ml x SSC + 16 ml EDTA, V f = 4L).

F. Oligonucleotides

An in situ analysis of the DNA sequences described herein was performed. The oligonucleotides used for this analysis were the following:

(4) DNA33221-1133 (PRO224)

p1: 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GCA GCG ATG GCA GCG ATG AGG-3 ' (SEQ ID NO: 74)

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p2: 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CAG ACG GGG CAG CAG GGA GTG-3 ' (SEQ ID NO: 75)

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G. Results

An in situ analysis was performed on the previous DNA sequences described herein. The results of these analyzes are as follows:

(3) DNA33221-1133 (PRO224) (LDLR homolog-1 TM)

The expression observed was limited to the endothelium vascular in a fetal spleen, adult spleen, fetal liver, Thyroid adult and lymph node (chimpanzee). A site was seen additional expression in developing spinal ganglia. He rest of the tissues were negative.

The fetal human tissues examined were: kidney (normal and terminal), adrenal, myocardium, aorta, spleen, ganglion lymphatic, pancreas, lung, skin, eye (including the retina), bladder and liver (normal, cirrhotic, acute insufficiency).

The nonhuman tissues examined were:

Chimpanzee tissue: salivary gland, stomach, Thyroid, parathyroid, skin, thymus, ovary, lymph node.

Rhesus monkey tissues: cerebral cortex, hippocampus, cerebellum, penis.

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Example 16 Use of PRO224 as a hybridization probe

The following procedure describes the use of a nucleotide sequence encoding PRO224 as a hybridization probe.

The DNA that comprises the coding sequence of PRO224 in full length or mature (as depicted in Figure 1, SEQ ID NO: 1) or a fragment thereof is used as a probe to screen homologous DNAs (such as variants encoders of the naturally occurring PRO224) in cDNA libraries of human tissue or genomic libraries of human tissue.

Hybridization and washing of the filters that contain any of the DNA of the libraries is carried out in the following conditions of high restriction. The hybridization of a radiolabelled probe obtained from the gene encoding a PRO224 polypeptide in the filters is carried out in a solution 50% formamide, 5x SSC, 0.1% SDS, sodium pyrophosphate 0.1%, 50 mM sodium phosphate, pH 6: 8, 2x Denhardt solution and 10% dextran sulfate at 42 ° C for 20 hours. The washing of the Filters are carried out in an aqueous solution of SSC 0.1x and SDS 0.1% at 42 ° C.

Next, the DNAs that can be identified they have the desired sequence identity with the natural sequence throughout its length of the coding DNA using standard techniques known in the state of the art.

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Example 17 Expression of PRO224 in E. coli

This example illustrates the preparation of a form of PRO224 without glucosilar by recombinant expression in E. coli .

The DNA sequence encoding PRO224 is initially amplified using selected PCR primers. The primers should contain sites in the restriction enzyme that correspond to the restriction enzyme sites 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 restriction enzymes and dephosphorylated. The amplified sequences of the PCR are then attached to the vector. The vector will preferably include sequences encoding an antibiotic resistance gene, a trp promoter, a poly-his leader (including the first six STII codons, poly-his sequence and enterokinase cleavage site), the PRO224 coding region, the terminator Lambda transcriptional and an argU gene.

The ligation mixture is then used to transform a strain of E. coli selected using the procedures described in Sambrook et al., Supra . Transformers 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.

Some clones can be grown throughout the night in a liquid culture medium such as a LB broth supplemented with antibiotics. The overnight crop can subsequently used to inoculate a larger scale culture. The cells are then cultured at an optical density desired, during which the expression promoter is activated.

After culturing the cells for several hours more, cells can be collected by centrifugation. He cell pellet obtained by centrifugation can solubilized using several agents known in the state of the technique, and then the PRO224 protein can be purified solubilized using a metal chelating column in conditions that allow tight protein binding.

The PRO224 can be expressed in E coli in a tagged poly-his form, using the following procedure. The DNA encoding the PRO224 is initially amplified using selected PCR primers. The primers will contain sites in the restriction enzyme that correspond to the sites in the restriction enzyme of the selected expression vector, and other useful sequences that provide a reliable and efficient initiation of translation, rapid purification in the metal chelating column and proteolytic elimination with enterokinase. The tagged poly-his sequences amplified by PCR are then linked to an expression vector, which is used to transform an E. coli host based on a strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (laclq) The transformers are first grown in LB containing 50 mg / ml carbenicillin at 30 ° C with stirring until an OD600 of 3-5 is obtained, then the cultures are diluted 50-100 times in CRAP medium ( prepared by mixing 3.57 g (NH 4) 2 SO 4, 0.71 g of sodium citrate • 2 H, 1.07 g KCl, 5.36 g of yeast extract Difco, 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 grown for approximately 20-30 hours at 30 ° C. with agitation Samples are extracted to verify expression by SDS-PAGE analysis, and the bulk of the culture is centrifuged to sediment the cells. gelated until purification and refolding.

The E. coli paste from 0.5 to 1 L fermentations (6-10 g sediments) is resuspended in 10 volumes of buffer (w / v) in 7 M guanidine, 20 mM Tris, pH 8. Sulfite is added solid sodium and sodium tetraionate to obtain the final concentrations of 0.1 M and 0.02 M, respectively, and the solution is kept under stirring overnight at 4 ° C. This step 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 buffer from a metal chelating column (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded into a 5 ml Ni 2 + -NTA Qiagen metal chelating column equilibrated in the buffer of the metal chelating column. The column is washed with an additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein are grouped and stored at 4 ° C. The protein concentration is calculated by its absorbance at 280 nm using the extinction coefficient calculated from its amino acid sequence.

Proteins are folded down diluting the slowly show in a freshly prepared fold buffer that Consists of: 20 mM tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, Cysteine S mM, 20 mM glycine and 1 mM EDTA. Withdrawal volumes are chosen so that the final protein concentration is between 50 and 100 micrograms / ml The withdrawal solution is gently stirred at 4 ° C for 12-36 hours. The withdrawal reaction it ends with the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before another purification of the protein, the solution is filtered through a 0.22 filter microns and acetonitrile is added until the final concentration of 2-10% The refolded protein is subjected to Pores RI / H reverse phase column chromatography using a 0.1% TFA mobile buffer with elution with a gradient of 10 to 80% acetonitrile. Samples of fractions with absorbance A 180 are analyzed in polysacrylamide gels SDS and the fractions containing homogeneous refolded protein are grouped Generally, appropriately folded species of most proteins are eluted at lower concentrations acetonitrile since these species are the most compact with their hydrophobic internal part that prevents interaction with the resin of reverse phase Aggregate species usually elute at higher concentrations of acetonitrile. In addition, to separate the misfolded forms of proteins of the desired form, the stage of Reverse phase also eliminates endotoxin from the samples.

Fractions containing the polypeptide PRO224 desired folding are grouped and acetonitrile removed using a gentle stream of nitrogen directed at the dissolution. Proteins are 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 resins (Pharmacia) balanced in the formulation buffer and filtered so sterile.

PRO224 was successfully expressed in E. coli in a labeled form (tagged) polyhis by the above procedure.

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Example 18 Expression of PRO224 in mammalian cells

This example illustrates the preparation of a form potentially glycosylated from PRO224 by expression recombinant in mammalian cells.

The vector, pRK5 (see EP 307,247, published March 15, 1989), is used as the expression vector. Optionally, PRO224 DNA is ligated to pRK5 with restriction enzymes selected to allow insertion of PRO224 DNA using ligation procedures such as those described in Sambrook et al., Supra . The resulting vector is called pRK5-PRO224.

In one embodiment, the selected host cells may be 293 cells. The human 293 cells (ATCC CCL 1573) are cultured to converge in tissue culture plates in a medium such as DMEM supplemented with fetal calf serum and optionally nutrients and / or antibiotics. . Approximately 10 µg of pRK5-PRO224 DNA is mixed with about 1 µg of DNA encoding the VA RNA RNA gene [Thimmappaya et al., Cell , 31 : 543 (1982)] and dissolved in 500 µl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl 2. To this mixture, 500 µL of 50 mM HEPES (pH 7.35), 280 mM NaCl, NaPO 4 are added dropwise 1.5 mM, and this allows the formation of a precipitate at 10 minutes at 25 ° C. The precipitate is suspended and added to the 293 cells and allowed to stand for about four hours at 37 ° C. The culture medium is extracted and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with a serum free medium, a fresh medium is added and the cells are incubated for approximately 5 days.

After approximately 24 hours of transfections, the culture medium is removed 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 12 hours of incubation, extract the conditioned medium, concentrate on a filter centrifugation and loaded on a 15% SDS gel. The processed gel can dry and let it form a film for a period of selected time to discover the presence of the polypeptide PRO224. Cultures containing transfected cells can undergo another incubation (in a serum-free medium) and the medium It is analyzed in selected bioanalysis.

In an alternative technique, PRO224 can be temporarily introduced into 293 cells using the dextran sulfate method described by Somparyrac et al., Proc. Natl Acad. Sci ., 12 : 7,575 (1981), 293 cells are cultured to a maximum density in a centrifuge flask and 700 µg of pRK5-PRO224 DNA is added. First, the centrifuge flask cells are concentrated 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 centrifuge flask containing tissue culture medium, 5 µg / ml bovine insulin and 0.1 µg / ml of bovine transferrin. After about four days, the conditioned medium is centrifuged and filtered to remove cells and residues. The sample containing the expressed PRO224 can then be concentrated and purified by any selected procedure, such as dialysis and / or column chromatography.

In another embodiment, it can be expressed in CHO cells. The pRK5-PRO224 can be transfected in CHO cells using known reagents such as CaPO or DEAE-dextran. As described previously, cell cultures can be incubated and the medium substituted by culture medium (alone) or by a medium that contain a radioactive marker such as "S-methionine." After determining the presence of a PRO224 polypeptide, the culture medium can be replaced by a serum free medium. Preferably, the cultures are incubated for about 6 days and then pick up the conditioned medium. Then you can concentrate and Purify the medium containing the expressed PRO224 polypeptide by any selected procedure.

The epitope tagged PRO224 (tagged) can also be expressed in host CHO cells. The PRO224 can be subcloned from the pRK5 vector. The subclone insert can undergo CPR to merge in frame with a selected tag epitope such as a poly-his tag in a baculovirus expression vector. The poly-his tagged PRO224 insert can then be subcloned into an SV40 directed vector containing a selection marker such as DHFR for the selection of stable clones. Finally, CHO cells can be transfected (as described above) with an SV40 targeted vector. Marking can be carried out, as described above, to verify the expression. The culture medium containing the labeled tagged PRO224 expressed by any selected method can then be concentrated and purified by means of any Ni 2+ chelate affinity chromatography.

PRO224 can also be expressed in cells CHO and / or COS through a transient expression procedure or in CHO cells by another stable expression procedure.

Stable expression in CHO cells is carried out using the following procedure. Proteins are expressed as an IgG (immunoadhesin) construct, in which the coding sequences of the soluble forms (eg, extracellular domains) of the respective proteins are fused to a sequence of IgGI constant regions containing the hinge region, the domains CH2 and CH2 and / or as a tagged form (tagged) poly-his.

After PCR amplification, the respective DNAs are subcloned into a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology , Unit 3.16, John Wiley and Sons (1997). The CHO expression vectors are constructed to have the compatible 5 'and 3' restriction sites of the DNA of interest to allow convenient release of the cDNAs. The vector used in the expression in CHO cells is as described in Lucas et al., Nucl. Acids Res ., 24 : 9 (1,774-1,779 (1996) and uses the SV40 immediate promoter / enhancer to drive the expression of the cDNA of interest and dihydrofolate reductase (DHFR). The expression of DHFR allows selection for stable maintenance of the plasmid after transfection.

Twelve micrograms of the desired plasmid DNA are introduced into approximately 10 million CHO cells using the commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim). Cells are cultured as described in Lucas et al., Supra . Approximately 3x10-7 cells are frozen in a glass container for further cultivation and production as described below.

Glass containers containing DNA Plasmid are liquefied by placing them in a water bath and mixed in vortex. The content is pipetted into a centrifuge tube that It contains 10 ml of medium and centrifuged at 1,000 rpm for 5 minutes The supernatant is aspirated and the cells are resuspended. in 10 ml of selective medium (0.2 µm of PS20 filtered with 0.2 µm of 5% diafiltered fetal bovine serum). Then you distribute the cells in a 100 ml centrifuge containing 90 ml of selective media. After 1-2 days, the cells are transferred to a 250 ml centrifuge that It contains 150 ml of selective culture medium and is incubated at 37 ° C. After another 2-3 days, the seeds are sown 250 ml, 500 ml and 2,000 ml centrifuges with 3x103 cells / ml. Cellular media are replaced by fresh media. prepared by centrifugation and resuspension in a medium of production. Although any suitable CHO media can be used, in fact a means of production described in the U.S. Patent US 5,122,469, published June 16, 1992. 1.2x106 are seeded in a 3 l production centrifuge cells / ml. On day 0, the number of cells and the pH are determined. He day 1, the centrifuge is sampled and stirring is started with filtered air On day 2, the centrifuge is sampled, modified the temperature up to 33 ° C and 30 ml of glucose 500 g / l and 0.6 are taken ml of 10% antifoam (for example, emulsion of 35% polydimethylsiloxane; Dow Coming 365 Medical Grade Emulsion). During production, the pH is adjusted when necessary to keep it around 7.2. After 10 days, or until the viability decrease below 70%, the crop is harvested cell by centrifugation and filtering through a filter of 0.22 µm. The filtrate is stored at 4 ° C or charged with immediately in columns for purification.

In the case of poly-his tagged constructs, the proteins are purified using a Ni2 + -NTA column (Qiagen). Before purification, imidazole is added to the conditioned medium to a concentration of 5 mM. The conditioned medium is pumped into a 6 ml Ni 2 + -NTA column equilibrated in 20 mM Hepes, pH 7.4, a buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4 -5 ml / min at 4 ° C. After loading, the column is washed with an additional equilibration buffer and the protein is eluted with an equilibrium buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted in a storage buffer containing 10 mM Hepes. , 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine column (Pharmacia) and stored at -80 ° C.

Immunoadhesin constructs (containing Fc) are purified from conditioned media as follows. The conditioned medium is pumped into a 5 ml protein A column (Pharmacia) that has been equilibrated in a 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed thoroughly with an equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by extraction of 1 ml fractions in tubes containing 275 µl of 1M Tris buffer, pH 9. Subsequently, the highly purified protein is desalted in a storage buffer as described above for the proteins. tagged poly-his. Homogeneity is evaluated by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.

PRO224 was stably expressed in CHO cells by the procedure described above. In addition, PRO224 was expressed in CHO cells by transient expression procedure.

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Example 19 Expression of PRO224 in yeast

The following procedure describes the expression PRO224 recombinant in yeast.

First, the vectors of Yeast expression for intracellular production or secretion of PRO224 from the ADH2 / GAPDH promoter. The DNA that encodes the PRO224 and the promoter is inserted into suitable sites of the enzyme restriction in the plasmid selected to direct the expression Intracellular PRO224. For secretion, it can be cloned the DNA encoding PRO224 in the selected plasmid, together with the DNA encoding the ADH2 / GAPDH promoter, a signal peptide of the Natural PRO224 or other mammalian signal peptide, or, for example, a yeast alpha factor or a leader sequence / secretory signal of invertase, and connecting sequences (if necessary) for the PRO224 expression.

Then cells can be transformed from yeasts, such as yeast strain AB110, with plasmids of expression described above and cultured in media of selected fermentation. Yeast supernatants transformed can be analyzed by acid precipitation 10% trichloroacetic and separated by SDS-PAGE, followed by staining offspring with Coomassie blue staining.

The recombinant PRO224 can be isolated and be purified by subsequently removing yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO224 can also be purified using selected column chromatography resins.

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Example 20 Expression of PRO224 in insect cells infected by baculovirus

The following procedure describes the expression recombinant in insect cells infected by baculovirus.

The sequence encoding PRO224 is fused in the 5 'direction of a tag epitope contained within a baculovirus expression vector. Such epitopes include poly-his tag tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids can be used, including commercially available plasmid derived plasmids such as pVL1393 (Novagen). In summary, the sequence encoding the PRO224 or the desired part of the coding sequence of the PRO224 (such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular) is amplified by CPR with complementary primers up to the 5 'and 3' regions. The 5 'primer can incorporate flanking restriction enzyme sites (selected). The product is then digested with those restriction enzymes selected and subcloned into the expression vector.

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Recombinant baculovirus is generated by cotransfection of the anterior plasmid and Baculovirus DNA
Gold? (Pharmingen) in Spodoptera frugiperda ("Sf9") cells (ATCCCRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses are collected and used in other amplifications. Viral infection and protein expression are carried out as described by O'Reilley et al., Baculovirus expression vectors : A Laboratory Manual , Oxford: Oxford University Press (1994).

The poly-his labeled tagged PRO224 can then be purified, for example, by Ni2 + chelating affinity chromatography as follows. Extracts are prepared from virus-infected recombinant Sf9 cells as described by Rupert et al., Nature , 362 : 175-179 (1993). In summary, the Sf9 cells are washed, resuspended in a sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 10 NP-40 0.1%; 0.4 M KCl) and twice subjected to ultrasound exposure for 20 seconds on ice. The sonicated extract is removed by centrifugation, the supernatant is diluted 50 times in a loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0 filter , 45 mm. 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 loading buffer. The filtered cell extract is loaded into the column at 0.5 ml per minute. The column is washed until the initial state A 280 is recovered with a loading buffer, at which time the fraction collection begins. The column is then washed with a secondary wash buffer (50 mM phosphate: 300 mM NaCl, 10% glycerol, pH 6.0), which elutes the bound protein in a nonspecific manner. After reaching the initial state A 280 again, the column is tuned with an imidazole gradient of 0 to 500 mM in the secondary wash buffer. One ml fractions are collected and analyzed by SDS-PAGE and silver staining or by immunoblotting with Ni2 + -NTA-conjugated with alkaline phosphatase (Qiagen). Fractions containing the tagged PRO224 His_ {10} eluted are grouped and dialyzed against the loading buffer.

Alternatively, purification of PRO224 labeled (tagged) (or Fc tagged) of IgG can be performed using known chromatography techniques, among which include, for example, column chromatography protein A or protein G.

After PCR amplification, the respective coding sequences are subcloned into a baculovirus expression vector (pb.PH.lgG for IgG fusions and pb.PH.His.c for tagged poly-his tagged proteins), and the vector and Baculogold® Baculovirus® DNA (Pharmingen) are co-transfected into Spodoptera frugiperda ("Sf9") 105 (ATCC CRL 1711) cells, using lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polyonector regions to include the his or Fc tag sequences. The cells are cultured in Hink TNM-FH medium supplemented with 10% FBS (Hyclone). The cells are incubated for 5 days at 28 ° C. The supernatant is collected and subsequently used for the first viral amplification by infection of Sf9 cells in Hink TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. The cells are incubated for 3 days at 28 ° C. The supernatant is harvested and the expression of the constructs is determined in the expression vector of baculoviruses by batch binding of 1 ml of supernatant to 25 mL of Ni ^ {2 +} - NTA (QIAGEN) for etiquetads proteins (tagged) of histidine or with CL-4B beads on a Sepharose column of Protein A (Pharmacia) for tagged IgG proteins followed by SDS-PAGE analysis comparing with a known concentration of standard protein by Coomassie blue staining.

The first supernatant of amplification viral is used to infect a cell culture with agitation centrifuge (500 ml) of Sf9 cells cultured in medium ESF-921 (Expression Systems LLC) to an MOI about 0.1. The cells are incubated for 3 days at 28 ° C. The supernatant is collected and filtered. The union is repeated by lots and the SDS-PAGE analysis as many times as necessary until cell culture expression is confirmed with centrifugal agitation.

The conditioned medium of the transfected cells (0.5 to 3 L) is collected by centrifugation to remove the cells and filtered through 0.22 micron filters. For the tagged poly-his constructs, the protein construct is purified using a Ni2 + -NTA column (Qiagen). Before purification, imidazole is added to the conditioned medium to a concentration of 5 mM. The conditioned medium is pumped to a 6 ml Ni 2 + -NTA column equilibrated in 20 mM Hepes, pH 7.4, a buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4 -5 ml / min at 4 ° C. After loading, the column is washed with an additional equilibration buffer and the protein is eluted with the equilibrium buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted in a storage buffer containing 10 mM Hepes. , 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine column (Pharmacia) and stored at -80 ° C.

Immunoadhesin (containing Fc) protein constructs are purified from conditioned media as follows. The conditioned medium is pumped into a 5 ml protein A column (Pharmacia) that has been equilibrated in a phosphate buffer containing 20 mM Na and pH 6.8. After loading, the column is washed extensively with an equilibration buffer before elution with 100 mM citric acid, at pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions in tubes containing 275 ml of 1M Tris buffer, at pH 9. The highly purified protein is subsequently desalted in a storage buffer as described above for labeled proteins (tagged) poly-his. The homogeneity of the proteins is verified by SDS polyacrylamide gel electrophoresis (PEG) and amino acid sequencing of the terminal end by Edman degradation.

Alternatively, a procedure with modified baculovirus incorporating high-5 cells can be used. In this procedure, the DNA encoding the desired sequence is amplified with suitable systems, such as Pfu (Stratagene), or fused in the 5 'direction of a tag epitope contained in a baculovirus expression vector. Such epitope tags include poly-of His tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids can be used, including plasmids obtained from commercially available plasmids such as plE1-1 (Novagen). PlE1-1 and plE1-2 vectors have been designed for constitutive expression of recombinant proteins from the baculovirus ie1 promoter in stably transformed insect cells (I). Plasmids differ only in the orientation of the multiple cloning sites and contain all promoter sequences that are known to be important for IE1-mediated gene expression in uninfected insect cells, as well as the hr5 enhancer element, pIE1-1 and pIE1-2 include the translation initiation site and can be used to produce fusion proteins. In summary, the desired sequence or the desired part of the sequence (such as the sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5 'and 3' regions. The 5 'primer can incorporate flanking restriction enzyme sites (selected). The product is then digested with those restriction enzymes selected and subcloned into the expression vector. For example, derivatives of pIE1-1 may include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine tag (pb.PH.His) in the 3 'direction of the desired sequence. Preferably, the construction of the vector is sequenced for confirmation.

High-5 cells are grown to a confluence of 50% under the following conditions: 27 ° C, absence of CO2, NO pen / strep. For each 150 mm plate, 30 µl of the pIE-based vector containing the sequence is mixed with 1 ml of Ex-Cell medium (medium: Ex. Cell 401 + 1/100 l-Glu JRH Biosciences No. 14401- 78P (note: this medium is light sensitive)), and in a separate tube 100 µl of CellFectin (CellFECTIN (G ibcoBRL No. 10362-010) (mixed in vortex)) is mixed with 1 ml of Ex medium -Cell. The two solutions are combined and allowed to incubate at room temperature for 15 minutes. 8 ml of Ex-Cell medium is added to 2 ml of the DNA / CellFECTIN mixture and it is spread on high-5 cells that have been washed once with Ex-Cell medium. The plate is then incubated in the dark for 1 hour at room temperature. The DNA / CellFECTIN mixture is then aspirated and the cells are washed once with Ex-Cell to remove excess CellFECTIN, 30 ml of freshly prepared Ex-Cell medium is added and the cells are incubated for 3 days at 28 ° C . The supernatant is harvested and the expression of the sequence is determined in the expression vector of baculoviruses by batch binding of 1 ml of supernatant to 25 mL of Ni ^ {2 +} - NTA (QIAGEN) for labeled proteins (tagged) of histidine or with CL-4B Sepharose protein A (Pharmacia) beads for tagged IgG proteins followed by SDS-PAGE analysis compared to a known concentration of protein reference by Coomassie blue staining.

The conditioned medium of the transfected cells (0.5 to 3 L) is collected by centrifugation to remove the cells and filtered through 0.22 micron filters. For poly-his tagged constructs, the protein comprising the sequence is purified using a Ni2 + -NTA column (Qiagen). Before purification, imidazole is added to the conditioned medium to a 5 mM concentration. The conditioned medium is pumped to a 6 ml Ni 2 + -NTA column equilibrated in 20 mM Hepes, pH 7.4, a buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4 -5 ml / min at 48 ° C. After loading, the column is washed with an additional equilibration buffer and the protein is eluted with the equilibrium buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted in a storage buffer containing 10 mM Hepes. , 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine column (Pharmacia) and stored at -80 ° C.

Immunoadhesin constructs (containing Fc) are purified from conditioned media as follows. The conditioned medium is pumped into a 5 ml protein A column (Pharmacia) that has been equilibrated in a phosphate buffer containing 20 mM Na and with a pH of 6.8. After loading, the column is washed extensively with an equilibration buffer before elution with 100 mM citric acid, at pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions in tubes containing 275 ml of 1M Tris buffer, at pH 9. The highly purified protein is subsequently desalted in a storage buffer as described above for labeled proteins (tagged) poly-his. The homogeneity of the sequence is evaluated by SDS polyacrylamide gels and by amino acid sequencing of the N-terminus by degradation of Edman and other analytical procedures depending on what is desired or needed.

PRO224 was expressed using the procedure of anterior bculovirus using high-5 cells.

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Example 21 Preparation of antibodies that bind to PRO224

This example illustrates the preparation of monoclonal antibodies that can specifically bind to PRO224.

Techniques for producing monoclonal antibodies are known in the state of the art and are described, for example, in Goding, supra . Immunogens that may be used include purified PRO224, fusion proteins containing PRO224 and cells expressing recombinant PRO224 on the cell surface. Immunogen selection can be carried out by any person skilled in the art.

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Mice, such as Balb / c, are immunized with the immunogen PRO224 emulsified in complete adjuvant of Freund and injected subcutaneously or intraperitoneally in a amount of 1-100 micrograms. Alternatively, the Immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research Hamilton MT) and injected in the legs Rear of the animal. Then the immunized mice receive a booster shot 10-12 days later with additional immunogen emulsified in the selected adjuvant. After this, for several weeks, mice can also receive booster shots with immunization shots additional. Serum samples can be obtained periodically from mice using retroorbital bleeding for analysis ELISA to detect anti-PRO224 antibodies.

After detecting an appropriate degree of antibodies, animals "positive" for antibodies can receive a last intravenous injection of PRO224. After three or four days, the mice are sacrificed and the Spleen cells The spleen cells are then fused (using 35% polyethylene glycol) with a cell line of selected murine myeloma, such as P3X63AgU. 1, available in the ATCC, CRL No. 1597. Mergers generate hybridoma cells that at then they can be seeded in 96 cell culture plates wells containing 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 by ELISA in search of reactivity against PRO224. The determination of "positive" hybridoma cells that secrete antibodies desired monoclonal anti-PRO224 is part of The skills of the technique.

Positive hybridoma cells can be injected intraperitoneally in synergistic Balb / c mice to produce ascites that contains monoclonal antibodies anti-PRO224. Alternatively, the cells of hybridoma can be grown in tissue culture flasks or "roller bonles". The purification of the antibodies Monoclonal produced in ascites can be achieved by precipitation of ammonia sulfate, followed by chromatography of gel exclusion Alternatively, the affinity chromatography based on antibody binding to the protein A or protein G.

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Example 22 Purification of PRO224 polypeptides using antibodies specific

Natural or PRO224 polypeptides Recombinants can be purified by a variety of techniques standard in the field of protein purification. For example, the pro-PRO224 polypeptide, the PRO224 polypeptide mature or pre-PRO224 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO224 polypeptide of interest. In general, a immunoaffinity column is prepared by covalent binding of anti-PRO224 polypeptide antibody with a activated chromatographic resin.

Polyclonal immunoglobulins are prepared to starting immune serum either by precipitation with sulfate ammonia or by purification on an immobilized protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Also, the monoclonal antibodies are prepared from ascites fluid mouse by precipitation with ammonia sulfate or by chromatography on immobilized protein A. Immunoglobulin partially purified covalently binds to a resin chromatographic such as SEPHAROSE ™ activated with CnBr (Pharmacia LKB Biotechnology). The antibody binds to the resin, it block the resin and wash the resin obtained in accordance with the manufacturer's instructions

Said immunoaffinity column is used in the purification of the PRO224 polypeptide by preparing a fraction of cells containing the PRO224 polypeptide in a form soluble. This preparation is obtained by solubilization of the whole cell or a subcellular fraction obtained by differential centrifugation by adding detergent or by other procedures well known in the state of technique. Alternatively, the useful amount can be secreted in soluble PRO224 polypeptide containing a signal sequence in the medium where cells are grown.

A preparation containing the PRO224 polypeptide soluble by the immunoaffinity column and is wash the column under conditions that allow absorbance Preferred PRO224 polypeptide (eg, high buffers ionic strength in the presence of detergent). Then eludes the column in conditions that interrupt the union PRO224 antibody / polypeptide (for example, a low pH buffer such as about pH 2-3, or a concentration raised from a chaotropic such as urea or thiocyanate ion) and collected PRO224 polypeptide.

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Example 23 Drug screening

This invention is particularly useful for screening compounds by PRO224 polypeptides or a binding fragment thereof in any of a variety of drug screening techniques. The PRO224 polypeptide or fragment used in such a test may be free in the solution, be fixed to a solid support, be on the surface of a cell, or be located intracellularly. A drug screening method uses eukaryotic or prokaryotic host cells that are stably transformed with recombinant nucleic acids expressing the PRO224 polypeptide or fragment. The drugs are screened against such transformed cells in competitive binding analysis. Said cells, either in a viable or fixed form, can be used for standard binding analysis. For example, the formation of complexes between a PRO224 polypeptide or a fragment thereof and the agent being tested can be measured. Alternatively, the decrease in complex formation between the PRO224 polypeptide and its target cell or the target receptors caused by the agent being examined can be examined.
testing.

In fact, the present invention provides screening procedures for drugs or other agents that may affect a disease or disorder associated with a polypeptide PRO224. These procedures include contacting such agent with a PRO224 polypeptide or a fragment thereof and analyze: (i) in search of the presence of a complex between the agent and the PRO224 polypeptide or a fragment thereof, or (ii) in looking for the presence of a complex between the PRO224 polypeptide or fragment thereof and the cell, by procedures well known in the state of the art. In said binding analysis competitively, usually the PRO224 polypeptide, or fragment of the Same, is marked. After proper incubation, the PRO224 free polypeptide, or fragment thereof, of that present together, and the amount of free or unmarked marker complexes is a measure of the ability of the particular agent to bind to the PRO224 polypeptide or to interfere with the complex PRO224 polypeptide / cell.

Another technique for screening drugs provides a very productive screening for compounds that have an affinity of suitable binding to a polypeptide and is described in detail in WO 84/03564, published September 13, 1984. To put it briefly, large amounts are synthesized.  of different small peptide test compounds on a solid substrate, such as plastic needles or some other surface. As for the PRO224 polypeptide, the peptide test compounds with the PRO224 polypeptide and it wash. The bound PRO224 polypeptide is detected by procedures well known in the state of the art. He purified PRO224 polypeptide can also directly cover culture plates for use in screening techniques drugs mentioned above. In addition, they can be used non-neutralizing antibodies to trap peptide e immobilize it on a solid support.

This invention also contemplates the use of competitive drug screening analysis in which neutralizing antibodies capable of binding to the PRO224 polypeptide specifically compete with a test compound to join the PRO224 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 a PRO224 polypeptide.

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Example 24 Reasonable drug design

The objective of a reasonable drug design is to produce structural analogs of a biologically active polypeptide of interest (i.e., a PRO224 polypeptide) or of low molecular weight 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 PRO224 polypeptide or that enhance the function of the PRO224 polypeptide in vivo , or interfere with it (cf. Hodgson, Bio / Technology , 9 : 19-21 ( 1991)).

In one procedure, the three-dimensional structure of PRO224, or of a complex inhibitor of the PRO224 polypeptide, is determined by x-ray crystallography, by computer modeling or, more commonly, by a combination of both procedures. Both the form and loads of the PRO224 polypeptide must be determined to explain the structure and determine the active site or sites of the molecule. Less frequently, useful information about the structure of the PRO224 polypeptide can be obtained by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design molecules similar to the PRO224 polypeptide or to identify effective inhibitors. Useful examples of reasonable drug design include molecules that have better activity or stability, as demonstrated by Braxton and Wells, Biochemistry , 31 : 7,796-7,801 (1992), or that act as inhibitors, agonists, or peptide antagonists natural as demonstrated by Athauda et al., J. Biochem ., 113 : 742-746 (1993).

A specific antibody can also be isolated. of a target, selected by functional analysis, as described above, and then solve its structure crystalline This procedure, in principle, provides a "pharmacore" on which the subsequent design of drugs It is possible to completely dispense with crystallography of proteins by generating anti-idiotypic antibodies against a functional, pharmacologically active antibody. Like a mirror image of a mirror image, the site would be expected binding of the anti-idiotypic antibodies were an analogue of the original receiver Then the antibody could be used antidiotypic to identify and isolate peptides from banks of chemically or biologically produced peptides. Isolated peptides they would act then as the "pharmacore".

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Under the present invention, they can sufficient quantities of the PRO224 polypeptide are available to perform such analytical studies as ray crystallography X. In addition, knowledge of the amino acid sequence of the PRO224 polypeptide provided herein will guide those use computer modeling techniques instead, or In addition, X-ray crystallography.

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Example 25 In vitro antitumor analysis

The antiproliferative activity of PRO224 polypeptides was determined in the experimental in vitro disease-oriented identification of anticancer drugs from the National Cancer Institute (NCI), using a binding analysis based on sulforhodamine B staining (SRB), essentially as described Skehan et al., J. Natl. Cancer Inst ., 82 : 1,107-1,112 (1990). The 60 tumor cell lines used in this study ("the NCI panel"), as well as the conditions for their maintenance and in vitro culture have been described by Monks et al., J. Natl. Cancer Inst ., 83 : 757-766 (1991). The objective of this analysis is to initially evaluate the cytotoxic and / or cytostatic activity of test compounds against different types of tumors (Monks and others, supra : Boyd, Cancer: Princ. Pract. Oncol. Update , 3 (10) : 1 -12 [1989]).

They were collected with trypsin / EDTA (Gibco) cells of approximately 60 human tumor cell lines, it washed once, resuspended in IMEM and determined its viability. Cell suspensions were added by pipette (volume of 100 µl) to different 96 microtiter plates wells. Cell density at 6 days of incubation was lower than after 2 days of incubation to avoid overgrowth. The cultures were left for a 24-hour preincubation period at 37 ° C for stabilization. Twice higher dilutions at the intended test concentration were added in time zero in equal parts of 100 µl to the wells of the plate microtiter (1: 2 dilution). These compounds were evaluated in five semi-logarithmic dilutions (1,000 to 100,000 times). The incubation periods were two days and six days in one 5% CO2 atmosphere and 100% humidity.

After incubation, the medium was removed and the cells were fixed in 0.1 ml of 10% trichloroacetic acid at 40 ° C The plates were washed five times with deionized water, they were dried, stained for 30 minutes with 0.1 ml staining of 0.4% sulforhodamine B (Sigma) dissolved in 1% acetic acid, se washed four times with 1% acetic acid to remove the unbound dye, dried and within five minutes the dye with 0.1 ml of Tris base [tris (hydroxymethyl) aminomethane] 10 mM, pH 10.5. The absorbance (OD) of sulforhodamine B at 492 nm it was measured using a computer interface reader plate 96 well microtiter.

A test sample is considered to be positive if it shows at least one inhibitory effect of 40% growth at one or more concentrations. The results are they represent in the following table 4 where the abbreviations of the Tumor cell type are as follows:

NSCL = non-small cell lung carcinoma; SNC = Central Nervous System.

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TABLE 4

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twenty

twenty-one

Deposit of the material

The following materials have been deposited together with the American Type Culture Collection, 10801 Universit Blvd., Manassas, VA 20110-2209, USA (ATCC):

Material Dep. ATCC Date of presentation DNA32284-1307 209670 11 out of March 1998

This deposit was carried out under the provisions of the Budapest Treaty on Recognition International Deposit of Microorganisms for the purpose of Patent Procedure and its regulations (Treaty of Budapest). This guarantees the maintenance of a viable crop of deposit for 30 years from the date of deposit. The deposits will be made available by the ATCC under the terms of the Budapest Treaty, and will be subject to an agreement between Genentech, Inc .. and ATCC, which guarantee public availability permanent and unlimited offspring of the deposit culture since the publication of US Pat. relevant or from the Public filing of any US patent application or from abroad, whichever comes first, and guarantees the availability of offspring to one determined by the US Patent and Trademark Commissioner To be authorized thereto in accordance with 35 U.S.C. \ NAK 122 and the rules of the Commissioner accordingly (including 37 CFR \ NAK 1.14 with particular reference to 886 OG 638).

The assignee of this application for patent has agreed that if a culture of materials in deposit died or lost or destroyed having been cultivated in suitable conditions, the materials will be replaced from immediately upon notification by others of equals. Disponibility of the deposited material does not have to be understood as a authorization to practice the invention in contravention of the rights granted under the authority of any government of in accordance with its patent laws.

The previous written report is considered enough to allow a subject matter expert to put in practice the invention. The scope of the present invention is not seen. limited by the construction presented, since the realization presented is considered a simple illustration of some aspects of the invention and other constructions that are functionally equivalents are within the scope of this invention such as defined in the claims. The presentation of material of this is not an acceptance that the written description contained herein is inadequate to allow the practice of Any aspect of the invention, including the best manner of It is not intended to limit the scope of claims to the specific illustrations it represents. In In reality, there are several modifications that in addition to the represented and described herein will be clear to subject matter experts from the preceding description.

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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 in this respect.

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Claims (15)

1. Polypeptide that has at least 80% of amino acid sequence identity with respect to length total of:

(i)

amino acid sequence of residues 1 or X to 282 shown in Figure 12 (SEQ ID NO: 25), in which X is any amino acid residue from 26 to 35;

(ii)

the amino acid sequence of residues 1 or X to Y shown in Figure 12 (SEQ ID NO: 25), in which X is any amino acid residue of 26 to 35 and in which Y is any residue amino acid from 226 to 235; or

(iii)

the amino acid sequence encoded by the full length coding sequence of DNA deposited under ATCC accession number 209263,

to use in a procedure for the Tumor treatment in a mammal.

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2. Use of a polypeptide for the preparation of a medication for the treatment of tumor in a mammal, in the that the polypeptide is a polypeptide that has at least 80% of amino acid sequence identity with respect to length total of:

(i)

amino acid sequence of residues 1 or X to 282 shown in Figure 12 (SEQ ID NO: 25), in which X is any amino acid residue from 26 to 35;

(ii)

the amino acid sequence of residues 1 or X to Y shown in Figure 12 (SEQ ID NO: 25), in which X is any amino acid residue of 26 to 35 and in which Y is any residue amino acid from 226 to 235; or

(iii)

the amino acid sequence encoded by the full length coding sequence of DNA deposited under Accession number ATCC 209263.

         \ vskip1.000000 \ baselineskip
      

3. Polypeptide to use according to claim 1 or the use according to claim 2, wherein the tumor is a cancer. 4. Polypeptide for use or use according to claim 3, wherein the cancer is selected from the group that It consists of breast cancer, ovarian cancer, kidney cancer, cancer colorectal, prostate cancer, lung cancer, cancer of the central nervous system, melanoma and leukemia. 5. Polypeptide to use or use according any of the preceding claims, wherein the degree Identity is at least 90% or 95%. 6. Polypeptide to use or use according any of the preceding claims, wherein the polypeptide has

(i)

amino acid sequence of residues 1 or X to 282 shown in Figure 12 (SEQ ID NO: 25), in which X is any amino acid residue from 26 to 35, or

(ii)

the amino acid sequence of residues 1 or X to Y shown in Figure 12 (SEQ ID NO: 25), in which X is any amino acid residue of 26 to 35 and in which Y is any residue amino acid from 226 to 235; or

(iii)

the amino acid sequence encoded by the full length coding sequence of DNA deposited under ATCC accession number 209263,

         \ vskip1.000000 \ baselineskip
      

7. Polypeptide to use or use according any of the preceding claims, wherein X is 31. 8. Polypeptide to use or use according any of the preceding claims, wherein Y is 230. 9. Polypeptide for use according to any of claims 1 or 3 and 8, in combination with another agent growth inhibitor, a cytotoxic agent or an agent Chemotherapeutic 10. Use according to any of the claims 2 or 3 to 8, in which the medicine additionally comprises another growth inhibiting agent, a cytotoxic agent or an agent Chemotherapeutic 11. Polypeptide for use according to any of the preceding claims, wherein the mammal is a human.

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12. Composition comprising a polypeptide as defined in any one of claims 1 to 8 together with a pharmaceutically acceptable carrier for use in a treatment procedure as defined in any of the claims 1 to 4 or 11. 13. Composition to use according to claim 12, wherein the composition further comprises  another growth inhibitory agent, a cytotoxic agent or a chemotherapeutic agent 14. In vitro method for inhibiting the growth of a tumor cell, the method comprising exposing said tumor cell to an effective amount of a polypeptide as defined in any one of claims 1 to 11. 15. Method according to claim 14, in that the tumor is as defined in any of the claims 3 to 4.