KR20010084882A - Methods and Compositions for Inhibiting Neoplastic Cell Growth - Google Patents

Abstract

The present invention relates to methods and compositions for inhibiting neoplastic cell growth. In particular, the invention relates to antitumor compositions and methods for the treatment of tumors. The present invention also relates to a method for screening growth inhibition, for example, for identifying antitumor compounds. The invention also relates to novel polypeptides and nucleic acid molecules encoding such polypeptides. Also provided herein are vectors and host cells comprising the nucleic acid sequences, chimeric polypeptide molecules fused to a heterologous polypeptide sequence of the polypeptides of the invention, antibodies that bind to the polypeptides of the invention, and methods of producing the polypeptides of the invention .

Description

Methods and Compositions for Inhibiting Neoplastic Cell Growth

Malignant tumors (cancer) are the second leading cause of death in the United States after heart disease [Boring et al., CA Cancel J. Clin., 43 : 7 (1993)].

Cancer is a malignant cell that proliferates and forms a tumor mass, increases the number of abnormal or neoplastic cells derived from normal tissue, infiltrates adjacent tissue by the neoplastic tumor cells, and eventually spreads to local lymph nodes and ends through the blood or lymph system Generation (transition). In cancer stage, cells grow under conditions that normal cells do not grow. Cancer appears in a variety of forms with different degrees of invasion and invasiveness.

Despite recent advances in cancer therapy, there is a great need for new therapeutic agents that can inhibit neoplastic cell growth. Accordingly, an object of the present invention is to identify a compound capable of inhibiting the growth of a neoplastic cell, for example, cancer cells.

SUMMARY OF THE INVENTION [

A. Embodiment

The present invention relates to methods and compositions for inhibiting neoplastic cell growth. More particularly, the present invention relates to a method of treating tumors, including cancer, such as breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, renal cancer and central nervous system cancer, leukemia, melanoma, etc., in a mammalian patient, ≪ / RTI >

In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or an agonist thereof, To a composition useful for inhibiting neoplastic cell growth. In a preferred embodiment, the compositions of the present invention comprise growth inhibitory amounts of PPRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof. In another preferred embodiment, the composition comprises cytotoxic amounts of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof. Optionally, the compositions of the present invention may further comprise one or more growth inhibitors and / or cytotoxic and / or chemotherapeutic agents.

In a further aspect, the invention provides a method of treating a mammal comprising administering to a mammal a therapeutically effective amount of a mammal, including a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or an agonist thereof, To compositions useful for the treatment of tumors in animals. The tumor is preferably cancer.

In another aspect, the invention provides a method of treating or preventing cancer, comprising exposing an effective amount of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or an agonist thereof, Lt; RTI ID = 0.0 > cell < / RTI > cell growth. In certain embodiments, the agonist is selected from the group consisting of anti-PR179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti- PRO328, anti- PRO301, anti- PRO356, anti-PRO509 or anti-PRO866 agonist antibodies. In another embodiment, the agonist is a small molecule that mimics the biological activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. The method can be carried out in vitro or in vivo.

In another embodiment,

(a) containers,

(b) a neoplastic cell growth, for example, a proliferation inhibitor comprising, as an active substance, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO306, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or an agonist thereof A composition for inhibiting tumor cell growth, and

(c) the above PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, or said PRO179, PRO207, PRO320, PRO219, To refer to the use of an agonist thereof which may be an antibody capable of binding to a PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, a label attached to the container, or a package insert And the like. In one particular embodiment, the agonist is selected from the group consisting of anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti- PRO328, anti- PRO301, anti- Anti-PRO356, anti-PRO509 or anti-PRO866 agonist antibody. In another embodiment, the agonist is a small molecule that mimics PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Similar products comprising a therapeutically effective amount of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists described herein for tumor therapy are also within the scope of the invention . Include cytotoxic or chemotherapeutic agents as defined herein, such as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof, Are also within the scope of the present invention.

B. Additional Embodiments

In another embodiment of the invention, the invention provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide to provide.

In one aspect, the isolated nucleic acid molecule comprises (a) a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, a transmembrane protein with or without a signal peptide as disclosed herein PRO209, PRO220, PRO224, PRO329, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides having any other specifically defined fragment of the full length amino acid sequence as disclosed herein, Or (b) the complement of the DNA molecule (a) is at least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83 , More preferably at least about 84%, more preferably at least about 85%, even more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88% More preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably at least about 93%, even more preferably at least about 94% , More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99% of the nucleotide sequence do.

In another aspect, the isolated nucleic acid molecule is selected from the group consisting of: (a) the coding sequence of full length PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide cDNA, The coding sequences of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides lacking the signal peptide as disclosed herein, The coding sequence of the extracellular domain of the full-length PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, or any other A DNA molecule comprising a coding sequence of a specially defined fragment or (b) a sequence identity of a complement of the DNA molecule (a) of at least about 80%, preferably at least about 81% , More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86%, even more preferably at least about 87% More preferably at least about 88%, more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least about 92% More preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% Preferably at least about 99% nucleotide sequence.

(A) a DNA molecule encoding the same mature polypeptide encoded by any of the ATCC deposited human protein cDNAs, or (b) a complement of the DNA molecule (a) More preferably at least about 80%, more preferably at least about 82%, more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 80%, preferably at least about 80% , More preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90% More preferably at least about 91%, more preferably at least about 92%, even more preferably at least about 93%, even more preferably at least about 94%, even more preferably at least about 95% 96% or more, more preferably about 97% or more, more preferably about 98% or more, Preferably it relates to an isolated nucleic acid molecule comprising a nucleotide sequence at least about 99%.

In yet another aspect, the present invention provides a method for producing a polypeptide comprising a nucleotide sequence encoding a PRO179, a PRO207, a PRO320, a PRO219, a PRO221, a PRO224, a PRO328, a PRO301, a PRO526, a PRO362, a PRO356, a PRO509 or a PRO866 polypeptide encoding a transmembrane domain deleted or transmembrane domain- Or a complement of such a coding nucleotide sequence, and the transmembrane domain (s) of such a polypeptide are disclosed herein. Thus, soluble extracellular domains of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides described herein are contemplated.

Other embodiments include, for example, an antisense oligonucleotide probe, or an anti-PR179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti- PRO224, anti- PRO328, anti- PRO279, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, or PRO369, which may optionally encode a polypeptide comprising a binding site for anti-PRO362, anti-PRO356, anti- Or a complement thereof, which may be used as a hybridization probe for a coding fragment of a PRO356, PRO359, PRO356, PRO509 or PRO866 polypeptide, wherein the coding sequence of the PRO176, PRO206, PRO509 or PRO866 polypeptide, Lt; / RTI > Such nucleic acid fragments are generally at least about 20 nucleotides in length, preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 60 nucleotides Preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 100, even more preferably at least about 110, even more preferably at least about 120 More preferably at least about 130, more preferably at least about 140, more preferably at least about 150, even more preferably at least about 160, even more preferably at least about 170, More preferably at least about 190, more preferably at least about 200, more preferably at least about 250, even more preferably at least about 300, even more preferably at least about 350 More preferably about 400, , More preferably at least about 450, more preferably at least about 500, even more preferably at least about 600, even more preferably at least about 700, even more preferably at least about 800, About 900 or more, more preferably about 1000 or more, wherein the term " about " in this context means the nucleotide sequence length ± 10% of the length. The new fragments of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide coding nucleotide sequences can be obtained by using any of the well- PRO206, PRO356, PRO509 or PRO866 polypeptide coding nucleotide sequences were aligned with other known nucleotide sequences and any PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO221, PRO221, It will be appreciated that the PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide coding nucleotide sequence fragment (s) may be determined in a routine manner by determining whether it is novel. All of these PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide coding nucleotide sequences are contemplated herein. Also provided are fragments of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, preferably anti-PRO179, PRO179, which includes the binding site for anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti- PRO276, anti- PRO362, anti- PRO356, anti- , PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide fragments are also contemplated.

In yet another embodiment, the invention provides isolated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 encoded by any of the isolated nucleic acid sequences identified above. Or PRO866 polypeptide.

In particular aspects, the present invention provides an isolated polypeptide comprising a full-length amino acid sequence as described herein, an amino acid sequence lacking a signal peptide as described herein, an extracellular domain of a transmembrane protein with or without a signal peptide as described herein, Sequence identity with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides having any other specifically defined fragment of the full length amino acid sequence as described herein , More preferably at least about 80%, preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85% , Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 9 More preferably at least about 90%, more preferably at least about 90%, even more preferably at least about 90%, even more preferably at least about 90%, even more preferably at least about 95% PRO209, PRO320, PRO219, PRO221 comprising an amino acid sequence of preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99% , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides.

In a further aspect, the invention encompasses a nucleic acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 81% sequence identity with an amino acid sequence encoded by any of the ATCC deposited human protein cDNAs as described herein More preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86%, even more preferably at least about 87% , More preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least about 92% Is at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% , More preferably about 99% , PRO209, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides comprising the amino acid sequence of SEQ ID NO.

In a further aspect, the invention provides an isolated nucleic acid molecule comprising a full-length amino acid sequence as described herein, an amino acid sequence lacking a signal peptide as described herein, an extracellular domain of a transmembrane protein with or without a signal peptide as described herein, PRO209, PRO220, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides having any of the other specifically defined fragments of the full length amino acid sequence as described herein, In comparison, about 80% or more positive, preferably about 81% or more positive, more preferably about 82% or more positive, more preferably about 83% or more positive, more preferably about 84% or more positive, Is preferably at least about 85% positive, more preferably at least about 86% positive, even more preferably at least about 87% positive, The crab is preferably at least about 88% positive, more preferably at least about 89% positive, even more preferably at least about 90% positive, even more preferably at least about 91% positive, even more preferably at least about 92% positive, , More preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% PRO209, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 comprising an amino acid sequence that is at least about 98% positive, more preferably at least about 99% Or PRO866 polypeptide.

In particular aspects, the present invention provides isolated PRO179, PRO207, PRO320, PRO219, PRO221 encoded by the nucleotide sequence coding for said amino acid sequence as described above herein and having no N-terminal signal peptide and / or initiation methionine , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Also described are methods of making the isolated PRO polypeptides described above, wherein the methods comprise contacting a cell comprising a vector comprising a suitable coding nucleic acid molecule with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, , PRO279, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides from these cell cultures under conditions suitable for expression of the PRO369, PRO362, PRO356, PRO509 or PRO866 polypeptide Recovery.

In another aspect, the invention provides isolated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides wherein the transmembrane domain is deleted or the transmembrane domain is inactivated . Also described are methods of making the isolated PRO polypeptides described above, wherein the methods comprise contacting a cell comprising a vector comprising a suitable coding nucleic acid molecule with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, , PRO279, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides from these cell cultures under conditions suitable for expression of the PRO369, PRO362, PRO356, PRO509 or PRO866 polypeptide Recovery.

In another embodiment, the invention relates to agonists of native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides as defined above. In certain embodiments, the agonist is selected from the group consisting of anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti- PRO328, anti-PRO301, anti- PRO356, anti-PRO509 or anti-PRO866 agonist antibody or small molecule.

In a further embodiment, the present invention provides a method of treating or preventing a PRO179, PRO207, PRO320, PRO219, PRO209, PRO216, PRO216, PRO216, PRO236, PRO366, PRO509 or PRO866 polypeptide by contacting a candidate molecule with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO279, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, which include monitoring the biological activity mediated by the PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. , ≪ / RTI > PRO356, PRO509 or PRO866 polypeptides. Preferably, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO362, PRO356, PRO509 or PRO866 polypeptides.

In yet a further embodiment, the invention provides a method of treating a subject suffering from a disease or condition selected from the group consisting of PRO179, PRO209, PRO220, PRO224, PRO232, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, Anti-PRO209, anti-PRO209, anti-PRO221, anti-PRO224, anti-PRO328, Anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 agonist antibodies together with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.

Another embodiment of the present invention is a pharmaceutical composition comprising an anti-PRO179, an anti-PRO207, an anti-PRO320 or an anti-PRO179, a PRO209, a PRO206, a PRO219, a PRO221, a PRO224, a PRO328, a PRO301, , Anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO276, anti-PRO362, anti-PRO356, anti-PRO509 or anti- PRO866 agonist antibodies Or an agonist thereof as described above, or an anti-PRO179, anti-PRO207, anti-PRO209, anti-PRO207, The use of an anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti- will be.

In another embodiment of the invention, the invention provides a vector comprising DNA encoding any of the polypeptides described herein. Also provided is a host cell comprising such an arbitrary vector. By way of example, the host cell may be a CHO cell, E. coli , yeast or Baculovirus-infected insect cells. A method of producing any of the polypeptides described herein is also provided, comprising culturing the host cell under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

In another embodiment, the invention provides chimeric molecules comprising any polypeptide described herein fused to a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules include any of the polypeptides described herein fused to an epitope tag sequence or an Fc region of an immunoglobulin.

In another embodiment, the invention also provides an antibody that specifically binds to any of the above or below-described polypeptides. Optionally, the antibody is a monoclonal antibody, a humanized antibody, an antibody fragment or a single chain antibody.

In another embodiment, the invention provides an oligonucleotide probe or antisense probe useful for isolating the nucleotide sequence of a genome and a cDNA, wherein said probe can be derived from any of the above or the following nucleotide sequences.

The present invention relates to methods and compositions for inhibiting neoplastic cell growth. In particular, the invention relates to antitumor compositions and methods of treating tumors. The present invention also relates to a method for screening growth inhibition, i.e., an antitumor compound.

Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of the cDNA containing the nucleotide sequence encoding the native sequence PRO179, wherein the nucleotide sequence (SEQ ID NO: 1) is a clone referred to herein as DNA16451-1078. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

2 is the amino acid sequence (SEQ ID NO: 2) of the native sequence PRO179 polypeptide derived from the coding sequence of SEQ ID NO: 1 shown in FIG.

Figure 3 shows the nucleotide sequence (SEQ ID NO: 6) of the cDNA containing the nucleotide sequence encoding the native sequence PRO207, wherein the nucleotide sequence (SEQ ID NO: 6) is a clone referred to herein as DNA30879-1152. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

4 is the amino acid sequence (SEQ ID NO: 7) of the native sequence PRO207 polypeptide derived from the coding sequence of SEQ ID NO: 6 shown in FIG.

Figure 5 shows the nucleotide sequence (SEQ ID NO: 9) of the cDNA containing the nucleotide sequence encoding the native sequence PRO320, wherein the nucleotide sequence (SEQ ID NO: 9) is a clone referred to herein as DNA32284-1307. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

6 is the amino acid sequence (SEQ ID NO: 10) of the native sequence PRO320 polypeptide derived from the coding sequence of SEQ ID NO: 9 shown in FIG.

Figure 7 shows the nucleotide sequence (SEQ ID NO: 14) of the cDNA containing the nucleotide sequence encoding the native sequence PRO219, wherein the nucleotide sequence (SEQ ID NO: 14) is a clone referred to herein as DNA32290-1164. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

8 is the amino acid sequence (SEQ ID NO: 15) of the native sequence PRO219 polypeptide derived from the coding sequence of SEQ ID NO: 14 shown in FIG.

Figure 9 shows the nucleotide sequence (SEQ ID NO: 19) of the cDNA containing the nucleotide sequence encoding the native sequence PRO221, wherein the nucleotide sequence (SEQ ID NO: 19) is a clone referred to herein as DNA33089-1132. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

10 is the amino acid sequence (SEQ ID NO: 20) of the native sequence PRO221 polypeptide derived from the coding sequence of SEQ ID NO: 19 shown in FIG.

Figure 11 shows the nucleotide sequence (SEQ ID NO: 24) of the cDNA containing the nucleotide sequence encoding the native sequence PRO224, wherein the nucleotide sequence (SEQ ID NO: 24) is a clone referred to herein as DNA33221-1133. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

12 is the amino acid sequence (SEQ ID NO: 25) of the native sequence PRO224 polypeptide derived from the coding sequence of SEQ ID NO: 24 shown in FIG.

Figure 13 shows the nucleotide sequence (SEQ ID NO: 29) of the cDNA containing the nucleotide sequence encoding the native sequence PRO328, wherein the nucleotide sequence (SEQ ID NO: 29) is a clone referred to herein as DNA40587-1231. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

14 is the amino acid sequence (SEQ ID NO: 30) of the native sequence PRO328 polypeptide derived from the coding sequence of SEQ ID NO: 29 shown in FIG.

Figure 15 shows the nucleotide sequence (SEQ ID NO: 34) of the cDNA containing the nucleotide sequence encoding the native sequence PRO301, wherein the nucleotide sequence (SEQ ID NO: 34) is a clone referred to herein as DNA40628-1216. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

16 is the amino acid sequence (SEQ ID NO: 35) of the native sequence PRO301 polypeptide derived from the coding sequence of SEQ ID NO: 34 shown in FIG.

Figure 17 shows the nucleotide sequence (SEQ ID NO: 42) of the cDNA containing the nucleotide sequence encoding the native sequence PRO526, wherein the nucleotide sequence (SEQ ID NO: 42) is a clone referred to herein as DNA44184-1319. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

18 is the amino acid sequence (SEQ ID NO: 43) of the native sequence PRO526 polypeptide derived from the coding sequence of SEQ ID NO: 42 shown in FIG.

Figure 19 shows the nucleotide sequence (SEQ ID NO: 47) of a cDNA containing the nucleotide sequence encoding the native sequence PRO362, wherein the nucleotide sequence (SEQ ID NO: 47) is a clone referred to herein as DNA45416-1251. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

20 is the amino acid sequence (SEQ ID NO: 48) of the native sequence PRO362 polypeptide derived from the coding sequence of SEQ ID NO: 47 shown in FIG.

Figure 21 shows the nucleotide sequence (SEQ ID NO: 54) of the cDNA containing the nucleotide sequence encoding the native sequence PRO356, wherein the nucleotide sequence (SEQ ID NO: 54) is a clone referred to herein as DNA47470-1130-P1. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

22 is the amino acid sequence (SEQ ID NO: 55) of the native sequence PRO356 polypeptide derived from the coding sequence of SEQ ID NO: 54 shown in FIG.

Figure 23 shows the nucleotide sequence (SEQ ID NO: 59) of the cDNA containing the nucleotide sequence encoding the native sequence PRO509, wherein the nucleotide sequence (SEQ ID NO: 59) is a clone referred to herein as DNA50148-1068. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

24 is the amino acid sequence (SEQ ID NO: 60) of the native sequence PRO509 polypeptide derived from the coding sequence of SEQ ID NO: 59 shown in FIG.

Figure 25 shows the nucleotide sequence (SEQ ID NO: 61) of the cDNA containing the nucleotide sequence encoding the native sequence PRO866, wherein the nucleotide sequence (SEQ ID NO: 61) is a clone referred to herein as DNA53971-1359. In addition, the positions of the start and stop codons are indicated in bold and underlined, respectively.

26 is the amino acid sequence (SEQ ID NO: 62) of the native sequence PRO866 polypeptide derived from the coding sequence of SEQ ID NO: 61 shown in FIG.

The term "PRO179", "PRO207", "PRO320", "PRO219", "PRO221", "PRO224", "PRO328", "PRO301", "PRO526", "PRO362", "PRO356" As used herein, the term " PRO866 " polypeptide refers to the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides and PRO179, PRO207, PRO320, PRO219, PRO221, , PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variants (further defined herein). The polypeptides may be isolated from a variety of sources, such as human tissue types or other sources, or may be produced recombinantly and / or synthetically, such as, for example, .

Natural sequence PRO221, natural sequence PRO328, natural sequence PRO301, natural sequence PRO221, natural sequence PRO221, natural sequence PRO221, PRO206 " or " native sequence PRO866 " are intended to encompass PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, A polypeptide having the same amino acid sequence as the PRO362, PRO356, PRO509 or PRO866 polypeptide. Such native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides may be isolated from nature or prepared by recombinant and / or synthetic methods. The term " native sequence " PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides specifically includes PRO179, PRO209, PRO320, PRO219, PRO221, PRO224, PRO328, (E. G., A differentially spliced form) of a naturally occurring truncated or secreted form (e. G., An extracellular domain sequence), PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides. And naturally occurring allelic variants. In one embodiment of the invention, the native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are shown in Figure 2 (SEQ ID NO: 2) , SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: PRO199, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, and PRO328 shown in FIG. 20, SEQ ID NO: 48, FIG. 22, SEQ ID NO: PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. 14 (SEQ ID NO: 30), 12 (SEQ ID NO: 25), 14 (SEQ ID NO: 30) , PRO209, PRO207, and PRO209 shown in FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 2 (SEQ ID NO: 2), and SEQ ID NO: 4 (SEQ ID NO: 2), although PRO220, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides are shown to start with the methionine residue designated as amino acid position 1 in the figure 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, Other methionine residues located upstream or downstream from amino acid position 1 in SEQ ID NO: 43, SEQ ID NO: 48, FIG. 22, SEQ ID NO: 55, , PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or It is conceivable and possible that it can be used as the starting amino acid residue of the PRO866 polypeptide.

&Quot; Extracellular domain " or " ECD " of a polypeptide disclosed herein refers to a polypeptide form that is essentially free of transmembrane and cytoplasmic domains. Typically, the polypeptide ECD will comprise less than 1% of the transmembrane and / or cytoplasmic domains, preferably less than 0.5% of said domains. It is to be understood that all transmembrane domain (s) identified in the polypeptides of the present invention have been identified in accordance with criteria routinely used in the art to identify hydrophobic domain types. Although the exact borderline of the transmembrane domain may vary, most are present within about five amino acid residues at either end of the transmembrane domain as first identified herein and shown in the accompanying drawings. Likewise, in one embodiment of the invention, the extracellular domain of the polypeptide of the invention comprises amino acid 1 to amino acid X of the mature amino acid sequence, wherein X is within 5 of either of the extracellular domain / transmembrane domain boundaries Lt; / RTI >

The approximate positions of the " signal peptides " of the various PRO polypeptides described herein are shown in the accompanying drawings. It should be borne in mind, however, that the C-terminal boundary of the signal peptide may vary, but most of it is that at any interface of the signal peptide C-terminal first identified herein, the amino acid is no more than about 5, C-terminal boundaries can be identified according to criteria commonly used in the art for identifying types of amino acid sequence elements (see, for example, Nielsen et al., Prot. Eng. 10: 1-6 ) And von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). In addition, it should be appreciated that in some cases the cleavage of the signal sequence of the secretory polypeptide is not entirely identical, resulting in one or more secreted polypeptides. These mature polypeptides in which signal peptides are cleaved within about 5 amino acid residues at either interface of the C-terminal end of the signal peptide identified herein, and polynucleotides encoding them, are encompassed by the present invention.

&Quot; PRO179 variant polypeptide " means a polypeptide comprising (a) an amino acid sequence of residues 1 or about 17 to 460 of the PRO179 polypeptide of Figure 2 (b) an amino acid sequence of X to 460 of the PRO179 polypeptide of Figure 2 Wherein X is any amino acid residue of any of positions 12 to 21 of Figure 2 (SEQ ID NO: 2); or (c) is about 80% homologous to the amino acid sequence of another fragment of the specifically derived amino acid sequence of Figure 2 (Excluding the PRO179 polypeptide of the native sequence) having the amino acid sequence identity of SEQ ID NO.

(A) the amino acid sequence of residue 1 or about 41 to 249 of the PRO207 polypeptide of Figure 4 (SEQ ID NO: 7), (b) the amino acid sequence of X to 249 of the PRO207 polypeptide of Figure 4 Wherein X is an optional amino acid residue of 36 to 45 of Figure 4 (SEQ ID NO: 7)) or (c) a specifically derived amino acid sequence of about 80% identity to the amino acid sequence of another fragment of the amino acid sequence of Figure 4 (Exclusive of PRO207 polypeptide of native sequence) having the amino acid sequence identity of SEQ ID NO.

"PRO320 variant polypeptide" refers to a polypeptide comprising: (a) an amino acid sequence of residue 1 or about 22 to 338 amino acid residues of the PRO320 polypeptide of Figure 6 (b) X333 of the PRO320 polypeptide of Figure 6 (SEQ ID NO: 10) And X is any amino acid residue of 17 to 26 of Figure 6 (SEQ ID NO: 10)), or (c) a polypeptide having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of Figure 6 Means an active PRO320 polypeptide as defined below (other than a PRO320 polypeptide of native sequence) having amino acid sequence identity.

"PRO219 variant polypeptide" means a polypeptide comprising: (a) an amino acid sequence of residue 1 or about 24-1005 of the PRO219 polypeptide of Figure 8; (b) an amino acid sequence of X to 1005 of the PRO219 polypeptide of Figure 8 (SEQ ID NO: 15) Wherein X is any amino acid residue from 19 to 28 of Figure 8 (SEQ ID NO: 15); or (c) an amino acid sequence that is about 80% identical to the amino acid sequence of another fragment of the specifically derived amino acid sequence of Figure 8 (Excluding the PRO219 polypeptide of the native sequence) having the amino acid sequence identity of SEQ ID NO.

"PRO221 mutant polypeptide" means a polypeptide comprising (a) an amino acid sequence of residue 1 or about 34 to 259 of the PRO221 polypeptide of Figure 10, (b) an amino acid sequence of X to 259 of the PRO221 polypeptide of Figure 10 (SEQ ID NO: 20) (C) an amino acid sequence of 1 or about 34 to X of Figure 10 (SEQ ID NO: 20), wherein X is an amino acid sequence of SEQ ID NO: (SEQ ID NO: 20), or (d) an amino acid sequence having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 10 (Except for the PRO221 polypeptide of native sequence) as defined herein.

(A) the amino acid sequence of residue 1 or about 31-282 of the PRO224 polypeptide of Figure 12 (SEQ ID NO: 25), (b) the amino acid sequence of X282 of the PRO224 polypeptide of Figure 12 (SEQ ID NO: 25) Wherein X is any amino acid residue from 26 to 35 of Figure 12 (SEQ ID NO: 25); (c) one or about 31 to X of amino acid sequence of Figure 12 (SEQ ID NO: 25) (SEQ ID NO: 25), or (d) a specifically derived amino acid sequence having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the amino acid sequence of FIG. 12 Refers to an active PRO224 polypeptide (excluding the native sequence PRO224 polypeptide) defined.

"PRO328 variant polypeptide" refers to a polypeptide comprising (a) an amino acid sequence of residue 1 or about 23 to 463 amino acid residues of the PRO328 polypeptide of Figure 14, (b) X to 463 of the PRO328 polypeptide of Figure 14 (SEQ ID NO: 30) , And X is any amino acid residue at positions 18 to 27 of Figure 14 (SEQ ID NO: 30)), or (c) at least about 80% or more of the amino acid sequence of another fragment of the specifically derived amino acid sequence of Figure 14 Means an active PRO328 polypeptide as defined below (excluding the PRO328 polypeptide of native sequence) having amino acid sequence identity.

(A) the amino acid sequence of residue 1 or about 28 to 299 of the PRO301 polypeptide of Figure 16 (SEQ ID NO: 35), (b) the amino acid sequence of X to 299 of the PRO301 polypeptide of Figure 16 (C) an amino acid sequence of 1 or about 28 to X of FIG. 16 (SEQ ID NO: 35), wherein X is an amino acid sequence of SEQ ID NO: (SEQ ID NO: 35), or (d) a specifically derived amino acid sequence having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the amino acid sequence of FIG. 16 Quot; means an active PRO301 polypeptide as defined (except for the native PRO301 polypeptide).

(A) the amino acid sequence of residue 1 or about 27 to 473 of the PRO526 polypeptide of Figure 18 (SEQ ID NO: 43), (b) the amino acid sequence of X to 473 of the PRO526 polypeptide of Figure 18 Wherein X is any amino acid residue 22 to 31 of Figure 18 (SEQ ID NO: 43)), or (c) about 80% amino acid sequence identity with another sequence of another fragment of the specifically derived amino acid sequence of Figure 18 (SEQ ID NO: (Excluding the PRO526 polypeptide of the native sequence), as defined below, having the amino acid sequence identity of SEQ ID NO: < RTI ID = 0.0 >

(A) the amino acid sequence of residue 1 or about 20 to 321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48), (b) the amino acid sequence X to 321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48) Wherein X is an optional amino acid residue from 15 to 24 of Figure 20 (SEQ ID NO: 48); (c) one or about 20 to X amino acid sequences of Figure 20 (SEQ ID NO: 48) (SEQ ID NO: 48), or (d) a specifically derived amino acid sequence having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the amino acid sequence of FIG. 20 (SEQ ID NO: 48) Quot; means an active PRO362 polypeptide (excluding the PRO362 polypeptide of native sequence) as defined.

(A) the amino acid sequence of residue 1 or about 27 to 346 of the PRO356 polypeptide of Figure 22 (SEQ ID NO: 55), (b) the amino acid sequence of X to 346 of the PRO356 polypeptide of Figure 22 Wherein X is an optional amino acid residue of 22 to 31 of Figure 22 (SEQ ID NO: 55)) or (c) a specifically derived amino acid sequence of another fragment of the amino acid sequence of Figure 22 (SEQ ID NO: 55) (Excluding the PRO356 polypeptide of the native sequence), as defined below, having the amino acid sequence identity of SEQ ID NO.

&Quot; PRO509 variant polypeptide " means a polypeptide comprising (a) the amino acid sequence of residue 1 or about 37-283 of the PRO509 polypeptide of Figure 24 (b) the amino acid sequence of X to 283 of the PRO509 polypeptide of Figure 24 (SEQ ID NO: 60) (C) an amino acid sequence of 1 or about 37 to X of FIG. 24 (SEQ ID NO: 60), wherein X is an amino acid sequence of SEQ ID NO: (SEQ ID NO: 60), or (d) a specifically derived amino acid sequence having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the amino acid sequence of FIG. 24 (SEQ ID NO: 60) Refers to the active PRO509 polypeptide (excluding the native sequence PRO509 polypeptide) defined.

(A) the amino acid sequence of residue 1 or about 27 to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: 62), (b) the amino acid sequence of X to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: Wherein X is any amino acid residue 22 to 31 of Figure 26 (SEQ ID NO: 62)) or (c) specifically derived amino acid sequence of another fragment of the amino acid sequence of Figure 26 (SEQ ID NO: 62) (Excluding the PRO866 polypeptide of the native sequence) having the amino acid sequence identity of SEQ ID NO.

Variants of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variants include, for example, N or C terminus of the native sequence as well as one or more amino acids PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides with residues added or deleted.

Typically, a PRO179 variant polypeptide comprises (a) a residue 1 or about 17 to 460 of the PRO179 polypeptide of Figure 2 (b) a polypeptide of XVI to 460 of the PRO179 polypeptide of Figure 2 (SEQ ID NO: 2) (SEQ ID NO: 2)), or (c) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 2 (SEQ ID NO: 2) is at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about , More preferably at least 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, even more preferably at least about 99%.

Typically, the PRO207 variant polypeptide comprises (a) residue 1 or about 41 to 249 of the PRO207 polypeptide of Figure 4, (b) X to 249 of the PRO207 polypeptide of Figure 4 (SEQ ID NO: 7) (SEQ ID NO: 7)), or (c) a specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 4 (SEQ ID NO: 7) of at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 9 , More preferably at least 5%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, and even more preferably at least about 99%.

Typically, the PRO320 variant polypeptide comprises (a) residue 1 or about 22 to 338 of the PRO320 polypeptide of Figure 6 (b) Figure 6 (SEQ ID NO: 10) X to 338 of the PRO320 polypeptide, (SEQ ID NO: 10)), or (c) a specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 6 (SEQ ID NO: 10) of at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94% , More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, even more preferably at least about 99%.

Typically, a PRO219 variant polypeptide comprises (a) residue 1 or about 24-1005 of the PRO219 polypeptide of Figure 8, (b) X of the PRO219 polypeptide of Figure 8 (SEQ ID NO: 15) (SEQ ID NO. 15)), or (c) a specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 8 (SEQ ID NO: 15) of at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94% About 95% or more, more preferably is a is from about 98% or more, more preferably about 96% or more, more preferably at least about 97%, more preferably to at least about 99%.

Typically, the PRO221 mutant polypeptide comprises (a) residues 1 or about 34 to 259 of the PRO221 polypeptide of Figure 10, (b) X to 259 of the PRO221 polypeptide of Figure 10 (SEQ ID NO: 20) (SEQ ID NO: 20), (c) one or about 34 to X of Figure 10 (SEQ ID NO: 20), wherein X is any of amino acids 199 to 208 of Figure 10 Amino acid) or (d) an amino acid sequence identity of at least about 80%, more preferably at least about 81%, more preferably at least about 85% amino acid sequence identity to another fragment of the specifically derived amino acid sequence of Figure 10 (SEQ ID NO: 20) More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87% , Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96% About 97% or more, more preferably about 98% or more, and more preferably about 99% or more.

Typically, the PRO224 variant polypeptide comprises (a) residue 1 or about 31 to 282 of the PRO224 polypeptide of Figure 12 (b), X to 282 of the PRO224 polypeptide of Figure 12 (SEQ ID NO: 25) (SEQ ID NO: 25), (c) one or about 31 to X of Figure 12 (SEQ ID NO: 25), wherein X is any of 226 to 235 of Figure 12 Amino acid) or (d) an amino acid sequence identity of at least about 80%, more preferably at least about 81%, more preferably at least about 85% amino acid sequence identity to another fragment of the amino acid sequence of Figure 12 (SEQ ID NO: 25) More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87% , Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96% 97% or more, more preferably about 98% or more, and more preferably about 99% or more.

Typically, the PRO328 variant polypeptide comprises (a) residue 1 or about 23 to 463 of the PRO328 polypeptide of Figure 14, (b) X to 463 of the PRO328 polypeptide of Figure 14 (SEQ ID NO: 30) (SEQ ID NO: 30)) or (c) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 14 (SEQ ID NO: 30) is at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94% Will be at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and even more preferably at least about 99%.

Typically, the PRO301 mutant polypeptide comprises (a) residues 1 or about 28 to 299 of the PRO301 polypeptide of Figure 16 (SEQ ID NO: 35), (b) X to 299 of the PRO301 polypeptide of Figure 16 (SEQ ID NO: 35), (c) one or about 28 to X of Figure 16 (SEQ ID NO: 35), wherein X is any of the amino acid residues 23 to 32 of SEQ ID NO: Amino acid) or (d) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 16 (SEQ ID NO: 35) is at least about 80%, more preferably at least about 81%, more preferably at least about More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87% , Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96% 97% or more, more preferably about 98% or more, and more preferably about 99% or more.

Typically, the PRO526 mutant polypeptide comprises (a) residues 1 or 27 to 473 of the PRO526 polypeptide of Figure 18, (b) X473 of the PRO526 polypeptide of Figure 18 (SEQ ID NO: 43) (SEQ ID NO: 43)) or (c) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 18 (SEQ ID NO: 43) is at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94% Will be at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and even more preferably at least about 99%.

Typically, a PRO362 variant polypeptide comprises (a) residues 1 or about 20 to 321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48), (b) X to 321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48) (SEQ ID NO: 48), (c) one or about 20 to X of Figure 20 (SEQ ID NO: 48), wherein X is any of residues 276 to 285 of Figure 20 Amino acid) or (d) an amino acid sequence identity of at least about 80%, more preferably at least about 81%, more preferably at least about 85% amino acid sequence identity to another fragment of the specifically derived amino acid sequence of Figure 20 (SEQ ID NO: 48) More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87% , Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96% 97% or more, more preferably about 98% or more, and more preferably about 99% or more.

Typically, a PRO356 mutant polypeptide comprises (a) residue 1 or about 27 to 346 of the PRO356 polypeptide of Figure 22, (b) X to 346 of the PRO356 polypeptide of Figure 22 (SEQ ID NO: 55) (SEQ ID NO: 55)) or (c) an amino acid sequence identity of at least about 80% with another fragment of the specifically derived amino acid sequence of Figure 22 (SEQ ID NO: 55) More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94% About 95% or more, more preferably is a is from about 98% or more, more preferably about 96% or more, more preferably at least about 97%, more preferably to at least about 99%.

Typically, the PRO509 variant polypeptide comprises (a) an amino acid sequence of residue 1 or about 37-283 of the PRO509 polypeptide of Figure 24 (SEQ ID NO: 60), (b) an amino acid sequence of X- 283 of the PRO509 polypeptide of Figure 24 (SEQ ID NO: 60), wherein X is any amino acid residue from 32 to 41 in FIG. 24 (SEQ ID NO: 60); Or (d) an amino acid sequence identity of at least about 80%, more preferably at least about 81%, with another fragment of the specifically derived amino acid sequence of Figure 24 (SEQ ID NO: 60) , More preferably at least about 82%, more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% About 87% or more, more preferably about 88% or more, more preferably about 89% or more, and even more preferably about 9 More preferably at least about 91%, more preferably at least about 92%, even more preferably at least about 93%, even more preferably at least about 94%, even more preferably at least about 95% , More preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, and even more preferably at least about 99%.

Typically, the PRO866 variant polypeptide comprises (a) a residue 1 or about 27 to 331 of the PRO866 polypeptide of Figure 26, (b) X to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: 62) (SEQ ID NO: 62)) or (c) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of Figure 26 (SEQ ID NO: 62) is at least about 80% More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91% Is at least 92%, more preferably at least about 93%, more preferably at least about 94% Will be at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and even more preferably at least about 99%.

The variant polypeptides may be selected from the group consisting of native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, , PRO509 and PRO866 polypeptides. Typically, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variant polypeptides are polypeptides of about 10 or more amino acids in length, More usually at least about 40 amino acids, more often at least about 50 amino acids, more often at least about 60 amino acids, more often at least about 70 amino acids, more often at least about 80 amino acids , More usually at least about 90 amino acids, more often at least about 100 amino acids, more often at least about 150 amino acids, more often at least about 200 amino acids, more often at least about 250 amino acids, More than 300 amino acids or more.

Table 1 below provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code is routinely applicable for use on UNIX operating systems to provide an ALIGN-2 sequence comparison computer program.

In addition, Tables 2a to 2d use the methods described below to determine amino acid sequence identity (%) (Tables 2a and 2b) and nucleic acid sequence identity (%) (Tables 2c and 2d) using an ALIGN-2 sequence comparison computer program Wherein PRO represents the amino acid sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides of interest in the household of interest, Quot; refers to the amino acid sequence of the polypeptide compared to the " PRO " polypeptide of interest, and " PRO- DNA " refers to the PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, Quot; PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866- coding nucleic acid sequences, " comparative DNA " refers to the nucleotide sequence of a nucleic acid molecule compared to the " PRO- Quot ;, " Y " and " Z " Naemyeo other, "N", "L" and "V" represents a nucleotide of a different household, respectively.

&Quot; Amino acid sequence identity (%) " for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide sequences identified herein are PRO179, PRO207, PRO320, PRO219, No conservative substitutions were made as part of sequence identity after introducing a gap to align the sequences of PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 and to obtain the maximum sequence identity if necessary Is defined as the percentage of amino acid residues of the same candidate sequence as the amino acid residues of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide sequences. Alignment to determine amino acid sequence identity (%) may be performed using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software in a variety of ways known in the art . Those skilled in the art will be able to determine parameters suitable for measuring alignment, including any algorithms necessary to maximally align over the entire length of the compared sequences. However, for purposes of this disclosure, amino acid sequence identity values (%) can be obtained using the sequence comparison computer program ALIGN-2 as described below, and the complete source code for the ALIGN-2 program is shown in Table 1 . The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., And the source code shown in Table 1 is maintained in the user documentation of the US Copyright Office (Washington, DC 20559), which is registered under US Copyright Registration No. TXU510087 . The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be edited from the source code presented in Table 1. The ALIGN-2 program can be edited and used in a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For purposes of the present application, the amino acid sequence identity (%) of a given amino acid sequence A (given amino acid sequence B, B, or B to a given amino acid sequence identity% Quot; or < / RTI > a given amino acid sequence A containing or containing)

X / Y x 100

Where X is the number of amino acid residues recorded as being equally matched by the sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that the amino acid sequence identity (%) of A to B is not equal to the amino acid sequence identity (%) of B to A if the length of amino acid sequence A is not equal to the length of amino acid sequence B. As an example of calculating the amino acid sequence identity (%) using this method, Tables 2a and 2b show how to calculate the amino acid sequence identity (%) of the amino acid sequence represented by " comparison protein " will be.

Unless otherwise stated, all amino acid sequence identity% values used herein are obtained as described above using an ALIGN-2 sequence comparison computer program. However, amino acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST-2 (Altschul et al., Nucleic Acids Res . 25 : 3389-3402 (1997)). The NCBI-BLAST-2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.). NCBI-BLAST-2 uses several search parameters, such as unmasked = yes, strand = all, expected occurrence = 10, minimum low complexity length = 15/5, multi-pass e - value = 0.01, a constant for multi-pass = 25, a drop-off for final gap alignment = 25, and a scoring matrix = BLOSUM62.

When NCBI-BLAST-2 is used for amino acid sequence comparison, the amino acid sequence identity (%) of a given amino acid sequence A (given in amino acid sequence B, B and / or B) to a given amino acid sequence B, Which may or may not be expressed as a given amino acid sequence A that has or comprises a constant amino acid sequence identity (%) to the amino acid sequence of SEQ ID NO.

X / Y x 100

Where X is the number of amino acid residues recorded as being equally matched by the sequence alignment program NCBI-BLAST-2 in the A and B alignment of the sequence alignment program NCBI-BLAST-2, and Y is the total amino acid residue number of B . It will be appreciated that the amino acid sequence identity (%) of A to B is not equal to the amino acid sequence identity (%) of B to A if the length of amino acid sequence A is not equal to the length of amino acid sequence B.

The amino acid sequence identity (%) can also be obtained using a WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266 : 460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to default values. Those that are not set to default values, that is, adjustable parameters, are set to the following values: Overlap Span = 1, Overlap Fraction = 0.125, Word Limit (T) = 11, and Score Matrix = BLOSUM62. To achieve the object of the present invention, the amino acid sequence identity value (%) is determined by (a) comparing the amino acid sequence of the target PRO polypeptide having the sequence derived from the native PRO polypeptide with the target amino acid sequence The number of identical amino acid residues matched to the amino acid sequence of the target polypeptide (which may be the PRO variant polypeptide as the PRO polypeptide) may be determined as WU-BLAST-2 and divided by the total number of amino acid residues of the target PRO polypeptide. For example, a polypeptide comprising the amino acid sequence A having an amino acid sequence identity of 80% or more with the amino acid sequence B ", the amino acid sequence A is the target amino acid sequence to be compared, and the amino acid sequence B is the amino acid sequence of the target PRO polypeptide .

(A) a nucleic acid sequence encoding residue 1 or about 17 to 460 of the PRO179 polypeptide of Figure 2 (SEQ ID NO: 2), (b) a nucleic acid sequence encoding a PRO179 polypeptide of SEQ ID NO: (C) a nucleic acid sequence encoding an amino acid sequence of X to 460 of PRO179 polypeptide, wherein X is any amino acid residue of positions 12 to 21 of Figure 2 (SEQ ID NO: 2), or (c) Quot; means a nucleic acid molecule encoding an active PRO179 polypeptide as defined below that has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO179 variant polynucleotides comprise (a) a nucleic acid sequence encoding residue 1 or about 17 to 460 of the PRO179 polypeptide of Figure 2 (SEQ ID NO: 2), (b) X to 460 of the PRO179 polypeptide of Figure 2 (C) a specific fragment of the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 2) that is specifically derived from a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 12 to 21 of Figure 2 More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about 92% More preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% Will be at least about 98%, more preferably at least about 99%. PRO179 polynucleotide variants do not encompass the entire native PRO179 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 41 to 249 of the PRO207 polypeptide of Figure 4 (SEQ ID NO: 7), (b) a nucleic acid sequence encoding a PRO207 variant polynucleotide of 4 (SEQ ID NO: 7), or (c) a nucleic acid sequence encoding a specifically-derived amino acid sequence of amino acids X to 249 of the PRO207 polypeptide, wherein X is any amino acid residue 36 to 45 of Figure 4 (SEQ ID NO: 7) As used herein means a nucleic acid molecule encoding an active PRO207 polypeptide as defined below having a nucleic acid sequence identity of at least about 80% with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO207 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 41 to 249 of the PRO207 polypeptide of Figure 4 (b) a polypeptide comprising a polypeptide having the amino acid sequence of X to 249 of the PRO207 polypeptide of Figure 4 (C) a specific fragment of the amino acid sequence of Figure 4 (SEQ ID NO: 7) specifically encoded by a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 36 to 45 of Figure 4 (SEQ ID NO: 7) More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about 92% More preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% Will be at least about 98%, more preferably at least about 99%. PRO207 polynucleotide variants do not encompass the entire native PRO207 nucleotide sequence.

The "PRO320 mutant polynucleotide" or "PRO320 variant nucleic acid sequence" includes (a) a nucleic acid sequence encoding residue 1 or about 22 to 338 of the PRO320 polypeptide of Figure 6 (SEQ ID NO: 10), (b) (C) a nucleic acid sequence encoding an amino acid sequence from X to 338 of the PRO320 polypeptide, wherein X is any amino acid residue from 17 to 26 of Figure 6 (SEQ ID NO: 10), or (c) As used herein means a nucleic acid molecule encoding an active PRO320 polypeptide as defined below having a nucleic acid sequence identity of at least about 80% with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO320 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 22 to 338 of the PRO320 polypeptide of Figure 6, (b) a polypeptide having the amino acid sequence of X333 of the PRO320 polypeptide of Figure 6 (SEQ ID NO: 10) A nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 17 to 26 of Figure 6 (SEQ ID NO: 10), or (c) a specifically derived amino acid sequence of any fragment of the amino acid sequence of Figure 6 More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity to the nucleic acid sequence encoding the nucleic acid sequence of SEQ ID NO: , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about 9 More preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% , Preferably at least about 98%, more preferably at least about 99%. PRO320 polynucleotide variants do not encompass the entire native PRO320 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 24-1005 of the PRO219 polypeptide of Figure 8 (SEQ ID NO: 15), (b) a nucleic acid sequence encoding a PRO219 variant polynucleotide of 8 (SEQ ID NO: 15), or (c) a nucleic acid sequence encoding a specifically derived amino acid sequence of PRO219 polypeptide wherein X is an amino acid residue of any of amino acids 19 to 28 of Figure 8 (SEQ ID NO: 15) Means a nucleic acid molecule encoding an active PRO219 polypeptide as defined below having a nucleic acid sequence identity of at least about 80% with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO219 variant polynucleotides comprise (a) a nucleic acid sequence encoding residue 1 or about 24-1005 of the PRO219 polypeptide of Figure 8, (b) a polypeptide having the amino acid sequence of X to 1005 of the PRO219 polypeptide of Figure 8 A nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 19 to 28 of Figure 8 (SEQ ID NO: 15), or (c) a specific fragment of an amino acid sequence of specifically derived Figure 8 More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about < RTI ID = 0.0 > More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% , Preferably at least about 98%, more preferably at least about 99%. PRO219 polynucleotide variants do not encompass the entire native PRO219 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 34 to 259 of the PRO221 polypeptide of Figure 10 (SEQ ID NO: 20), (b) a nucleic acid sequence encoding a PRO221 polypeptide of (C) a nucleic acid sequence encoding an amino acid sequence from X to 259 of the PRO221 polypeptide, wherein X is any amino acid residue 29 to 38 of Figure 10 (SEQ ID NO: 20); Wherein X is any amino acid from 199 to 208 of Figure 10 (SEQ ID NO: 20), or (d) a nucleic acid sequence encoding another fragment of the specifically derived amino acid sequence of Figure 10 (SEQ ID NO: 20) ≪ RTI ID = 0.0 > and / or < / RTI > having at least about 80% nucleic acid sequence identity to a nucleic acid molecule encoding an active PRO221 polypeptide as defined below. Typically, the PRO221 mutant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 34 to 259 of the PRO221 polypeptide of Figure 10, (b) a nucleic acid sequence encoding a PRO221 polypeptide of X to 259 of Figure 10 (SEQ ID NO: 20) (C) one or about 34 to X of Figure 10 (SEQ ID NO: 20), wherein X is an amino acid residue selected from the group consisting of SEQ ID NO: (SEQ ID NO: 20), or (d) a nucleic acid sequence identity with a nucleic acid sequence encoding a specific fragment of a specifically derived amino acid sequence of FIG. 10 (SEQ ID NO: 20) More preferably at least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85% , Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88% More preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, even more preferably at least about 93%, even more preferably at least about 94% , More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, and even more preferably at least about 99%. PRO221 polynucleotide variants do not encompass the entire native PRO221 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 31 to 282 of the PRO224 polypeptide of Figure 12 (SEQ ID NO: 25), (b) a nucleic acid sequence encoding a PRO224 polypeptide of (C) a nucleic acid sequence encoding the amino acid sequence of X282 of the PRO224 polypeptide, wherein X is any amino acid residue from 26 to 35 of Figure 12 (SEQ ID NO: 25), (c) Wherein X is any amino acid from 226 to 235 of Figure 12 (SEQ ID NO: 25), or (d) a nucleic acid sequence encoding another fragment of the amino acid sequence of Figure 12 (SEQ ID NO: 25) ≪ RTI ID = 0.0 > and / or < / RTI > having at least about 80% nucleic acid sequence identity to a nucleic acid molecule encoding an active PRO224 polypeptide as defined below. Typically, the PRO224 variant polynucleotide comprises (a) a nucleic acid sequence encoding residues 1 or about 31-282 of the PRO224 polypeptide of Figure 12 (b) a polypeptide comprising the amino acid sequence of X282 of the PRO224 polypeptide of Figure 12 (SEQ ID NO: 25) (C) one or about 31 to X of Figure 12 (SEQ ID NO: 25), wherein X is an amino acid residue selected from the group consisting of SEQ ID NO: (SEQ ID NO: 25)) or (d) a nucleic acid sequence identity with a nucleic acid sequence encoding a specific fragment of the amino acid sequence of FIG. 12 (SEQ ID NO: 25) More preferably at least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85% , Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88% , More preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably at least about 93% , More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, and even more preferably at least about 99%. PRO224 polynucleotide variants do not encompass the entire native PRO224 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 23 to 463 of a PRO328 polypeptide of Figure 14 (SEQ ID NO: 30), (b) a nucleic acid sequence encoding a PRO328 variant polynucleotide of 14 (SEQ ID NO: 30), or (c) a nucleic acid sequence encoding the amino acid of X to 463 of PRO328 polypeptide, wherein X is any amino acid residue of 18 to 27 of Figure 14 (SEQ ID NO: 30) Quot; means a nucleic acid molecule that encodes an active PRO328 polypeptide as defined below that has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO328 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 23 to 463 of the PRO328 polypeptide of Figure 14 (SEQ ID NO: 30), (b) a polypeptide having the amino acid sequence of X to 463 of the PRO328 polypeptide of Figure 14 (C) a specific fragment of the amino acid sequence of FIG. 14 (SEQ ID NO: 30) that is specifically derivatized, wherein the amino acid sequence of SEQ ID NO: More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about < RTI ID = 0.0 > , More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% More preferably at least about 98%, more preferably at least about 99%. PRO328 polynucleotide variants do not encompass the entire native PRO328 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 28 to 299 of the PRO301 polypeptide of Figure 16 (SEQ ID NO: 35), (b) a nucleic acid sequence encoding a PRO301 variant polynucleotide or PRO301 variant nucleic acid sequence of Figure 16 (C) a nucleic acid sequence encoding the amino acid sequence of residues X to 299 of the polypeptide, wherein X is any amino acid residue from 23 to 32 of Figure 16 (SEQ ID NO: 35); Wherein X is any amino acid from amino acid 230 to amino acid 239 of Figure 16 (SEQ ID NO: 35), or (d) a polypeptide that specifically codes for any fragment of the amino acid sequence of Figure 16 Refers to a nucleic acid molecule that encodes an active PRO301 polypeptide as defined below that has at least about 80% nucleic acid sequence identity to a nucleic acid sequence of SEQ ID NO: 2. Typically, the PRO301 mutant polynucleotide comprises (a) a residue of the PRO301 polypeptide of Figure 16 1 or about 28 to 299 (B) a nucleic acid encoding a nucleic acid sequence encoding a PRO301 polypeptide of Figure 16 (SEQ ID NO: 35), wherein X is an amino acid residue at positions 23 to 32 of Figure 16 (SEQ ID NO: 35) (C) one or about 28 to X of Figure 16 (SEQ ID NO: 35), wherein X is any amino acid of amino acid 230 to amino acid 239 of Figure 16 (SEQ ID NO: 35); or (d) More preferably at least about 80%, more preferably at least about 82%, more preferably at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding any fragment of the amino acid sequence of Figure 16 (SEQ ID NO: 35) Is at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88% , More preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91% , More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96% About 97% or more, more preferably about 98% or more, and more preferably about 99% or more. PRO301 polynucleotide variants do not encompass the entire native PRO301 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 27 to 473 of the PRO526 polypeptide of Figure 18 (SEQ ID NO: 43), (b) a nucleic acid sequence encoding a PRO526 polypeptide of 18 (SEQ ID NO: 43), or (c) a nucleic acid sequence encoding a specifically derived 18 amino acid sequence of SEQ ID NO: 43, wherein X is an amino acid residue of X to 473 of the PRO526 polypeptide, wherein X is any amino acid residue from 22 to 31 of FIG. Means a nucleic acid molecule encoding an active PRO526 polypeptide as defined below having a nucleic acid sequence identity of at least about 80% with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO526 mutant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 27 to 473 of the PRO526 polypeptide of Figure 18, (b) a nucleic acid sequence encoding a PRO526 polypeptide of X473 of Figure 18 (SEQ ID NO: 43) (C) a specific fragment of the amino acid sequence of Figure 18 (SEQ ID NO: 43) specifically encoded by a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 22 to 31 of Figure 18 (SEQ ID NO: 43) More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about < RTI ID = 0.0 > , More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% More preferably at least about 98%, more preferably at least about 99%. PRO526 polynucleotide variants do not encompass the entire native PRO526 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 20 to 321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48), (b) a nucleic acid sequence encoding a PRO362 variant polynucleotide of (C) a nucleic acid sequence encoding an amino acid sequence from X to 321 of the PRO362 polypeptide, wherein X is any amino acid residue from 15 to 24 of Figure 20 (SEQ ID NO: 48), (c) Wherein X is any amino acid from amino acid 276 to amino acid 285 of Figure 20 (SEQ ID NO: 48), or (d) a polypeptide that encodes any fragment of the specifically derived amino acid sequence of Figure 20 (SEQ ID NO: 48) Means a nucleic acid molecule that encodes an active PRO362 polypeptide as defined below that has at least about 80% nucleic acid sequence identity to a nucleic acid sequence. Typically, the PRO362 variant polynucleotides comprise (a) a nucleic acid sequence encoding residue 1 or about 20 to 321 of the PRO362 polypeptide of Figure 20 (b) a polypeptide having the amino acid sequence of X to 321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48) (C) one or about 20 to X of Figure 20 (SEQ ID NO: 48), wherein X is an amino acid residue selected from the group consisting of SEQ ID NO: 20 (SEQ ID NO: 48)) or (d) a nucleic acid sequence homologous to a nucleic acid sequence encoding any fragment of the specifically derived amino acid sequence of FIG. 20 (SEQ ID NO: 48) More preferably at least about 80%, more preferably at least about 81%, even more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85% , More preferably at least about 86%, more preferably at least about 87% More preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least about 92%, even more preferably at least about 93% , More preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% Will be at least about 99%. PRO362 polynucleotide variants do not encompass the entire native PRO362 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 27 to 346 of the PRO356 polypeptide of Figure 22 (SEQ ID NO: 55); (b) a nucleic acid sequence encoding a PRO356 variant polynucleotide of 22 (SEQ ID NO: 55)), or (c) a nucleic acid sequence encoding a specifically-derived amino acid sequence of the PRO356 polypeptide, wherein X is an amino acid residue of any of amino acids 22 to 31 of Figure 22 Quot; means a nucleic acid molecule encoding an active PRO356 polypeptide as defined below that has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO356 mutant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 27 to 346 of the PRO356 polypeptide of Figure 22 (SEQ ID NO: 55), (b) a polypeptide having the amino acid sequence of X to 346 of the PRO356 polypeptide of Figure 22 (C) a specific fragment of the amino acid sequence of FIG. 22 (SEQ ID NO: 55) specifically encoded by a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 22 to 31 of Figure 22 (SEQ ID NO: 55) More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about < RTI ID = 0.0 > , More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% More preferably at least about 98%, more preferably at least about 99%. PRO356 polynucleotide variants do not encompass the entire native PRO356 nucleotide sequence.

The "PRO509 variant polynucleotide" or "PRO509 variant nucleic acid sequence" includes (a) a nucleic acid sequence encoding residue 1 or about 37-283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60) (C) a nucleic acid sequence encoding an amino acid sequence from X to 283 of the PRO509 polypeptide, wherein X is an optional amino acid residue in positions 32 to 41 of Figure 24 (SEQ ID NO: 60), (c) (X) is any amino acid from amino acid 200 to amino acid 209 of Figure 24 (SEQ ID NO: 60), or (d) is a polypeptide that encodes any fragment of the specifically derived amino acid sequence of Figure 24 Means a nucleic acid molecule that encodes an active PRO509 polypeptide as defined below that has at least about 80% nucleic acid sequence identity to a nucleic acid sequence. Typically, the PRO509 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 37 to 283 of the PRO509 polypeptide of Figure 24 (SEQ ID NO: 60), (b) a polypeptide having the amino acid sequence of X to 283 of the PRO509 polypeptide of Figure 24 (C) one or about 37 to X of FIG. 24 (SEQ ID NO: 60), wherein X is an amino acid residue selected from the group consisting of SEQ ID NO: (SEQ ID NO: 60), or (d) a nucleic acid sequence identity with a nucleic acid sequence encoding a specific fragment of a specifically derived amino acid sequence of FIG. 24 (SEQ ID NO: 60) More preferably at least about 80%, more preferably at least about 81%, even more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85% , More preferably at least about 86%, more preferably at least about 87% More preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least about 92%, even more preferably at least about 93% , More preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% Will be at least about 99%. PRO509 polynucleotide variants do not encompass the entire native PRO509 nucleotide sequence.

(A) a nucleic acid sequence encoding residue 1 or about 27 to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: 62), (b) a nucleic acid sequence encoding a PRO866 variant polynucleotide of 26 (SEQ ID NOS: 62), (c) a nucleic acid sequence encoding amino acids X to 331 of the PRO866 polypeptide, wherein X is any amino acid residue 22 to 31 of Figure 26 (SEQ ID NO: 62) Quot; means a nucleic acid molecule that encodes an active PRO866 polypeptide as defined below that has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of SEQ ID NO: Typically, the PRO866 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 27 to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: 62), (b) a polypeptide having the amino acid sequence of X to 331 of the PRO866 polypeptide of Figure 26 (C) a specific fragment of the specifically derived amino acid sequence of Figure 26 (SEQ ID NO: 62), or a fragment of the amino acid sequence of SEQ ID NO: More preferably at least about 81%, more preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84% of nucleic acid sequence identity with a nucleic acid sequence encoding a nucleic acid sequence having at least 80% , More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% Is at least about 90%, more preferably at least about 91%, more preferably at least about < RTI ID = 0.0 > , More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% More preferably at least about 98%, more preferably at least about 99%. PRO866 polynucleotide variants do not encompass the entire native PRO866 nucleotide sequence.

Variant polynucleotides of the order of about 30 or more nucleotides in length, more often more than about 60 nucleotides, more commonly, of the order of < RTI ID = 0.0 > More preferably at least about 120 nucleotides, more often at least about 150 nucleotides, more often at least about 180 nucleotides, more often at least about 210 nucleotides, more often at least about 240 nucleotides, more usually at least about 120 nucleotides, more often at least about 150 nucleotides, More often more than about 300 nucleotides, more often more than about 450 nucleotides, more often more than about 600 nucleotides, more often more than about 900 nucleotides or more .

&Quot; Nucleic acid sequence identity (%) " for the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptide coding nucleic acid sequences identified herein are PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptide coding nucleic acid sequences and introducing gaps to obtain maximum sequence identity (% Is defined as the percentage of nucleotides in the same candidate sequence as the nucleotides of the PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 nucleic acid sequences. Alignment for measuring nucleic acid sequence identity (%) may be performed using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software in a variety of ways known in the art . Those skilled in the art will be able to determine parameters suitable for measuring alignment, including any algorithms necessary to maximally align over the entire length of the compared sequences. However, in order to accomplish the object of the present invention, the nucleic acid sequence identity value (%) can be obtained using the sequence comparison computer program ALIGN-2 as described below, and the complete source code for the ALIGN- . The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., And the source code shown in Table 1 is maintained in the user documentation of the US Copyright Office (Washington, DC 20559), which is registered under US Copyright Registration No. TXU510087 . The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or may be edited from the source code described in Table 1. The ALIGN-2 program can be edited and used in the UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For purposes of the present application, nucleic acid sequence identity (%) (given nucleic acid sequence D, D, or constant nucleic acid sequence identity% to D) of a given nucleic acid sequence C to D, Lt; RTI ID = 0.0 > C) < / RTI > is calculated as follows:

W / Z x 100

Where W is the number of nucleotides recorded equally by the sequence alignment program ALIGN-2 in the alignment of the sequence alignment program ALIGN-2 of C and D, and Z is the total number of nucleotides of D. It will be appreciated that if the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, then the nucleic acid sequence identity of C to D is not equal to the nucleic acid sequence identity (%) of D to C. As an example of nucleic acid sequence identity (%) calculation, Tables 2c and 2d show how to calculate the nucleic acid sequence identity (%) of a nucleic acid sequence referred to as " comparison DNA " .

Unless specifically stated otherwise, all nucleic acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described above. However, nucleic acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST-2 [Altschul et al., Nucleic Acids Res . 25 : 3389-3402 (1997)). The NCBI-BLAST-2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.). NCBI-BLAST-2 uses several search parameters, such as unmasked = yes, strand = all, expected occurrence = 10, minimum low complexity length = 15/5, multi-pass e - value = 0.01, a constant for multi-pass = 25, a drop-off for final gap alignment = 25, and a scoring matrix = BLOSUM62.

When NCBI-BLAST-2 is used for nucleic acid sequence comparison, the nucleic acid sequence identity (%) of a given nucleic acid sequence C to D and / or D (given in nucleic acid sequence D, D, or D Which may or may not be expressed as a given nucleic acid sequence C, which has or comprises a constant nucleic acid sequence identity (%) to the nucleotide sequence of SEQ ID NO:

W / Z x 100

Where W is the number of nucleotides that are equally recorded by the sequence alignment program NCBI-BLAST-2 in the NCBI-BLAST-2 program alignment of C and D, and Z is the total number of nucleotides of D. It will be appreciated that if the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, then the nucleic acid sequence identity of C to D is not equal to the nucleic acid sequence identity (%) of D to C.

In addition, the nucleic acid sequence identity (%) is obtained using the WU-BLAST-2 computer program [Altschul et al., Methods in Enzymology 266 : 460-80 (1996)]. Most of the WU-BLAST-2 search parameters are set to default values. The parameters that were not set to default values, i.e., the adjustable parameters, were set to the following values: Overlap span = 1, overlap fraction = 0.125, word limit (T) = 11, and score matrix = BLOSUM62. In order to achieve the object of the present invention, the nucleic acid sequence identity value (%) is determined by (a) the nucleic acid molecule compared with the nucleic acid sequence of the target PRO polypeptide-encoding nucleic acid molecule having the sequence derived from the native PRO polypeptide- BLAST-2, which is the same nucleotide sequence as the PRO mutant polynucleotide as the sequence compared to the polypeptide-encoding nucleic acid molecule, and then (b) the total nucleotides of the target PRO polypeptide- Get divided by number. For example, an isolated nucleic acid molecule comprising a nucleic acid sequence A having a nucleic acid sequence identity of 80% or more with the nucleic acid sequence B ", wherein the nucleic acid sequence A is a sequence of a target nucleic acid to be compared and the nucleic acid sequence B is a PRO polypeptide Is the nucleic acid sequence of the target nucleic acid molecule to be encoded.

In another embodiment, variant polynucleotides of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 comprise a nucleotide sequence coding for the full length PRO179 polypeptide of Figure 2 10, a nucleotide sequence encoding a full-length PRO320 polypeptide of Figure 6 (SEQ ID NO: 10), a nucleotide sequence encoding a full-length PRO209 polypeptide of Figure 8 (SEQ ID NO: The nucleotide sequence encoding the full-length PRO221 polypeptide of Figure 14 (SEQ ID NO: 20), the nucleotide sequence encoding the full-length PRO224 polypeptide of Figure 12 (SEQ ID NO: 25), the nucleotide sequence encoding the full- ), The nucleotide sequence encoding the full-length PRO301 polypeptide of Figure 18 (SEQ ID NO: 43), the nucleotide sequence encoding the full-length PRO526 polypeptide of Figure 18 The nucleotide sequence encoding the full length PRO362 polypeptide of Figure 20 (SEQ ID NO: 48), the nucleotide sequence encoding the full length PRO356 polypeptide of Figure 22 (SEQ ID NO: 55), the nucleotide sequence encoding the full length PRO509 polypeptide of Figure 24 PRO279, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, which can hybridize to the nucleotide sequence encoding the full-length PRO866 polypeptide of Figure 26 (SEQ ID NO: 62), preferably under stringent hybridization and washing conditions. , PRO362, PRO356, PRO509 or PRO866 polypeptides, respectively. The variant polypeptides include PRO179, PRO209, PRO220, PRO220, PRO224, PRO224, 0.0 > PRO509 < / RTI > or PRO866 variant polynucleotides.

With respect to amino acid sequence identity comparisons performed as described above, the term " positive " includes not only the same but also the amino acid residues of the compared sequence having similar characteristics. The amino acid residue in which the positive value of the target amino acid residue is recorded is the same as the target amino acid residue or the target amino acid residue is preferably substituted (defined in Table 3 below).

For purposes of the present application, a given amino acid sequence B has a positive value (%) of a given amino acid sequence A for B and / or B (given amino acid sequence B has a positive positive value (%) for B and / Or may be expressed as a given amino acid sequence A containing)

X / Y x 100

Where X is the number of amino acid residues that record the positive value defined above in the alignment of the sequence alignment program ALIGN-2 of A and B and Y is the total number of amino acid residues of B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the positive value of A for B is not equal to the positive value of B for A.

&Quot; Isolated " as used to describe the various polypeptides disclosed herein means that the polypeptide has been identified and has been isolated and / or recovered from its natural environmental elements. Preferably, the isolated polypeptide is unbound with all components bound in nature. Pollution factors of the polypeptide's natural environment are substances that generally interfere with the diagnostic or therapeutic use of the polypeptide, including enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is (1) purified to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) coomassie blue or preferably silver Can be purified to yield only one band on SDS-PAGE under non-reducing or reducing conditions. The isolated polypeptide may be a polypeptide that is present in the system within the recombinant cell because it will not have at least one element of the natural environment, such as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, . However, usually isolated polypeptides will be obtained by one or more purification steps.

&Quot; isolated " nucleic acid molecules or anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219 encoding PRO109, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Quot; isolated nucleic acid molecule encoding an anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO276, anti-PRO362, anti- PRO356, anti- PRO509 or anti- PRO866 antibody is PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866-coding nucleic acids or anti- PR179-, Anti-PRO202-, anti-PRO204-, anti-PRO208-, anti-PRO301-, anti-PRO276-, anti- Means a nucleic acid molecule identified and separated from one or more contaminant nucleic acid molecules that are typically joined in the natural source of the PRO866-encoding nucleic acid. Preferably, the isolated nucleic acid is free from all components bound in its natural state. PRO209-, PRO220-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866- encoding nucleic acid molecules or isolated anti- , Anti-PRO207-, anti-PRO320-, anti-PRO219-, anti-PRO221-, anti-PRO224-, anti-PRO328-, anti-PRO301-, anti- , Anti-PRO509- or anti-PRO866-encoding nucleic acid molecules are different from those found in nature. Thus, the isolated nucleic acid molecule may be present in the native cells in the presence of the PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866- Encoding nucleic acid molecule or a nucleic acid molecule encoding an anti-PR179-, anti-PRO207-, anti-PRO320-, anti-PRO219-, anti- -PRO362-, anti-PRO356-, anti-PRO509- or anti-PRO866-encoding nucleic acid molecules. However, it is contemplated that, for example, a polynucleotide that is located at a different chromosomal location from a natural cell and that is typically present at a position on the chromosome of the anti-PRO179, anti-PRO179, PRO209, Cells expressing the anti-PR207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti- PRO526, anti- PRO362, anti- , PRO209-, PRO220-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866-coding nucleic acid molecules or anti-PRO179-, Anti-PRO209-, anti-PRO219-, anti-PRO221-, anti-PRO224-, anti-PRO328-, anti-PRO301-, anti-PRO626-, anti- The anti-PRO509- or anti-PRO866-encoding nucleic acid molecule can be an isolated nucleic acid molecule or an anti-PRO179 encoding a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide , Anti-PRO207, anti-PRO320, anti-PRO2 Is contained in an isolated coding nucleic acid molecule encoding an anti-PR192, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibody.

The term " regulatory sequence " is a DNA sequence necessary for the expression of coding sequences operably linked in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. For example, the DNA of a full-length or secretory leader is operably linked to the DNA of the polypeptide when expressed as a precursor protein in the form before secretion of the polypeptide, and the promoter or enhancer affects the transcription of the polypeptide sequence , The ribosome binding site is operably linked to the coding sequence when it is placed to facilitate translation. Generally, " operably linked " means that the DNA sequences to be ligated are located adjacent to each other, and in the case of a secretory leader, not only located adjacent but also within the same reading frame. However, the enhancer does not need to be located contiguously. Linking is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

The term " antibody " is used in its broadest sense and specifically includes a single anti-PRO179, anti-PRO207, anti-PRO320, anti- PRO219, anti- -PRO526, anti-PRO362, anti-PRO356, anti-PRO509 and anti-PRO866 monoclonal antibodies (including agonist antibodies), anti- Anti-PRO209, anti-PRO207, anti-PRO207, anti-PR202, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti- Anti-PR320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti- Fragments of the anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti- PRO362, anti- ). As used herein, the term " monoclonal antibody " refers to the same individual antibody that comprises a population of substantially allogeneic antibodies, i.e., a population, except for possible spontaneous mutations that may be present in minor amounts.

The " stringency " of the hybridization reaction is readily determinable by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. Hybridization generally depends on the reannealing ability of the denatured DNA when the complementary strands are in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, the higher the relative temperature, the more stringent the reaction conditions, while the lower the relative temperature, the less stringent the reaction conditions. For more information and description of the severity of the hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology , Wiley Interscience Publishers (1995).

"Strict conditions" or "high stringency conditions" as defined herein means (1) conditions of low ionic strength and high temperature during washing, eg, at a temperature of 50 ° C., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (2) 50 mM sodium phosphate buffer (pH 6.5) / 0.1% bovine serum albumin / 0.1% containing formamide, for example, 42 캜, 750 mM sodium chloride and 75 mM sodium citrate at the time of hybridization (3) 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 0.1% sodium chloride, and 50% (v / v) formaldehyde prepared using 0.1% Pycol / 0.1% polyvinylpyrrolidone. ), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ug / ml), 0.1% SDS, and 10% dextran sulfate 0.2 x SSC (sodium chloride / sodium citrate) at 42 占 and 50% formaldehyde at 55 占 폚. Draw washing, and to perform a high-stringency wash with 0.1 x SSC containing EDTA at a 55 ℃.

The term " moderate stringency conditions " refers to hybridization conditions as described in Sambrook et al., Molecular Cloning : A Laboratory Manual, New York: Cold Spring Harbor Press (1989) Temperature, ionic strength, and% SDS). Examples of moderate stringency conditions include 20% formamide, 5 x SSC (150 mM sodium chloride, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate and 20 mg 0.0 > 37 C < / RTI > and then the filter is washed with 1 x SSC at about < RTI ID = 0.0 > 37-50 C. < / RTI > Those skilled in the art will appreciate how to adjust the temperature, ionic strength, and the like necessary to accommodate factors such as probe length and the like.

The term " epitope tagged ", as used herein, refers to a chimera comprising a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide fused to a " tag polypeptide "Lt; / RTI > polypeptide. The tag polypeptide is sufficiently short to have sufficient residues to provide an epitope to such an extent that the antibody can be made but not interfere with the activity of the polypeptide being fused. It is desirable that the tag polypeptide is so unique that the antibody to itself does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and typically have about 8 to 50 amino acid residues (preferably about 10 to 20 amino acid residues).

The term " immunoadhesin " as used herein refers to an antibody-like molecule that combines the effector function of an immunoglobulin constant domain with the binding specificity of a heterologous protein (" adhesin "). Structurally, the immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with an amino acid sequence having the desired binding specificity that is not (i. E., Heterologous) antigen recognition and binding site of the antibody. The adhesin portion of the immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of the receptor or ligand. Immunoglobulin constant domain sequences in immunoadhesins include any immunization such as IgG-1, IgG-2, IgG-3, IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM It can be obtained from globulin.

&Quot; Active " or " active ", as used herein, refers to the biological and / or immunological nature of natural or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides Refers to the form (s) of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Refers to an ability to induce the production of an antibody against an antigenic epitope of native or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. (Inhibition or stimulation) by natural or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, Is used to refer to the ability to induce the production of antibodies against an antigenic epitope of natural or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 .

&Quot; Biologically active " in antibodies or other agonists that can be identified by the screening assays disclosed herein refers to the ability of the molecule to elicit one or more of the effects listed herein, together with the definition of " therapeutically effective amount & . In certain embodiments, " biological activity " is an ability to inhibit neoplastic cell growth or proliferation. A preferred biological activity is inhibition, including delayed or complete arrest of growth of target tumor (e.g., cancer) cells. Another desirable biological activity is the cytotoxic activity that results in the death of target tumor (e.g., cancer) cells. Another biological activity is the induction of apoptosis in target tumor (eg, cancer) cells.

The term " immunological activity " means the immunological cross reactivity of one or more epitopes of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides.

As used herein, " immunologically cross reactive " means that the candidate polypeptide is a polyclonal antibody raised against known active PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Is capable of competitively inhibiting the quantitative biological activity of the antisera and the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides having the above activities. The antisera are prepared in a conventional manner by subcutaneous injection of chlorine or rabbits with, for example, analogs of known activity in a complete prodrug adjuvant, and boosting by intraperitoneal or subcutaneous injection in the complete prod. The immunological cross-reactivity is preferably " specific ", which means that the corresponding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, The binding affinity for a PRO362, PRO356, PRO509 or PRO866 polypeptide is significantly higher (preferably about 2 times or more, more preferably about 4 times or more, more preferably about 2 times or more, Quot; means about 6 times or more, and most preferably about 8 times or more).

&Quot; Tumor " as used herein refers to all neoplastic cell growth and proliferation of malignant or benign and all pre-cancerous and cancerous cells and tissues.

&Quot; Cancer " and " cancer " refer to or define the physiological state of a mammal having the typical characteristics of unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, sarcoma, sarcoma, and leukemia. More specific examples of the cancer include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastric cancer, pancreatic cancer, glioma, liver cancer, bladder cancer, liver cancer, Endometrial cancer, salivary gland carcinoma, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer.

&Quot; Treatment " means treatment performed to prevent the occurrence of a disease or to change the pathology of the disease. Thus, " treatment " refers to both treatment treatment and inhibition or prevention measures. Those requiring treatment include those who have already been sick, as well as those who must prevent disease. In the treatment of tumors (e.g., cancer), the therapeutic agent may directly reduce the pathology of cancer cells or may make the tumor cells more susceptible to treatment with other therapeutic agents, for example radiation and / or chemotherapeutic agents have.

"Pathology" of cancer includes all phenomena that damage the health of the patient. This includes, but is not limited to, abnormal or unregulated cell growth, metastasis, normal function disruption of adjacent cells, secretion of abnormal levels of cytokines or other secretions, inhibition or deterioration of inflammation or immune response, and the like.

An " effective amount " of the polypeptide or agonist disclosed herein in relation to inhibition of neoplastic cell growth is an amount capable of inhibiting the growth of target cells to some extent. The term includes growth inhibition, cell inhibition and / or cytotoxic effect and / or apoptosis of the target cell. The "effective amount" of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists for inhibiting neoplastic cell growth can be determined empirically and routinely have.

(1) inhibition of tumor growth to some extent, including delayed and complete growth arrest of tumor growth, (2) reduction in the number of tumor cells, (3) inhibition of tumor growth, ), (4) inhibition of tumor cell invasion into the surrounding organs (i.e., decrease, delay or complete stop), (5) inhibition of metastasis (And not necessarily cause) an antitumor immune response that can lead to degeneration or rejection of the disease, and / or (7) a reduction in one or more symptoms associated with the disease to some extent. A " therapeutically effective amount " of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or an agonist for tumor therapy can be determined empirically in a conventional manner.

The "growth inhibiting amount" of a polypeptide, or an agonist thereof, of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, It can be inhibited in vitro or in vivo. The "growth inhibiting amount" of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or their agonists for inhibiting neoplastic cell growth is determined empirically in a conventional manner You can decide.

A " cytotoxic amount " of a polypeptide, or an agonist thereof, of a cell, particularly a tumor, such as a cancer cell, It is the amount that can be produced in or in vivo. The "cytotoxic amount" of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists for inhibiting neoplastic cell growth is determined empirically in a conventional manner You can decide.

As used herein, " cytotoxic agent " refers to a substance that inhibits or inhibits the function of cells and / or destroys cells. The term encompasses toxins or fragments thereof having an enzymatic activity of a radioactive isotope (e.g., I ¹³¹ , I ¹²⁵ , Y ^90, and Re ¹⁸⁶ ), a chemotherapeutic agent and a toxin, such as a bacterial, fungal, .

A " chemotherapeutic agent " is a chemical compound useful in the treatment of tumors, e.g., cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, For example, paclitaxel (trade name, Taxol, Bristol Myers Squibb on Colloquy, New Jersey, USA) and dock laundry cell (Taxotere, Long Plan Lorea, Anthony, France) , Methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifospamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, Minomycin, aminopterin, dactinomycin, mitomycin, esperamicin (see U.S. Patent No. 4,675,187), melphalan and other related nitrogen mustards. Also included within the definition of such chemotherapeutic agents are hormonal agents that modulate or inhibit hormone action on tumors such as tamoxifen and onafristone.

As used herein, " growth inhibitory agent " refers to a compound or composition that inhibits the in vitro or in vivo growth of cells, particularly tumors, such as cancer cells. Thus, growth inhibitors are substances that significantly reduce the percentage of target cells in the S phase. Examples of growth inhibitors include substances that block cell cycle progression (at times other than the S phase), such as those that induce G1 arrest and M group arrest. Exemplary M-blockers include Vincass (vincristine and vinblastine), Taxol, and Topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Also, substances that stop G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara- do. Further information can be found in Murakami et al., The Molecular Basis of Cancer , Mendelsohn and Israel, eds., "Cell cycle regulation, oncogens, and antineoplastic drugs" (WB Saunders: Philadelphia, Lt; / RTI >

&Quot; Cytokine " is a generic name for proteins that act on other cells as intercellular mediators, released by a single cell community. Examples of cytokines are lymphokines, monokines and typical polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, rilacsin, prolelaxacin, follicle stimulating hormone (FSH) Glycoprotein hormones such as thyroid stimulating hormone (TSH) and luteinizing hormone (LH), liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-? And?, Mullerian inhibitory substance, Mouse gonad stimulating cell-related peptide, inhivin, actibin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor such as NGF-β; Platelet growth factor; Transforming growth factors (TGF), such as TGF-a and -beta, insulin like growth factor-1 and -Π, erythropoietin (EPO), bone inducers, interferons, CSF (G-CSF), interleukin-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte- IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- , IL-12; Tumor necrosis factors, such as TNF-a or TNF-beta; And other polypeptide factors including LIF and kit ligand (KL). The term cytokine as used herein includes biologically active equivalents of proteins and native sequence cytokines from natural sources or from recombinant cell cultures.

As used herein, the term " prodrug " refers to a precursor or derivative form of a pharmaceutical active substance that is less cytotoxic to tumor cells than the parent drug and can be converted enzymatically or into more active forms [ Wilman, "Prodrugs in Cancer Chemotherapy" , Biochemical Society Transactions, 14: 375-382, 615th Meeting Belfast (1986) and Stella et al,. "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Deliver, Borchardt et al ., (ed.), pp. 247-276, Humana Press (1985)). Prodrugs of the present invention include, but are not limited to, phosphate containing prodrugs, thiophosphate containing prodrugs, glycosylated prodrugs or optionally substituted phenylacetamide containing prodrugs, 5-fluorocytosine and other 5-fluoro uridine prodrugs But is not limited to. Cytotoxic drugs that may be derivatized with a prodrug for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

The term " agonist " is used in its broadest sense and refers to all molecules that mimic the biological activity of native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides do. Suitable agonist molecules specifically include fragments or amino acid sequence variants of the agonist antibody or fragment of the antibody, native PRO179, PRO207, PRO220, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, Peptides, small organic molecules, and the like. A method for identifying an agonist of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide comprises contacting tumor cells with a candidate agonist, And measuring the temperature of the liquid.

&Quot; Chronic " administration, as opposed to acute administration, refers to administration of the agent in a continuous form to maintain the initial therapeutic effect (activity) over a prolonged period of time. &Quot; Intermittent " administration is rather a substantially periodic treatment that is not performed continuously without interruption.

Therapeutic "mammals" are classified as mammals, including humans, livestock and breeding animals, and zoo, competition or pets such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits and the like It refers to all animals that become. A preferred mammal is a human.

Administration " in combination " with one or more other therapeutic agents includes simultaneous (concurrent) or sequential administration in any order.

As used herein, " carrier " is a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to the cell or mammal exposed to the dose and concentration employed. Physiologically acceptable carriers are often pH buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins; Other hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, Such as mannitol or sorbitol, salt forming counter ions such as sodium, and / or nonionic surfactants such as TWEEN TM, polyethylene glycol (PEG) and PLURONICS, Trade name).

&Quot; Natural antibody " and " natural immunoglobulin " are glycoproteins of about 150,000 daltons of heterotetramer, generally consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one disulfide covalent bond, and the number of disulfide bonds depends on the different immunoglobulin isoforms of the heavy chain. Also, each heavy and light chain has disulfide bonds in the chain at regular intervals. Each heavy chain has a variable domain (V _H ) at one end and a number of constant domains linked to this domain. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end, The constant domain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that certain amino acid residues form a shared region between the light and heavy chain variable domains.

The term " variable " is believed to be used in the binding and specificity of each particular antibody to a particular antigen, and the sequence of a particular portion of the variable domain between the antibodies is significantly different. However, the variability is not evenly distributed throughout the variable domains of the antibody. Variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions of both light and heavy variable domains. The portion having a very high degree of conservation in the variable domain is referred to as a framework region (FR). The variable domains of the native heavy and light chains include four framework regions that primarily select the sequence of the beta -sheet by three CDRs that form a loop connecting or, optionally, forming a part of its structure do. The CDRs within each chain are kept very close to each other by the FR region and contribute to the formation of the antigen binding site of the antibody along with the CDRs of the other chain (Kabat et al., NIH Publ. No. 91-3242, Vol. , pages 647-669 (1991)). The constant domains are not directly involved in antigen binding of the antibody, but exhibit antibody-mediated functions in multiple effector functions, such as antibody-dependent cytotoxicity.

As used herein, " hypervariable region " refers to an amino acid residue of an antibody that plays an important role in antigen binding. (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and residues 31 (L3) in the heavy chain variable domain Amino acid residues of amino acid residues 35-35 (H1), 50-65 (H2) and 95-102 (H3) [Kabat et al., Sequences of Proteins of Immunological Interests , 5th Ed. Public Health Services, National Institute of Health, Bethesda, MD. (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and residues 26-32 in the heavy chain variable region 32 (H1), 53-55 (H2) and 96-101 (H3)) [Clothia and Lesk, J. Mol. Biol ., 196 : 901-917 (1987). The " framework " or " FR " moiety is a region residue other than the above defined and variable region residues.

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

Papain digestion of the antibody produces two identical antigen binding fragments, the so-called " Fab " fragments with one antigen binding site, and the remaining named " Fc " fragments reflecting their ability to crystallize readily. Treatment of pepsin produces an F (ab ') ₂ fragment that has two antigen binding sites and is still capable of cross-linking to the antigen.

&Quot; Fv " is a minimal antibody fragment comprising a complete antigen recognition and antigen binding site. This region consists of one light chain variable domain and one light chain variable domain dimer tightly noncovalently bound. With this configuration, the three CDRs of phosphorus at each variable degree interact to define an antigen binding site on the surface of the V _H -V _L dimer. Overall, six CDRs confer antigen binding specificity to the antibody. However, a single variable domain (or half of the Fv comprising only CDRs specific to only three antigens) is also less susceptible to binding than the entire binding site, but can bind to the antigen by recognizing the antigen.

In addition, the Fab fragment contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab fragments differ from Fab fragments in that several residues are added to the carboxy terminus of the heavy chain CH1 domain, which contains at least one cysteine derived from the antibody hinge region. Fab'-SH herein refers to Fab 'in which the cysteine residue (s) of the constant domain retain a free thiol group. The F (ab ') ₂ antibody fragment was originally made up of several pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The "light chain" of an antibody (immunoglobulin) derived from any vertebrate species can be classified as one of two distinctly different types referred to as kappa (?) And lambda (?) Based on the amino acid sequence of its constant domain have.

Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, some of which are subclassed (isoforms), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2 .

As used herein, the term " monoclonal antibody " refers to the same individual antibody that comprises a population of substantially allogeneic antibodies, i.e., a population, except for possible spontaneous mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, each monoclonal antibody is directed against a single determinant for the antigen, as opposed to conventional antibodies (polyclonal antibodies) that typically comprise other antibodies to different determinants (epitopes). In addition to their specificity, monoclonal antibodies are synthesized by hybridoma cultures and have the advantage that they are not contaminated by other immunoglobulins. The modifier " monoclonal " refers to the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and is not to be construed as requiring antibody production in any particular way. For example, the monoclonal antibodies used in the present invention can be prepared by a hybridization magic or recombinant DNA method originally described in Kohler et al., Nature , 256 : 495 (1975) [US Pat. No. 4,816,567] . Also, " monoclonal antibodies " are described, for example, in Clackson et al., Nature , 352 : 624-628 (1991) and Marks et al., J. Mol. Biol ., 222 : 581-597 (1991).

The monoclonal antibody herein specifically refers to a heavy chain and / or a portion of a light chain that is identical or distinct from the sequence corresponding to an antibody derived from a particular species or an antibody belonging to a particular antibody group or subclass, (Immunoglobulin) that is identical or homologous to an antibody derived from another species or an antibody belonging to another antibody group or subclass or a fragment of such an antibody, (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA , 81 : 6851-6855 (1984)].

Quot; humanized " forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e. G., Fv, Fab, Fab ', F (ab') ₂ or other antigen binding small sequence of antibody). In most cases, a humanized antibody is one in which the residue of the complementarity determining region (CDR) of the recipient is replaced with a residue from the CDR of a non-human species (donor antibody) such as a mouse, rat or rabbit with the desired specificity, affinity and ability Human immunoglobulin (acceptor antibody). In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the donor antibody or in the CDR or framework sequences introduced. These modifications make it possible to improve and maximize antibody performance. In general, a humanized antibody will comprise substantially all of one or more, typically two or more, variable domains, wherein all or substantially all of the CDR regions correspond to regions of non-human immunoglobulin, and all or substantially all of the FR regions Corresponds to the region of the human immunoglobulin consensus sequence. In addition, the humanized antibody will optimally contain at least a portion of an immunoglobulin constant region (Fc), generally at least a portion of a human immunoglobulin region (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)). Humanized antibodies include PRIMATIZED (TM) antibodies in which the antigen binding region of the antibody is derived from an antibody produced by immunizing a short tail monkey with the desired antigen.

A "single chain Fv" or "sFv" antibody fragment comprises the V _H and V _L domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide also includes a polypeptide linker between the V _H and V _L domains such that the sFv forms the necessary structure for antigen binding. For sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113 , Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "dia body (diabody)" in the same polypeptide chain (V _H -V _L) in the light chain variable domain (V _L) small antibody fragments with two antigen-binding site comprising a heavy chain variable domain (V _H) connected to a . By using a linker that is too short to pair two domains in the same chain, the domain is forcedly paired with the complementary domain of the other chain to create two antigen binding sites. Diabodies are described, for example, in EP 404,097, WO 93/11611 and Hollinger et al., Proc. Natl. Acad. Sci. USA , 90 : 6444-6448 (1993).

An " isolated " antibody is an antibody that has been identified and isolated and / or recovered from elements of its natural environment. Contaminants in his natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones and other monovalent or non-hydrophobic solutes. In a preferred embodiment, the antibody comprises (1) more than 95% by weight, most preferably greater than 99% by weight of the antibody as measured by the Lowry method, or (2) using a spinning cup sequencer To a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid, or (3) by means of SDS-PAGE under reducing or nonreducing conditions using coomassie blue, or preferably silver staining It will be refined. Isolated antibodies include antibodies in situ within recombinant cells since one or more components of the natural environment of the antibody will not be present. However, the isolated antibody will generally be purified by one or more purification steps.

&Quot; Label " when used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a " labeled " antibody. The label can be detected by itself (radioactive isotope label or fluorescent label) or, if it is an enzyme label, it can catalyze the chemical change of the detectable substrate compound or composition.

&Quot; Solid phase " means a non-aqueous matrix to which an antibody of the invention may be attached. Examples of solid phases include partially or fully formed glasses (e.g., microporous glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrenes, polyvinyl alcohols, and silicones. In certain embodiments, depending upon the context, the solid phase may comprise a well of the assay plate, and in other embodiments, the solid phase is a purification column (e. G., Affinity chromatography column). The term also includes non-continuous solid phases of discrete particles such as those described in U.S. Patent No. 4,275,149.

&Quot; Liposomes " are used to deliver a drug (e. G., PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or antibodies directed thereto) It is a small vesicle made up of several types of useful lipids, phospholipids and / or surfactants. The components of the liposomes are typically arranged in bilayer form similar to the lipid alignment of biological membranes.

&Quot; Small molecule " is defined herein as having a molecular weight of about 500 daltons or less.

II. Compositions and Methods of the Invention

A. The full length PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides

The present invention relates to newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866. In particular, cDNAs encoding the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides were identified and isolated as described in more detail in the Examples below.

A cDNA clone encoding the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356 and PRO866 polypeptides was deposited with the ATCC as described in the Examples below [Clone PRO509 Those skilled in the art can easily determine the actual nucleotide sequence of the clone by analyzing the sequence of the deposited clone using common methods in the art. General techniques can be used to determine the expected amino acid sequence from the nucleotide sequence. In the case of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides described herein and the nucleic acid encoding the same, It is confirmed that it is regarded as a reading frame that can be confirmed well.

B. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variants

In addition to the full length native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptides described herein, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, , PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide variants. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 mutants are selected from the group consisting of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, , PRO356, PRO509 or PRO866 DNA and may be produced by the synthesis of a target PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Those skilled in the art will appreciate that amino acid changes such as changes in the number or position of glycosylation sites or changes in membrane anchoring properties will result in post-translational modifications of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, It will be understood that the processing may be changed.

The full-length native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, Variations in several domains of PRO526, PRO362, PRO356, PRO509 or PRO866 can be produced using conservative and non-conservative mutagenesis techniques and guidance, for example, as described in U.S. Patent No. 5,364,934. PRO219, PRO221, PRO224, PRO328, PRO302, PRO236, PRO326, PRO209 or PRO866 in comparison with the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, Substitution, deletion or addition of one or more codons encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, which alter the amino acid sequence of PRO362, PRO356, PRO509 or PRO866 Or insertion. Optionally, the mutation is generated by substituting one or more amino acids in one or more domains of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with any other amino acid. The amino acid residues that can be inserted, substituted or deleted without deleterious effects on the desired activity can be obtained by substituting known sequences of PRO179, PRO207, PRO220, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 Can be determined by comparing the sequence of a homologous protein molecule and minimizing the number of amino acid sequence changes produced in regions of high identity. Amino acid substitutions can be the substitution of one amino acid with another amino acid having similar structure and / or chemical properties, e. G., Replacement of lysine with serine, i. E. Conservative substitution of amino acids. Insertion or deletion may optionally occur at about 1 to 5 amino acids. Acceptable variations can be determined by systematically inserting, deleting, or substituting amino acids within the sequence and testing the activity of the resulting variant displayed by full-length or mature native sequences.

Provided herein are PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptide fragments. For example, when compared to full-length native proteins, these fragments may be truncated at the N-terminus or at the C-terminus and internal residues may be deleted. Some fragments lack amino acid residues that are not essential for the desired biological activity of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides of the present invention.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 fragments can be produced by any of many conventional techniques. The target peptide fragments can be chemically synthesized. Other methods include enzymatic degradation methods, for example by treating the protein with an enzyme known to cleave the protein at a site defined by a particular amino acid residue, or by cleaving the DNA with a suitable restriction enzyme, such as PRO179, PRO207, PRO320, PRO219, PRO221 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 fragments and isolating this target fragment. However, another suitable technique involves isolating and amplifying a DNA fragment encoding a target polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that define the target end of a DNA fragment are used as 5 ' and 3 ' primers in PCR. Preferably, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptide fragments are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: (SEQ ID NO: 10), SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: PRO279, PRO220, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 (SEQ ID NO: Or one or more biological and / or immunological activities with the PRO866 polypeptide.

In a specific embodiment, conservative substitutions of the subject are shown in Table 3 with the title of preferred substituents. When the biological activity is changed by such substitution, a more substantial change, which is named as a substitution example in Table 3 below or is described in more detail below for the type of amino acid, is introduced and the product screened.

Substantial modifications of the function or immunological identity of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be achieved by (a) (B) preserving the charge or hydrophobicity of the molecule at the target site, or (c) selecting substitutions that significantly modify its effect of retaining most of the side chains. Naturally occurring residues can be divided into the following groups according to their common branching characteristics:

(1) hydrophobicity: norleucine, met, ala, val, leu, with,

(2) Neutral hydrophilic: cys, ser, thr,

(3) Acid: Asp, glu,

(4) Basicity: asn, gln, his, lys, arg,

(5) residues affecting the chain orientation: gly, pro and

(6) aromatic; trp, tyr, phe.

Non-conservative substitutions will replace the same kind of component with another kind. In addition, the thus substituted moiety can be introduced into the conservative substitution moiety or, more preferably, can be introduced into the remaining (non-conserved) moiety.

Variations can be made using methods known in the art such as oligonucleotide-mediated (locating) mutagenesis, alanine scanning and PCR mutagenesis. Location-directed mutagenesis [Carter et al., Nucl. Acids Res. , ≪ / RTI > 13 : 4331 (1986); Zoller et al., Nucl. Acids Res. , 10: 6487 (1987)], cassette mutagenesis [Wells et al, Gene, 34 :.. 315 (1985)], restriction selection mutagenesis [Wells et al, Philos. Trans. R. Soc. London SerA, 317: 415 (1986 )] or PRO179 of the present invention was carried out on the DNA cloning technology other known, PRO207, PRO320, PRO219, PRO221 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 A polypeptide mutant DNA can be produced.

In addition, one or more amino acids can be identified along the contiguous sequence using a scanning amino acid assay. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Typically, alanine is the preferred scanning amino acid because it is less likely to alter the backbone sequence of the variant, removing side chains outside the beta-carbon [Cunningham and Wells, Science , 244 : 1081-1085 (1989)]. In addition, alanine is preferred because it is usually the most common amino acid. Also, alanine is frequently found in both buried and exposed sites (Creighton, The Proteins , (WH Freeman & Co., NY); Chothia, J. Mol. Biol. , ≪ / RTI > 150 : 1 (1976)]. An isoteric amino acid can be used if the alanine substitution does not produce a suitable amount of the variant.

C. Variations of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866

Covalent modifications of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 are included within the scope of the present invention. One form of covalent bond modification is an organic derivatizing agent capable of reacting with a selected side chain or N terminal or C terminal residue of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 And the target amino acid residues of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. The derivatization using a bifunctional agonist can be carried out using, for example, anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, , PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or the like on the water-insoluble support matrix or surface for use in the anti-PRO356, anti-PRO509 or anti- It is useful for cross-linking PRO866 or vice versa. Commonly used crosslinking agents are, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4- Homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and Methyl-3 [(p-azidophenyl) dithio] propioimidate.

Other variations include deamidation of the glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of the proline and lysine, phosphorylation of the hydroxyl group of the seryl or threonyl residue, lysine, Methylation of alpha-amino groups of arginine and histidine side chains [TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & 79-86 (1983)], acetylation of the N-terminal amine and amidation of the C-terminal carboxyl group.

Other types of covalent modifications of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides included within the scope of the present invention include changes in the native glycosylation pattern of the polypeptide . A "change in the native glycosylation pattern" refers to the deletion of one or more carbohydrate moieties found in the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 (Or by deletion of the glycosylation by a chemical and / or enzymatic method) and / or by using the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, Quot; means the addition of one or more glycosylation sites that are not present in PRO866. The term also includes qualitative changes in the glycosylation of natural proteins, including changes in the nature and proportions of the various carbohydrate residues present.

The addition of glycosylation sites to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be achieved by altering the amino acid sequence. The change may be made, for example, by the addition or substitution of one or more serine or threonine residues in the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 For linked glycosylation sites). The amino acid sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 is particularly useful for the production of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224 , PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can be optionally altered through changes at the DNA level by mutating in a preselected base.

Other means of increasing the number of carbohydrate moieties on the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides include coupling the glycosides to the polypeptides chemically or enzymatically I will. Such methods are described, for example, in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem. , pp. 259-306 (1981).

Removal of the carbohydrate moieties present in the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be accomplished by chemical or enzymatic or amino acid residues that function as glycosylation targets Lt; / RTI > by mutation of the coding codon. Chemical deglycosylation techniques are well known in the art and are described, for example, in Hakimuddin, et al., Arch. Biochem. Biophys. , 259 : 52 (1987) and Edge et al., Anal. Biochem. , 118 : 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using a variety of endoglycosidases and exoglycosidases [Thotakura et al., Meth. Enzymol. , ≪ / RTI > 138 : 350 (1987)).

Other classes of covalent modifications of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 are described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417 , PRO209, PRO320, PRO219, PRO221, PRO224, PRO221, or PRO227 in one of various non-proteinaceous polymers such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene in the manner described in US Pat. No. 4,791,192 or 4,179,337, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides.

In addition, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides of the present invention may also be used in combination with other heterologous polypeptides or amino acid sequences, such as PRO179, PRO207, PRO320, PRO219, PRO221 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866.

In one embodiment, such a chimeric molecule comprises a tag polypeptide that provides an epitope to which the anti-tag antibody can selectively bind and a tag polypeptide that provides an epitope in which the anti- Or a fusion of PRO866 polypeptides. The epitope tag is generally located at the amino or carboxyl terminus of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. The presence of the epitope tagged forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides is detected using an antibody against the tagged polypeptide . In addition, introduction of an epitope tag results in an increase in the affinity of the PRO179, the PRO207, the PRO320, the PRO219, the PRO221, the PRO224, the PRO328, the PRO301, the PRO526, the PRO362, and the PRO362 by affinity purification using an affinity matrix using another kind of affinity matrix, PRO356, PRO509 or PRO866 polypeptides can be easily purified. A variety of tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-his-gly-tag, flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. , 8: 2159-2165 (1988)] , c-myc tag and hence of 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [Evan et al, Molecular and Cellular Biology, 5:. 3610-3636 (1985)] , And the herpes simplex virus protein glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering , 3 (6): 547-553 (1990)]. Other tag polypeptides include Flag-peptides [Hopp et al., BioTechnology , 6 : 1204-1210 (1988)], KT3 epitope peptides [Martin et al., Science , 255 : 192-194 (1992) Epitope peptides [Skinner et al., J. Biol. Chem. , 266 : 15163-15166 (1991)] and the T7 gene 10 protein tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA , 87 : 6393-6397 (1990).

In another embodiment, chimeric molecules comprise fusions of specific regions of immunoglobulins or immunoglobulins with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. In the case of a bimodal form of chimeric molecule (also referred to as " immunoadhesin "), the fusant may be the Fc region of an IgG molecule. The Ig fusions are preferably designed such that the site of one or more of the variable regions in the Ig molecule is replaced by a soluble (deletion or deletion) region of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides Active membrane trans- < / RTI > domains). In a particularly preferred embodiment, the immunoglobulin fusions comprise the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. For methods of producing immunoglobulin fusions, see U.S. Patent No. 5,428,130, issued June 27, 1995.

D. Preparation of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866

Described below is the production of PRO179 by culturing cells transfected or transfected with a vector containing nucleic acids predominantly PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 nucleic acids. , PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Of course, other methods known in the art can be used to produce PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide sequences or fragments thereof. For example, the sequences or fragments thereof included in the above definition of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be obtained by direct peptide synthesis (Stewart et al., Solid-Pharmaceutical Peptide Synthesis , WH Freeman Co., San Francisco, CA (1969), Merrifield, J. Am. Chem. Soc. , ≪ / RTI > 85 : 2149-2154 (1963)). In vitro protein synthesis can be performed by manual or automated methods. Automated synthesis can be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. The various PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide fragments can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full length PRO179, PRO207, PRO320 , PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides.

1. Isolation of DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866

The coding DNA of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide may be used in combination with the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, , PRO509 or PRO866 mRNA and can be obtained from a cDNA library prepared from tissues which are thought to express it at a detectable level. Thus, human PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding DNA can be conveniently obtained from cDNA libraries prepared from human tissue as described in the Examples . The coding genes may also be obtained from the genomic library or by known synthetic methods (e. G., Automated nucleic acid synthesis), such as, for example, < RTI ID = 0.0 > PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, can do.

The library comprises a probe designed to identify a gene of interest or a protein encoded by the gene (for example, the desired PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides Or an oligonucleotide consisting of about 20 to 80 bases). Screening of the cDNA or genomic library using the selected probe is performed using standard methods as described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) can do. . PRO179, PRO207, PRO320, PRO219 , PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 other means for isolating the coding gene is to use PCR method [Sambrook et al, supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Haror Laboratory Press, 1995).

The following examples illustrate techniques for screening cDNA libraries. The oligonucleotide sequence selected as the probe should be of sufficient length and sufficiently distinct sequence to minimize false results. The oligonucleotides are preferably labeled so that they can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art and include the use of radiolabeled, biotinylated or enzymatic labels such as ³² P-labeled ATP. Hybridization conditions, including moderate stringency and high stringency is provided in the literature (Sambrook et al., Supra).

The sequence identified in the library screening method may be aligned with a public database such as GenBank or other known sequences available and available from other proprietary databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of a molecule or across a full-length sequence can be determined using methods known in the art and disclosed herein.

Nucleic acid having protein coding sequence and the first screening a cDNA or genomic library selected using conventional methods of primer extension described in the literature (Sambrook et al., Supra) to detect the estimated amino acid sequence and the precursor, if necessary, as disclosed herein, , < / RTI > by processing an intermediate of mRNA that has not been reverse transcribed with cDNA.

2. Selection and Transformation of Host Cells

The host cell is transfected or transformed with the expression or cloning vector described herein for the production of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, In a conventional nutrient medium modified for selection of a transfectant or gene amplification encoding a target sequence. Those skilled in the art can select culture conditions such as medium, temperature, pH, etc., without performing unnecessary experiments. In general, principles, protocols, and techniques for maximizing the productivity of cell cultures are described in Mammalian Cell Biotechnology: a Practical Approach , M. Butler, ed. (IRL Press, 1991) and Sambrook et al., May be found in the above document.

Eukaryotic transfection methods and prokaryotic transformation methods, such as CaCl ₂ , CaPO ₄ , liposome-mediated methods and electroporation, are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques suitable for the cell. Calcium treatment or electroporation using the calcium chloride method described in the literature (Sambrook et al., Supra) is generally used for prokaryotic cells. Infection with Agrobacterium tumefaciens has been shown to inhibit the growth of certain plant cells as described in Shaw et al., Gene , 23 : 315 (1983), and WO 89/05859, published Jun. 29, ≪ / RTI > For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology , 52 : 456-457 (1978) can be used. Common features of mammalian cell host system transfection are described in U.S. Patent No. 4,399,216. Transformation into yeast is generally described in Van Solingen et al., J. Bact. , 130 : 949 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) , 76 : 3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, protoplast fusion with bacterial protoplasts, or polycations, such as polybrene and polyornithine, may also be used. See Keown et al., Methods in Enzymology , 185 : 527-537 (1990) and Mansour et al., Nature , 336 : 348-352 (1988) for a variety of techniques for transforming mammalian cells do.

Suitable host cells for cloning or expressing DNA in the vector herein are prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells include true bacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, e. E. coli. ≪ / RTI > A variety of this. Coli strains, e. E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31, 537); Coli strains W3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are readily available. Other suitable prokaryotic host cells include Escherichia, e. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, For example, marcescans and Shigella, and Bacillus, e. G. Subtilis and non-subtilis. Licheniformis (e.g., B. riciniformis 41P described in DD 266,710, published April 12, 1989), Pseudomonas such as p. Aeruginosa and Streptomyces. ≪ / RTI > These examples are merely illustrative and not restrictive. The strain W3110 is a particularly preferred host or parental host since it is a host strain common to recombinant DNA product fermentation. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 can be modified to result in a mutation of the gene encoding the endogenous protein of the host, and an example of such a host is E. coli having the complete genotype tonA . E. coli W3110 strain 1A2, with complete genotype tonA ptr3 . E. coli W3110 strain 9E4, complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r . E. coli W3110 strain 27C7 (ATCC 55,244), complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan ^r . E. coli W3110 strain 37D6, strain 37D6 with a non-kanamycin resistant degP deletion mutant. E. coli W3110 strain 40B4, and periplasmic protease variants disclosed in U.S. Patent No. 4,946,783, issued Aug. 7, Coli strains. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotic cells, eukaryotic microbes such as filamentous fungi or yeast are suitable as cloning or expression hosts for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding vectors. Saccharomyces cerevisiae is a lower eukaryotic host microorganism commonly used. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature , 290: 140 [1989]; EP 139,383, published May 2, 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology , 9: 968-975 (1991)); Lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Baceriol. , 737 [1983]), Kay. Fragilis (ATCC 12,424), Kay. Bulgaricus (ATCC 16,045), Kay. Wickeramii (ATCC 24,178), Kay. Waltii (ATCC 56,500), Kay. Drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology , 8: 135 (1990)), Kay. Thermotolerans and K. Marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol. , 28: 265-278 [1988]); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA , 76: 5259-5263 [1979]); Schwanniomyces such as, for example, Siebenia mume occidentalis (EP 394,538, published October 31, 1990); And filamentous fungi such as Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991) and Aspergillus host, e.g., Listen to this. 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 AA. Niger (Kelly and Hynes, EMBO J. , 4: 475-479 [1985]). Methyl auxotrophic yeast is suitable and has a genus consisting of Hansenula, Candida, Kloeckera, Pichia, Carolomyces, Torulopsis and Rhodotorula But not limited to, yeast that can grow on methanol. A list of specific species that are examples of these types of yeast can be found in [C. Anthony, The Biochemistry of Methylotrophs , 269 (1982).

Suitable host cells for expression of the glycosylated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding nucleic acids are derived from multicellular organisms. Examples of invertebrate cells are insect cells and plant cells such as Drosophila S2 and Spodoptera Sf9. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 lines (COS-7, ATCC CRL 1651) transformed by SV40, 293 cells subcloned for growth in human embryonic kidney lines (293 cells or suspension culture, Graham et al., J Gen. Virol. , 36: 599 (1997)), Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA , 77: 4216 (1980)), mouse Sertoli cells TM4, Mather, Biol Reprod, 23 :.. 243-251 (1980)), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065) and mouse mammary tumor (MMT 060562, ATCC CCL51) . One skilled in the art can readily select suitable host cells.

3. Choosing and Using Replicable Vectors

(For example, cDNA or genomic DNA) can be cloned (amplification of DNA) or cloned for expression (for example, Vector. Various vectors can be easily obtained. For example, the vector may be in the form of a plasmid, a cosmid, a viral particle, or a phage. Suitable nucleic acid sequences may be inserted into the vector by a variety of methods. Generally, DNA is inserted into the appropriate restriction endonuclease site (s) using techniques known in the art. The vector component generally includes, but is not limited to, a signal sequence, a replication origin, one or more marker genes, an enhancer component, a promoter, and a transcription termination sequence. The preparation of suitable vectors comprising one or more of the above components employs standard ligation techniques known to those skilled in the art.

Not only can they be produced by direct recombinant methods, but they also include specific cleavage sites at the N-terminus of the mature protein or polypeptide, Or a fusion polypeptide with a heterologous polypeptide, which may be another polypeptide or signal sequence having the same. In general, the signal sequence may be a component of a vector or it may be part of the PRO179, PRO207, PRO206, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding DNA inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader (Saccharomyces and Kluyveromyces alpha-factor leader (US Patent 5,010,182)) or an acid phosphatase Reader, Mr.. It may be a signal sequence as described in C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990) or WO 90/13646 published Nov. 15, 1990. In mammalian cell expression, signal sequences from secreted polypeptides of mammalian signal sequences, e. G. The same or related species, and viral secretory leader can be used to direct secretion of the protein.

Both the expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are known for a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 fits most Gram negative bacteria, the 2μ plasmid origin fits yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) clone vectors in mammalian cells useful.

Expression and cloning vectors will typically contain a selectable gene, also referred to as a selectable marker. A representative selection gene, for example, in the case of Bacillus, is a gene encoding D-alanine racemase that (a) is a protein that confers resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, b) proteins that complement nutritional deficiencies, or (c) proteins that supply important nutrients that are not available from complex media.

Examples of suitable selectable markers for mammalian cells are those that enable the identification of cells capable of accommodating PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding nucleic acids For example, DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is a CHO cell line lacking DHFR activity, see Urlaub et al, Proc. Natl. Acad. Sci. USA , 77: 4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (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 selection markers for mutant strains of yeast lacking the ability to grow into tryptophan (e.g., ATCC 44076 or PEP4-1) [Jones, Genetics , 85: 12 (1977)].

Expression and cloning vectors contain promoters operably linked to a coding nucleic acid sequence, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, which direct mRNA synthesis. Promoters recognized by a variety of potential host cells are known. Promoters suitable for use in prokaryotic hosts include the? -Lactamase and lactose promoter systems [Chang et al., Nature , 275: 615 (1978); Goeddel et al., Nature , 281: 544 (1979)], alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nucleic acid Res. , ≪ / RTI > 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA , 80: 21-25 (1983). The promoter used in the bacterial system may also be a Shine-Dalgano (SD) operably linked to DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Sequence. ≪ / RTI >

Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem. , 255: 2073 (1980)] or other sugar degrading enzymes [Hess et al., J. Adv. Enzyme Reg. , ≪ / RTI > 7: 149 (1968); Holland, Biochemistry , 17: 4900 (1978)) such as enolase, glyceraldehyde-3-phosphate dihydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 -Phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosulfate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters that are inducible promoters with additional transcriptional advantages that are regulated by growth conditions include alcohol dehydrogenase 2, isocytocrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde- 3-phosphate dehydrogenase, and promoter regions for enzymes that act on maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

The PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 transcript from the vector in the mammalian host cell can be obtained from viruses such as polyoma virus, (UK) 2,211,504, published on July 5, 2006), adenovirus (e.g., adenovirus 2), cow papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkey virus 40 From the genome, from a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, which is obtained from a heat-shock promoter.

Transcription of DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are generally cis-acting components of DNA of about 10 to 300 bp that act on the promoter to increase transcription. Many enhancer sequences are known to result from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, an enhancer will usually be used which is derived from eukaryotic viruses. Examples include the SV40 enhancer (bp 100-270) on the back of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the back of the origin of replication, and the adenovirus enhancer. The enhancer may be spliced to the vector at the 5 'or 3' position into the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding sequences, It is preferable to be located at a site.

In addition, expression vectors used in eukaryotic host cells (polynuclear cells derived from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) may contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically obtained from eukaryotic cells, or 5 'and sometimes 3' untranslated regions of viral DNA or cDNA. These regions contain nucleotide fragments that are transcribed as polyadenylation fragments in the untranslated portion of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding mRNA.

Other methods, vectors and host cells suitable for application in the synthesis of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 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 performed using appropriately labeled probes based on the sequences provided herein, for example Northern blotting, northern blotting to quantify transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization. In addition, an antibody capable of recognizing a specific duplex including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes can be used. In other words, an antibody can be labeled and an assay can be performed that binds the duplex to the surface to detect the presence of antibody bound to the duplex when duplexes are formed on the surface.

Gene expression can also be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and analysis of cell culture fluids or body fluids to directly quantify gene products. Antibodies useful in immunohistochemical staining and / or assays of a sample fluid may be monoclonal or polyclonal antibodies and may be prepared in any mammal. Conveniently, the synthetic peptides or PRO179, PRO207, PRO207, PRO202, PRO202, PRO224, PRO329, PRO207, PRO224, PRO328, PRO301, PRO526, PRO366, PRO356, PRO509 or PRO866 polypeptides, based on the native sequences provided herein, , PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 encoding DNA and encoding a specific antibody epitope.

5. Purification of the polypeptide

The forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides may be recovered from the culture medium or from the bacterial culture for host cells. When bound to the membrane, it can be released from the membrane by using a suitable detergent solution (e.g. Triton-X 100) or by cleavage of the enzyme. Cells used for the expression of the coding nucleic acid encoding the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide can be used in the freeze-thaw cycle, ultrasonication, mechanical grinding, And may be pulverized by various physical or chemical means, for example.

It may be desirable to purify PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 from the recombinant cell protein or polypeptide. Examples of suitable purification methods include fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as chromatography on DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, Protein A Sepharose column for the removal of contaminants such as IgG, and Protein A Sepharose column for removal of contaminants such as < RTI ID = 0.0 > PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, Is a metal chelating column for binding epitope tagged forms. A variety of protein purification methods can be used, such methods are well known in the art and are described, for example, in Deutscher, Methods in Enzymology , 182 (1990); Scopes, Protein Purification: Principles and Practice , Springer-Verlag, New York (1982). The purification step (s) selected will depend, for example, on the properties of the production method used and the specific PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 produced .

E. Antibodies

Some of the drug candidate substances for use in the compositions and methods of the present invention are those that mimic the biological activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides and Antibody fragment.

1. polyclonal antibody

Methods for producing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be produced in mammals, for example, by injection of one or more immunizing agents and, if desired, adjuvants. In general, the immunizing agent and / or adjuvant will be injected into the mammal by subcutaneous or intraperitoneal injection. The immunizing agent may include PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fusion fragments thereof. It may be useful to conjugate an immunizing agent to a protein known to be immunogenic in an immunized mammal. Examples of such immunogenic proteins include keyhole limpet hemocyanin, serum albumin, sorbitol globulin, and soybean trypsin inhibitor. Examples of adjuvants that may be used include prodrug complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose decolinomicolate). Immunization methods may be selected by those skilled in the art without undue experimentation.

2. Monoclonal antibody

In addition, the antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods as described in Kohler and Milstein, Nature , 256 : 495 (1975). In the hybridoma method, a mouse, hamster or other appropriate host animal is usually immunized with an immunizing agent to induce a lymphocyte capable of producing or producing an antibody that specifically binds to the immunizing agent. In addition, the lymphocytes can be immunized in vitro.

The immunizing agent will generally comprise PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fusion proteins thereof. In general, peripheral blood lymphocytes (" PBL ") are used when cells of human origin are desired, whereas spleen cells or lymph node cells are used when a non-human mammalian source is desired. Subsequently, a suitable fusion agent, such as polyethylene glycol, is used to fuse lymphocytes and immortalized cell lines to form hybridoma cells (Goding, Monoclonal Antibodies: Principle and Practice , Academic Press, (1986) pp. 59-103]. Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Generally, a mouse or mouse myeloma cell line is used. The hybridoma cells may preferably be cultured in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cell is deficient in the hypoxanthine guanine phospholibosyl transferase (HGPRT or HPRT) enzyme, the hybridoma medium will typically include hypoxanthine, aminopterin and thymidine (" HAT medium ") These substances inhibit the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that efficiently fuse, support a stable production of a high level of antibody by the selected antibody-producing cells, and are sensitive to media such as HAT medium. A more preferred immortalized cell line is, for example, a murine myeloma cell line available from the Salk Institute Cell Distribution Center (San Diego, CA) and the American Type Culture Collection (Manassas, Va., USA) . In addition, human myeloma cell lines and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133 : 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The presence of monoclonal antibodies to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can then be analyzed in the culture medium in which the hybridoma cells are cultured . Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by in vivo binding assays, for example, immunoassay by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) . Such techniques and assays are well known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis [Munson and Pollard, Anal. Biochem., 107: 220 (1980).

After identifying the desired hybridoma cells, the clones were subcloned by limiting dilution and cultured by the standard method [Goding, supra ]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Hybridoma cells can also be cultivated in vivo as multiple tumors in an animal.

The monoclonal antibody secreted by the subclone may be purified by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography And can be isolated and purified from a culture medium or a plurality of liquids.

In addition, monoclonal antibodies can be prepared by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be readily transformed by using a conventional method (for example, by using an oligonucleotide probe capable of specifically binding to a gene encoding a heavy chain and a light chain of a mouse antibody) And can be sequenced. The hybridoma cells of the present invention serve as a desirable source of such DNA. Once isolated, the DNA may be inserted into an expression vector and then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, resulting in monoclonal The antibody can be synthesized. In addition, DNA, for example, to replace the coding sequence for human heavy and light chain constant domains in place of the homologous sequence of the murine or (U.S. Patent Nos. 4,816,567;. Morrison et al, supra), or a non-immunoglobulin polypeptide Can be modified by covalently attaching all or part of the coding sequence to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides may be substituted for the constant domains of the antibodies of the invention, or substituted for the variable domains of one antigen-binding site of the antibody of the invention to generate chimeric dipeptidic antibodies.

The antibody may be a monovalent antibody. Methods for making monovalent antibodies are known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. To prevent heavy chain cross-linking, the heavy chain is typically truncated at any point in the Fc region. Alternatively, the related cysteine residue is substituted or deleted with another amino acid residue to prevent cross-linking.

In vitro methods Also suitable for the production of antibodies. The degradation of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using techniques commonly known in the art.

3. Human and humanized antibodies

In addition, the antibody of the present invention may comprise a humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies include immunoglobulin chains comprising minimal sequence derived from chimeric immunoglobulin, non-human immunoglobulin, or fragments thereof (e.g., Fv, Fab, Fab ' , F (ab ') ₂ or another antigen binding sequence of an antibody). Humanized antibodies are human immunoglobulins (CDRs) in which residues of the complementarity determining region (CDR) of the receptor have been replaced with residues of CDRs of non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and ability Acceptor antibody). In some cases, the Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise an acceptor antibody or a residue that is not found in the CDR or framework sequences to be introduced. In general, a humanized antibody will comprise substantially all of one or more, usually two or more, variable domains, wherein all or substantially all of the CDR regions correspond to regions of non-human immunoglobulin and all or substantially all FR regions are human Corresponds to the region of the immunoglobulin consensus sequence. In addition, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-327 (1988); And Presta, Curr. Op. Struct. Biol. 2 : 593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced thereto from non-human origin. These non-human amino acid residues are often referred to as " import " residues and are typically obtained from the " import " Humanization can be carried out essentially as described by Winter et al., Jones et al., Nature , 321 : 522-525 (1986); by substituting rodent CDRs or CDR sequences for the corresponding sequences of human antibodies. Riechmann et al., Nature , 332 : 323-329 (1988); Verhoeyen et al., Science , 239 : 1534-1536 (1988). Thus, such " humanized " antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which less than a substantially complete human variable domain is replaced by a corresponding sequence from a non-human species. Indeed, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues have been replaced with residues of similar moieties in rodent antibodies.

In addition, human antibodies can be prepared using a variety of techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol. , 227 : 381 (1991), Marks et al., J. Mol. Biol. , 222 : 581 (1991)). Also, techniques such as Coulter et al. And Boerner et al. Can also be used to prepare 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, by introducing a human immunoglobulin locus into a transgenic animal, e.g., a mouse in which the endogenous immunoglobulin gene is partially or totally inactivated Human antibody production has been observed following antigen administration, which is very similar to that observed in humans in all respects including gene rearrangement, assembly and antibody listing. (Marks et al., Bio / Technology 10 , 779-783 (1992), Lonberg et al., ≪ RTI ID = 0.0 > al., Nature 368 , 856-859 (1994), Mo rrison, Nature 368, 812-13 (1994 ), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995)).

4. Bispecific antibodies

Specific antibody is a monoclonal antibody, preferably a human or humanized antibody, having binding specificity for two or more different antigens. One of the binding specificities is for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 and the other for any other antigen, Protein, receptor or receptor subunit.

Methods for producing bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-light-chain pairs wherein the two heavy chains have different specificities (Millstein and Cuello, Nature , 305 : 537-539 (1983)]. Due to the random arrangement of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has an accurate bispecific structure. Purification of the correct molecule is generally carried out in an affinity chromatographic step. A similar method is described in WO 93/08829 (published May 13, 1993) and Traunecker et al., EMBO J. , 10 : 3655-3659 (1991).

The variable domain (antibody-antigen binding site) of the antibody with the desired binding specificity is fused to the immunoglobulin constant domain sequence. The fusion preferably takes place in an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CHl) is present in at least one of the fusion entities, which comprises a site essential for light chain binding. The immunoglobulin heavy chain fusions and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected with the appropriate host organism. For more details on producing bispecific antibodies see, for example, Suresh et al., Methods in Enzymology , 121 : 210 (1986).

According to another method disclosed in WO 96/27011, the covalent region between a pair of antibody molecules can be manipulated to maximize the proportion of the heterodimer recovered from the recombinant cell culture. A preferred shared region comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the shared region of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A complementary " cavity " of the same or similar size to a large side chain was generated in the shared region of the second antibody molecule by replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine). This provides a mechanism to increase the yield of the heterodimer higher than other undesired end-products such as homodimers.

Bispecific antibodies can be prepared from full-length antibodies or antibody fragments (e. G., F (ab ') ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, chemical conjugation can be used to produce bispecific antibodies. Brennan et al., Science 229: 81 (1985) describe a method of generating F (ab ') ₂ fragments by truncating intact antibodies by protein hydrolysis. These fragments are reduced in the presence of dithiol complexing agent sodium arsenite to stabilize the nearby dithiol and prevent the formation of intermolecular disulfide bonds. The resulting Fab 'fragment was then converted to a thionitrobenzoate (TNB) derivative. Thereafter, one of the Fab'-TNB derivatives was re-converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a material for selective immobilization of the enzyme.

this. The Fab 'fragments were directly recovered from the E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175 : 217-225 (1992)) discloses the preparation of a fully humanized bispecific antibody F (ab ') ₂ molecule. Each Fab ' Were separately secreted from E. coli and coupled by in vitro directed chemical methods to form bispecific antibodies. The bispecific antibodies thus formed were capable of binding to ErbB2 receptor overexpressing cells and normal human T cells, as well as initiating lytic activity of human cytotoxic lymphocytes against human breast cancer targets.

In addition, several techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have been described. For example, bispecific antibodies have been prepared using a leucine zipper [Kostelny et al., J. Immunol. 148 (5) : 1547-1553 (1992)). By gene fusion, the leucine zipper peptides of the Fos and Jun proteins were ligated to the Fab 'portion of two different antibodies. The hinge region of the antibody homodimer was reduced to form monomers and then reoxidized to form antibody heterodimers. In addition, this method can be used as a method for producing antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci. USA 90 : 6444-6448 (1993) provides another mechanism for making bispecific antibody fragments. This fragment contains a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) by a linker, which is too short to allow two domains on the same chain to pair with each other. Thus, the V _H and V _L domains of one fragment pair with the complementary V _L and V _H domains of the other fragment, resulting in two antigen binding sites. Another method of making bispecific antibody fragments using single chain Fv (sFv) dimers has been reported (Gruber et al., J. Immunol. 152: 5368 (1994)).

Antibodies with a bivalent or higher valency are also contemplated. For example, triple specific antibodies can be made (Tutt et al., J. Immunol. 147: 60 (1991)).

Exemplary bispecific antibodies can bind to two different epitopes of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides described herein. Or to focus on the intracellular defense mechanism for cells expressing the specific PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti- PRO362, anti- PRO356, anti- PRO509 or anti- PRO866 polypeptide arm Such as a T-cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or an Fc receptor (FcR) of IgG such as Fc? RI (CD64), Fc? RI (CD32) And Fc [gamma] RIII (CD16). Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing certain PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Such antibodies include, but are not limited to, PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866- binding arms and cytotoxic or radionuclide Chelating agents such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides and also binds to tissue factor (TF).

5. Heterozygous antibodies

Heterozygous antibodies are also included within the scope of the present invention. A heterozygous antibody consists of two covalently linked antibodies. Such antibodies are useful, for example, for targeting immune system cells to undesired cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089 Respectively. It is believed that the heterologous antibody can be prepared using methods known in the art of synthetic protein chemistry, including methods using cross-linking agents. For example, immunotoxins can be prepared by using disulfide exchange reactions or by forming thioether linkages. Reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and reagents such as those disclosed in, for example, U.S. Patent No. 4,676,980.

6. Operation of effector function

It may be desirable to modify the antibody of the invention to function as an effector, for example, to increase the effect of the antibody in the treatment of cancer. For example, cysteine residue (s) can be introduced into the Fc region to form interchain disulfide bonds within this region. Homotypic dimer antibody thus produced enhances internalization capacity and increases complement-mediated cell death and antibody-dependent cellular cytotoxicity (Carcon et al., J. Exp Med. , 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148 : 2918-2922 (1992)). Also, Wolff et al. Cancer Research , 53 : 2560-2565 (1993)) can be used to produce homodimeric antibodies with enhanced anti-tumor activity. Alternatively, antibodies with dual Fc regions can be engineered to enhance complement fungi and ADCC capabilities (Stevenson et al., Anti-Cancer Drug Design , 3 : 219-230 (1989)).

7. Immunoconjugates

The present invention also relates to the use of a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active bacterial, fungal, plant or animal toxin, or a fragment thereof) or a radioactive isotope ) ≪ / RTI >

Chemotherapeutic agents useful for the production of the immunoconjugate are described above. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), lysine A chain, Aleurites fordii protein, Dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), bitter melon (momordica charantia) inhibitor, Cicin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restricotisin, penomycin, enomycin, and tricothecenes. Several radionuclides can be used in the production of radiolabeled antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

Various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters , Dimethyladipimidate HCL), active esters (e.g., disuccinimidylsuberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., Diisocyanate), and bis (diazonium benzoyl) ethylenediamine), bis-diazonium derivatives such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2,6- An active fluorine compound (e.g., 1,5-difluoro-2,4-dinitrobenzene) can be used to make a conjugate of an antibody and a cytotoxic agent. For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science , 238 : 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is a representative chelating agent for conjugation of radioactive nucleotides and antibodies (see WO 94/11026).

In another embodiment, a " receptor " (e. G., Streptavidin) used for tumor preliminary targeting can be conjugated to an antibody, wherein the antibody-receptor conjugate is administered to the patient, Ligand " (e. G., Avidin) conjugated to a cytotoxic agent (e. G., A radionucleotide).

8. Immune liposome

The antibodies disclosed herein may also be formulated as immuno-liposomes. Liposomes containing this antibody can be prepared by methods known in the art, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985), Hwang et al., Proc. Natl Acad. Sci. USA , 77 : 4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposome was extruded through a filter having a predetermined pore size to obtain a liposome having a predetermined diameter. Martin et al., J. Biol. Chem. , 257 : 286-288 (1982)), a Fab 'fragment of an antibody of the present invention can be linked to a liposome via a disulfide-interchange reaction. Chemotherapeutic agents (e. G., Doxorubicin) are optionally included in liposomes [Gabizon et al., J. National Cancer Inst. , 81 (19) : 1484 (1989)).

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

The proteins disclosed herein were analyzed in a panel of 60 tumor cell lines currently used in in vitro study drug development screening for a disease of the National Cancer Institute (NCI). The purpose of this screening is to identify molecules that have cytotoxic and / or cytostatic activity against other types of tumors. NCI has screened 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)]. Tumor cell lines used in this study are described in Monks et al., Supra. Cell lines with greatly inhibited growth by the protein of the present application are listed in the examples.

The results showed that the tested proteins exhibited cytotoxic and cytotoxic activity in several cases and concentrations in various cancer cell lines, and thus are useful candidates for tumor therapy.

In addition, other cell based assays and animal models of tumors can be used to demonstrate NCI cancer screening results and to understand the occurrence and etiology of the identified protein and neoplastic cell growth herein. Although stable cell lines are preferred, primary cultures derived, for example, from tumors of transgenic mice (described below) can be used in the cell based assays herein. Techniques for deriving continuous cell lines from transgenic animals are well known in the art [e.g., Small et al., Mol. Cell. Biol. , 5 : 642-648 (1985)).

G. Animal model

Various well-known animal models can be used to further understand the role of the genes identified herein in the development and etiology of tumors and to test the efficacy of other agonists of natural polypeptides, including candidate therapeutics including antibodies and small molecule agonists . Due to the in vivo properties of the molecule, it is possible to predict the response, especially in human patients. The in vivo properties of the model make it possible to predict the response of the therapeutic agent, particularly in human patients. Animal models of tumors and cancers (e.g., breast, colon, prostate, lung, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents, for example, rat models. The model may be used to induce tumor cells into homologous mice using standard techniques, such as subcutaneous injection, tail vein injection, spleen grafting, intraperitoneal grafting, subcutaneous grafting or orthotopic grafting, [PCT Publication No. WO 97/33351 (published September 18, 1997)].

The most commonly used animal for tumor research is immunodeficient mice, especially nude mice. Nude mice with thymic hypoplasia have been extensively used for this purpose by the discovery that they work successfully as hosts for human tumor xenografts. The autosomal nu gene of zygote contains many nude mouse species of other similar genotypes such as 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, nude mice as well as various other animals with inherited immunological defects were mated and used as recipients of tumor xenografts. For more details, see, e.g., The Nude Mouse in Oncology Research , E. Boven and B. Winograd, eds. (CRC Press, Inc., 1991).

The animal-derived cells may be cultured in a known tumor / cancer cell line, such as any of the tumor cell lines listed above and, for example, the B104-1-1 cell line (a stable NIH-3T3 cell line transfected with the nu original oncogene ), ras transfected NIH-3T3 cells, Caco-2 (ATCC HTB-37) or moderately well-differentiated stage II human colon adenocarcinoma cell line HT-29 (ATCC HTB-38) . Samples of tumors or cancers can be obtained surgically from a patient using standard methods, frozen and stored in liquid nitrogen (Karmali et al., Br. J. Cancer , 48: 689-696 (1983)].

Tumor cells can be introduced into animals, such as nude mice, in a number of ways. The subcutaneous (sc) space of the mouse is very suitable for tumor implantation. Tumors can be transplanted subcutaneously as solid blocks, biopsy tubes using a trocar, or cell suspensions. In the case of solid block or trocar implantation, an appropriately sized tumor tissue fragment is introduced into the subcutaneous space. The cell suspension is prepared from a primary tumor or a stable tumor cell line and injected subcutaneously. In addition, tumor cells can be injected into subcutaneous grafts. At this location, the inoculum is deposited between the lower part of the skin connective tissue and the subcutaneous tissue. Animal models of breast cancer include, for example, rat neuroblastoma (neu oncogene firstly isolated cells) or neu-transformed NIH-3T3 cells by Drebin et al., Proc. Natl. Acad. Sci. USA , 83 : 9129-9133 (1986)).

Similarly, colon cancer animal models can produce colon cancer cells in animals in animals, such as nude mice, by subculture to induce tumor expression within the animal. An analogous transplantation model of human colon cancer in nude mice is described, for example, in Wang et al., Cancer Research , 54 : 4726-4728 (1994) and Too et al., Cancer Research , 55 : 681-684 . The model was based on the so-called METAMOUSE of a product commercially available from AntiCancer Inc. (San Diego, Calif., USA).

Tumors derived from animal models can be removed and cultured in vitro. Cells from the in vitro culture are then inoculated into the animal. The tumor may be used as a target for further testing or drug screening. In addition, tumors induced by subculture can be isolated and analyzed for differential expression of the gene of interest, after isolation of the RNA from the pre-transfused cells and after one or more cycles of inoculation. The subculture technique can be performed in any known tumor or cancer cell line.

For example, Meth A, CMS4, CMS5, CMS21 and WEHI-164 are chemically-induced fibrosarcoma of female BALB / c mice and provide an adjustable model system for studying the antitumor activity of various agents [Palladino et al , J. Immunol. 138 : 4023-4032 (1987)]. Briefly, tumor cells multiply in cell culture in vitro. The cell lines are pre-washed and resuspended in buffer at a cell density of 10 X 10 ⁶ to 10 X 10 ⁷ cells / ml. Animals are then subcutaneously injected with 10 to 100 [mu] l cell suspension to allow for one to three weeks to allow tumors to develop.

In addition, Lewis lung (3LL) carcinoma of mice, the most thoroughly studied experimental tumor, can be used as a tumor study model. The efficacy of this tumor model is associated with beneficial effects in the treatment of human patients diagnosed with pulmonary small cell carcinoma (SCLL). These tumors can be induced in normal mice by injecting tumor fragments from diseased mice or cells maintained in culture [Zupi et al., Br. J. Cancer 41 : suppl. 4, 309 (1980)], tumors can even begin with the injection of single cells and are evidence that many parts of the infected tumor cells survive. Further information on this tumor model can be found in Zacharski, Haemostasis 16 : 300-320 (1986).

One method of assessing the efficacy of a test compound in an animal model is to measure the size of the implanted tumor before and after treatment. Typically, the size of the implanted tumor is measured with a slide caliper in two or three dimensions. Because the measurement limited to two dimensions does not reflect the exact size of the tumor, it is generally converted to the corresponding volume using a mathematical formula. However, the measurement of tumor size is very inaccurate. The therapeutic effect of drug candidates can be better represented by treatment-induced growth retardation and specific growth retardation. Another important variable that indicates tumor growth is tumor volume times. Rygaard and Spang-Thomson, Proc. 6th Int. A computer program is available for the calculation and display of tumor growth, such as the program reported in Workshop on Immune-Deficient Animals , Wu and Sheng eds. (Basel, 1989, 301). However, treatment-induced necrosis and inflammatory responses actually result in an increase in tumor size, at least initially. Therefore, these changes should be carefully monitored by a combination of morphometry and flow cytometry.

Recombinant (transgenic) animal models can be manipulated using standard techniques for producing transgenic animals by introducing the coding regions of the genes identified herein into the genome of the animal of interest. Can be used without limitation in mice, rats, rabbits, guinea pigs, sheep, goats, pigs and non-human primates, such as baboons, chimpanzees and monkeys, as targets for transgenic manipulation. Known techniques for introducing transgene into the above animals include the precursor microinjection method (U.S. Patent No. 4,873,191), retrovirus-mediated gene transfer of embryos (e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA , 82 : 6148-615 (1985)], gene targeting in embryonic cells [Thompson et al., Cell , 56 : 313-32 (1989)], electroporation of the embryo [Lo, Mol. Cell. Biol. , ≪ / RTI > 3 : 1803-1814 (1983); Sperm-mediated gene transfer [Lavitrano et al., Cell. 57 : 717-73 (1989). For further information, see, for example, U.S. Patent No. 4,736,866.

For purposes of the present invention, a transgenic animal includes an animal (" mosaic animal ") having transgene only in a portion of the cell. The transgene can be integrated into a single transgene or concatamer, i. E. A head-to-head or head-to-tail arrangement. Selective introduction of transgene into certain cell types is described, for example, in Lasko et al . [ Proc. Natl. Acd. Sci. USA. 89 : 6232-636 (1992).

Expression of transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to confirm fusion of the transgene. MRNA expression levels can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR or immunocytochemistry. Animals can also be examined for signs of tumor or cancer development.

The efficacy of antibodies and other candidate drugs that specifically bind to the polypeptides identified herein can also be tested in the treatment of spontaneous animal tumors. A suitable target for such studies is oral squamous cell carcinoma of the cats (SCC). Cat oral SCC is the most common oral malignant tumor of the cats, as it accounts for 60% of the oral tumors reported in this species, very aggressive and malignant. Although the low incidence of metastasis reflects only a short survival period of cats with this tumor, it is rarely metastasized to the primary site. Because of the dissection of the cat's mouth, it is generally not possible to perform surgical operations on these tumors. Currently, there is no effective treatment for this tumor. Prior to the study, each cat will undergo a complete clinical examination, biopsy and computed tomography (CT). Cats diagnosed with sublingual squamous cell carcinoma are excluded from the study. These tumors can paralyze the tongue, and even if the tumor is removed by treatment, the animal can not consume food on its own. Repeat each cat for a long period of time. Photographs of the tumor are taken at the time of the review, both daily and later, during the treatment period. Computed tomography of each cat after treatment, followed by computed tomography and chest radiogram every 8 weeks. Data are compared to controls to assess differences in survival, response, and toxicity. Positive reactions preferably require evidence of tumor regression with improved quality of life and / or increased life span.

In addition, other naturally occurring animal tumors, such as dogs, cats and fibroblasts, adenocarcinomas, lymphomas, pemphigoid, and leiomyosarcoma can be tested. Among these mammals, dog and cat mammary carcinoma is a desirable model because it shows very similar forms and responses to humans. However, the use of these models is limited to the rare occurrence of this type of tumor in animals.

H. Screening method for drug candidates

Screening assays for drug candidate substances are intended to identify compounds, or signals through such receptors, that competitively bind to or form conjugates with the receptor (s) of the polypeptide identified herein. Such a screening assay will include a high throughput screening assay for the chemical library, which would be particularly suitable for identifying drug candidates for small molecules. Small molecules contemplated include peptides, preferably soluble peptides, (poly) peptide-immunoglobulin fusions, and polyclonal and monoclonal antibodies and antibody fragments, single chain antibodies, anti-idiotypic and chimeric antibodies, Antibodies, or fragments thereof, and humanized antibodies of human and antibody fragments, including, but not limited to, antibodies. The assays can be performed in a variety of formats, including well-characterized protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays.

In the binding assay, the interaction is binding, and the complex formed can be isolated or detected in the reaction mixture. In certain embodiments, the receptor or drug candidate of the polypeptide encoded by the gene identified herein is anchored to a solid phase, e. G., A microtiter plate, by covalent attachment or noncovalent attachment. Non-covalent attachment is generally achieved by coating the solid surface with a solution and drying. Alternatively, an immobilized antibody, e. G., A monoclonal antibody specific for the immobilized polypeptide, can be used to anchor it to the solid surface. The assay can be performed by adding a non-immobilizable component which can be labeled with a detectable label, to a coding surface comprising a fixed component, for example an anchored component. When the reaction is terminated, unreacted components are removed, for example by washing, and anchored complexes on the solid surface are detected. Where the original non-immobilized component comprises a detectable label, detection of the label immobilized on the surface indicates that the complex forming reaction has taken place. Where the original non-immobilized component does not include the label, for example, a labeled antibody that specifically binds to the immobilized complex can be used to detect the complex formation reaction.

If the candidate compound interacts with a specific receptor but does not bind, the interaction of the candidate compound with the polypeptide can be analyzed by well-known methods for detecting protein-protein interactions. Such assays include conventional methods, such as cross-linking, co-immunoprecipitation, and co-purification through gradient or chromatographic columns. Protein-protein interactions have also been described by Fields and co-workers (Fields and Song, Nature (London) , 340: 245-246 (1989), Chien et al., Proc. Natl. Acad. : 9578-9582 (1991)), yeast-expressed gene systems described in Chevray and Nathans, Proc. Natl. Acad. Sci. USA , 89: 5789-5793 (1991) can do. Many transcription activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other acting as a transcription-active domain. The yeast expression system (commonly referred to as " 2-hybrid system ") described in this document utilizes this property and uses two hybrid proteins, one of which is the target protein with the DNA-binding domain of GAL4 And the other is that the candidate active protein is fused with the active domain. Expression of the GAL1-lacZ reporter gene under the control of the GAL4-active promoter depends on the reconstitution of the GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected using a chromogenic substrate for beta -galactosidase. MATCHMAKER ^TM , which uses two-hybrid techniques to identify protein-protein interactions between two specific proteins, is available from Clontech. The system can also be extended to map protein domains involved in specific protein interactions, as well as to pinpoint the location of amino acid residues critical to this interaction.

I. Pharmaceutical composition

Agonist antibodies specifically binding to the polypeptides of the invention, the proteins identified herein, and other molecules identified by the screening assays disclosed herein may be administered in the form of pharmaceutical compositions for the treatment of various diseases, including cancer .

When an antibody fragment is used, a minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable-region sequence of the antibody, a peptide molecule capable of binding a target protein sequence can be designed. Such peptides may be chemically synthesized and / or produced by recombinant DNA techniques (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA , 90 : 7889-7893 (1993) Reference).

In addition, the formulations herein may comprise one or more active compounds required for the particular indication to be treated, preferably a compound having complementary activity that does not cause side effects with each other. Alternatively, or additionally, the composition may include an agent that enhances its function, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor. The molecule is suitably present in an amount effective for its intended purpose.

Therapeutic formulations of the polypeptides identified herein may be prepared by mixing the antibody having the desired purity with any pharmaceutically acceptable carrier, adduct or stabilizer [ Remington's Pharmaceutical Sciences , 16th edition, Osol, A. ed. (1980)] and prepared in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers, excipients or stabilizers should be such that the dose and concentration administered is non-toxic to the patient and can be any suitable buffer, such as buffers of phosphoric acid, citric acid and other organic acids; (For example, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, methyl or propyl paraben and the like), an antioxidant including ascorbic acid and methionine, Cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophobic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counter ions such as sodium; Metal complexes (e. G., Zn-protein complexes); And / or non-ionic surfactants such as TWEEN (trade name), PLURONICS (trade name) or polyethylene glycol (PEG).

In addition, the formulations herein may comprise one or more active compounds required for the particular indication to be treated, preferably a compound having complementary activity that does not cause side effects with each other. Alternatively, or additionally, the composition may include an agent that enhances its function, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor. The molecule is suitably present in an amount effective for its intended purpose.

The active ingredient may be administered orally or parenterally, for example, in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) For example, in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules. This technique is described in Remington ' s Pharmaceutical Sciences , 16th edition, Osol, A. ed. (1980).

Formulations used for intravenous administration should be sterile. This is easily accomplished by filtration through sterile filtration membranes before or after lyophilization and reconstitution.

The therapeutic compositions herein may be placed in vials having a container with a sterile inlet, such as a container for intravenous solution or with a cap which may be pierced with a hypodermic needle.

Sustained release formulations may also be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), L- And lactic acid-glycolic acid copolymer and looprolide acetate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOT (trade name) Spear), and poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules for more than 100 days, but some hydrogels release proteins for shorter periods of time. If the encapsulated antibody remains in the body for a long period of time, it can be denatured or aggregated as a result of exposure to moisture at 37 < 0 > C to reduce biological activity and alter immunogenicity. Rational strategies can be designed according to the relevant mechanisms for stabilization. For example, if it is found that the cohesion mechanism is an intramolecular SS bond formation through thio-disulfide interchange, it is possible to modify the sulfhydryl moiety, lyophilize from acidic solution, control moisture content, use appropriate additives, Can be stabilized by the development of a matrix composition.

J. Treatment

Polypeptides of the invention and their agonists, including antibodies, peptides and small molecule agonists, can be used for the treatment of a variety of tumors, such as cancers. Representative diseases to be treated include benign or malignant tumors such as renal cell carcinoma, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, ovarian cancer, colorectal cancer, pancreatic cancer, lung cancer, vulvar cancer, thyroid cancer, And various types of head and neck cancer), leukemia and lymphoma; Neurodegenerative diseases, astrocytic diseases, hypothalamic diseases, and other glandular diseases, macrophilic diseases, epithelial diseases, interstitial diseases and stomatal diseases; And inflammatory diseases, angiogenic diseases and immune diseases. The antitumor agents of the present invention (such as the polypeptides described herein and the agonists mimicking their activity, such as antibodies, peptides and small organic molecules) can be administered by any known method, for example intravenous administration as bolus, Preferably human, by continuous intravenous, intraperitoneal, intracerebral, subcutaneous, intraarticular, intra-synovial, intrathecal, oral, topical, or prolonged continuous route by inhalation route. Intravenous administration of the antibody is preferred.

Other therapies may be combined with administration of an anti-cancer agent such as an antibody of the present invention. For example, a patient treated with such an anti-cancer agent can also be subjected to radiation therapy. Alternatively, or additionally, the patient may be administered a chemotherapeutic agent. The manufacture and administration schedule of such chemotherapeutic agents may be according to the manufacturer's instructions or may be determined empirically by one of ordinary skill in the art. The preparation and administration schedule of such chemotherapeutic agents are described in Chemotherapy Service Ed., MCPerry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may be administered before or after administration of the anticancer agent of the present invention, or may be administered simultaneously. The anticancer agent of the present invention may be administered in combination with an anti-estrogen compound such as tamoxifen or an anti-progesterone compound such as onapristone (see EP 616812) in a known dose of the molecule.

It is also preferred to administer other tumor-associated antibodies, such as antibodies that bind to ErbB2, EGFR, ErbB3, ErbB4 or vascular endothelial factor (VEGF). Alternatively, or additionally, two or more antibodies that bind to the same or two or more different antigens disclosed herein may be co-administered to a patient. Sometimes, it may be beneficial to administer one or more cytokines to the patient. In a preferred embodiment, the antibody of the present invention may be administered concurrently with the growth inhibitory agent. For example, the anti-cancer agent of the present invention can be administered after the growth inhibitor is first administered. However, simultaneous administration or administration of the anticancer agent of the present invention may be considered. A suitable dosage of the growth inhibitor is the presently used dosage, and the dosage can be lowered due to the combined action (increase) of the growth inhibitor and the anticancer agent of the present invention.

For the prevention or treatment of the disease, a suitable dose of the anticancer agent herein will depend on the type of disease, the severity and course of the disease to be treated as defined above, whether the anticancer drug to be administered is a prophylactic or therapeutic target, Clinical history and response to medications, and discretion and primary care physician. The agent is suitably administered to the patient at a time or through a series of treatments. Animal experiments provide reliable guidance in determining effective doses in human therapy. The ratio adjustment of the effective amount of interspecies is described in Mordenti, J and Chappell, W. "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Uacobi et al., Eds., Pergamon Press, 96].

For example, depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg, 0.1-20 mg / kg) of the antibody may be administered to a patient by one or more separate administrations ≪ / RTI > A typical daily dose is from about 1 [mu] g / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administrations over several days depending on the symptoms, the treatment is continued until the desired disease symptoms are suppressed. However, other dosages may be effective. The progress of this therapy can be easily monitored by conventional techniques and analysis. Guidance and delivery methods for specific dosages are provided in the literature (see, for example, U.S. Patent Nos. 4,657,760, 5,206,344 or 5,225,212). It is contemplated that other agents may be effective for other therapeutic compounds and other diseases, and dosing methods that target one organ or tissue may require delivery methods other than, for example, another organ or tissue.

K. Products

In another embodiment of the present invention, there is provided an article of manufacture comprising a substance useful for the diagnosis or treatment of a disease described above. Products include containers and labels. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container may be formed of various materials such as glass or plastic. The container may contain a composition effective for the diagnosis or treatment of the disease, and may have a sterile inlet (e.g., the container may be an intravenous infusion bag, or may be a vial with a cap through which a hypodermic needle may be pierced) . The active substance of the composition is an antitumor agent of the present invention. Labels on or in the container indicate that the composition is used for the diagnosis or treatment of a selected disease. The article of manufacture may also comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and dextrose solution. The article may also include other materials desirable from a commercial standpoint and user's perspective, including other buffer solutions, diluents, filters, needles, syringes, and packaging inserts containing instructions for use.

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

All patents and documents cited herein are incorporated herein by reference in their entirety.

Commercial reagents mentioned in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of cells identified in the Examples and throughout the specification as ATCC Accession Numbers is the American Type Culture Collection (Manassas, Va.).

Example 1: Extracellular domain homology screening to identify novel polypeptides and cDNA encoding them

The EST database was investigated using an extracellular domain (ECD) sequence (including the secretory signal sequence, if present) from about 950 known secretory proteins from the Swiss-Prot public database . The EST database includes public databases (eg, Dayhoff, Genbank) and private databases (eg, LIFESEQ ™, Incyte Pharmaceuticals, Palo Alto, CA) . Surveys were performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)) as a comparison of the ECD protein sequences for the 6 frame translation of the EST sequence. A comparator with a BLAST score of 70 (or in some cases 90) or more that does not code a known protein was clustered and analyzed using the consensus DNA sequence " phrap " (Phil Green, University of Washington, Seattle, Washington, USA) .

Using this extracellular domain homology screening, consensus DNA sequences were assembled for other EST sequences identified using phrap. In addition, the consensus sequence obtained is often extended (but not always) using a repeated cycle of BLAST or BLAST-2 and phrap to elongate as consensus sequence as possible using a source of the EST sequence discussed above .

Oligonucleotides were then synthesized on the basis of the consensus sequences obtained as described above and used to identify cDNA libraries containing sequences of interest by PCR and to isolate clones of full length coding sequences for PRO polypeptides And used as a probe. The forward and reverse PCR primers are generally in the range of 20 to 30 nucleotides and are often designed to provide PCR products of about 100 to 1000 bp in length. The length of the probe sequence is typically 40-55 bp. In some cases, if the consensus sequence is greater than about 1 to 1.5 kbp, additional oligonucleotides are synthesized. To screen several libraries for full-length clones, DNA from these libraries was screened by PCR amplification according to the literature (Ausubel et al., Current Protocols in Molecular Biology ) using PCR primer pairs. Clones encoding the gene of interest were then isolated using the probe oligonucleotide and one of the primer pairs by using a positive library.

The cDNA library used to isolate cDNA clones was prepared by standard methods using commercially available reagents such as reagents from Invitrogen (San Diego, CA, USA). The cDNA was primed with oligo dT with the NotI site, ligated to the hemizin-treated SalI adapter at the blunt end, digested with NotI, appropriately sorted by size by gel electrophoresis, and cloned into a suitable cloning vector (e. g. pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see Holmes et al., Science , 253 : 1278-1280 (1991)).

Example 2: Isolation of cDNA clones encoding human PRO179

A cDNA clone (DNA16451-1078) encoding a native human PRO179 polypeptide was identified using yeast screening in a human fetal liver library predominantly representing the 5'end of the primary cDNA clone.

OLI114: 5'-CCACGTTGGCTTGAAATTGA-3 '(SEQ ID NO: 3)

OLI115: 5'-CCTTTAGAATTGATCAAGACAATTCATGATTTGATTCTCTATCTCCAGAG-3 '(SEQ ID NO: 4)

OLI116: 5'-TCGTCTAACATAGCAAATC-3 '(SEQ ID NO: 5)

Clone DNA 16451-1078 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 37 to 39 and an apparent stop codon at nucleotide positions 1417 to 1419 (Figure 1, SEQ ID NO: 1). The expected polypeptide precursor is 460 amino acids in length. The full-length PRO179 protein is shown in Figure 2 (SEQ ID NO: 2).

Analysis of the sequence of the full-length PRO179 shown in Fig. 2 (SEQ ID NO: 2) demonstrates the presence of several important polypeptide domains as shown in Fig. 2, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full-length PRO179 sequence (FIG. 2, SEQ ID NO: 2) revealed a signal peptide of about amino acid 1 to about amino acid 16, about 23 amino acids to about amino acid 27, about amino acid 115 to about amino acid 119, about amino acid 296 to about amino acid 300, Glycosylation sites of about amino acid 357 to about amino acid 361, cAMP and cGMP dependent protein kinase phosphorylation sites of about amino acid 100 to about amino acid 104 and about amino acid 204 to about amino acid 208, tyrosine kinase phosphorylation of about amino acid 342 to about amino acid 351 Region of about amino acid 279 to about amino acid 285, about amino acid 352 to about amino acid 358, and about amino acid 367 to about amino acid 373, about amino acid 120 to about amino acid 142, and about amino acid 127 to about amino acid 149, Prove the existence of a new zipper shape.

Clone DNA 16451-1078 was deposited with the ATCC on September 18, 1997 and deposited with accession number 209281. The estimated molecular weight of the full-length PRO179 protein shown in Figure 2 is about 53,637 daltons and the estimated pI is about 6.61.

Analysis of the Day-Hope database (version 35.45 Swiss Prob 35) of the full-length sequence shown in Figure 2 (SEQ ID NO: 2) revealed that two known human ligands (h-TIE-2L1 and h-TIE-2L2) of the TIE- Demonstrating the presence of fibrinogen-like domains exhibiting high degree of sequence homology. Abbreviation "TIE" is an acronym indicating a tyrosine kinase having a "Ig and EGF homology domains (t yrosine kinase containing I g and E GF homology domains), is coined to refer to a new family of receptor tyrosine kinases. Accordingly, PRO179 Was identified as a new member of the TIE ligand family.

Example 3: Isolation of cDNA clones encoding human PRO207

(EST) DNA library (e. G., LIFESEQ (registered trademark) (Incyte Pharmaceuticals, Palo Alto, Calif.)) A cDNA library was prepared by using mRNA isolated from human fetal kidney tissue (Clontech). Using this RNA, Life Technologies (Life Technologies, Gaithersbug In this procedure, the size of the double-stranded cDNA was greater than 1000 bp, and the SalI / NotI-linked cDNA was ligated to the XhoI (SEQ ID NO: 2) cDNA library using the reagent and protocol (Superscript Plasmid System) / Not I. The pRK5D was inserted into the sp6 transcription initiation site followed by the SfiI restriction enzyme site XhoI / NotI cDNA cloning site. This library was screened on the basis of EST by hybridizing with the synthetic oligonucleotide probe 5'-CCAGCCCTCTGCGCTACAACCGCCAGATCGGGGAGTTTATAGTCACCCGG-3 '(SEQ ID NO: 8).

The entire cDNA clone was sequenced. The nucleotide sequence of the full-length DNA30879-1152 is shown in Fig. 3 (SEQ ID NO: 6). Clone DNA 30879-1152 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 58 to 60 (FIG. 3, SEQ ID NO: 6) and an apparent stop codon at nucleotide positions 805 to 807. The expected polypeptide precursor is 249 amino acids in length.

Analysis of the sequence of the full-length PRO207 shown in Fig. 4 (SEQ ID NO: 7) demonstrates the presence of several important polypeptide domains as shown in Fig. 4, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full length PRO207 sequence (FIG. 4, SEQ ID NO: 7) showed that the signal peptide of about 1 to about amino acid 40, the N-glycosylation site of about amino acid 139 to about amino acid 143, about amino acid 27 to about amino acid 33, 29 to about amino acid 35, about amino acid 36 to about amino acid 42, about amino acid 45 to about amino acid 51, about amino acid 118 to about amino acid 124, about amino acid 121 to about amino acid 127, about amino acid 125 to about amino acid 131, The amidated site of the N-myristoylation site of about amino acid 134, about 10 to about amino acid 14 and about 97 to about amino acid 101, about 97 to about amino acid 35, of the prodrug lipoprotein lipid attachment site of about amino acid 35 . Clone DNA 30879-1152 was deposited with the ATCC on Oct. 10, 1997 and deposited with accession number 209358. The estimated molecular weight of the full-length PRO207 protein shown in Figure 4 is about 27,216 daltons and the estimated pI is about 9.61.

Based on the BLAST and FastA sequence alignment analysis of the full length PRO207 sequence shown in Figure 4 (SEQ ID NO: 7), it was found that PRO207 is a member of several members of the TNF cytokine family, particularly human lymphotoxin-beta (23.4%) and human CD40 ligand (19.8% ). ≪ / RTI >

Example 4: Isolation of cDNA clones encoding human PRO320

The consensus DNA sequence was assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA28739. Based on the DNA28739 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO320.

PCR primer pairs (forward and reverse) were synthesized:

The forward PCR primer 5'-CCTCAGTGGCCACATGCTCATG-3 '(SEQ ID NO: 11)

Reverse PCR primer 5'-GGCTGCACGTATGGCTATCCATAG-3 '(SEQ ID NO: 12)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from consensus sequence DNA 28739.

Hybridization probe

5'-GATAAACTGTCAGTACAGCTGTGAAGACACAGAAGAAGGGCCACAGTGCC-3 '(SEQ ID NO: 13)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO320 gene were then isolated with either the probe oligonucleotide and the PCR primers by using a positive library. The RNA for cDNA library preparation was isolated from the human fetal lung tissue (LIB025).

DNA sequence analysis of the isolated clone as described above gave the full-length DNA sequence of DNA32284-1307 (FIG. 5, SEQ ID NO: 9) and the protein sequence of PRO320 derived therefrom.

The entire coding sequence of DNA32284-1307 is shown in Figure 5 (SEQ ID NO: 9). Clone DNA 32284-1307 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 135 to 137 and an apparent stop codon at nucleotide positions 1149 to 1151. The expected polypeptide precursor is 338 amino acids in length. Analysis of the sequence of the full-length PRO320 shown in Figure 6 (SEQ ID NO: 10) demonstrates the presence of several important polypeptide domains, and the location for the important polypeptide domain is approximate as described above. Analysis of the full length PRO320 polypeptide shown in Figure 6 showed that the signal peptide of about amino acid 1 to about amino acid 21, the amidated portion of about 330 amino acids to about amino acid 334, about 109 amino acids to about amino acid 121, about amino acid 191 to about amino acid 203 And the aspartic acid and asparagine hydroxylation sites of about amino acid 236 to about amino acid 248, the EGF-like domain cysteine signal of about amino acid 80 to about amino acid 91, about amino acid 103 to about amino acid 125, about amino acid 230 to about amino acid 252, and about amino acid 185 ≪ / RTI > to about amino acid 207 of the calcium-binding EGF-like domain. Clone DNA32284-1307 was deposited with the ATCC on March 11, 1998 and assigned accession number 209670. The estimated molecular weight of the full length PRO320 protein shown in Figure 6 is about 37,143 daltons and the estimated pI is about 8.92.

Example 5: Isolation of cDNA clones encoding human PRO219

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA28729. Based on the DNA28729 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO219.

PCR primer pairs (forward and reverse) were synthesized:

The forward PCR primer 5'-GTGACCCTGGTTGTGAATACTCC-3 '(SEQ ID NO: 16)

The reverse PCR primer 5'-ACAGCCATGGTCTATAGCTTGG-3 '(SEQ ID NO: 17)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from consensus sequence DNA28729.

Hybridization probe

5'-GCCTGTCAGTGTCCTGAGGGACACGTGCTCCGCAGCGATGGGAAG-3 '(SEQ ID NO: 18)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO219 gene were then isolated with either the probe oligonucleotide and PCR primers by using positive libraries. The RNA for cDNA library preparation was isolated from the human fetal kidney tissue.

DNA sequence analysis of the isolated clones as described above gave the full-length DNA sequence of DNA32290-1164 (Fig. 7, SEQ ID NO: 14) and the protein sequence of PRO219 derived therefrom.

The complete coding sequence of DNA32290-1164 is shown in Figure 7 (SEQ ID NO: 14). Clone DNA 32290-1164 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 204 to 206 and an apparent stop codon at nucleotide positions 2949 to 2951. The expected polypeptide precursor is 1005 amino acids in length. Analysis of the sequence of the full-length PRO219 shown in Figure 8 (SEQ ID NO: 15) demonstrates the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full-length PRO219 polypeptide shown in Figure 8 showed that the signal peptide of about amino acid 1 to about amino acid 23, the N-glycosylation site of about amino acid 221 to about amino acid 225, about amino acid 115 to about amino acid 119, about amino acid 606 to about CAMP and cGMP dependent protein kinase phosphorylation sites of amino acids 610 and about amino acids 892 to about amino acid 896, about amino acid 133 to about amino acid 139, about amino acid 258 to about amino acid 264, about amino acid 299 to about amino acid 305, about amino acid 340 to about amino acid 346, about 453 to about amino acid 459, about amino acid 494 to about amino acid 500, about amino acid 639 to about amino acid 645, about amino acid 690 to about amino acid 694, about amino acid 752 to about amino acid 758, and about amino acid 792 to about amino acid 798 N-myristoylation site, about amino acid 314 to about About amino acid 560 to about amino acid 564, and about amidated amino acid 601 to about amino acid 605, about amino acid 253 to about amino acid 265, about amino acid 294 to about amino acid 306, about amino acid 335 to about amino acid 347, about amino acid 376 From about amino acid 388, from about amino acid 417 to about amino acid 429, from about amino acid 458 to about amino acid 470, from about amino acid 540 to about amino acid 552, and from about amino acid 581 to about amino acid 593, aspartic acid and asparagine hydroxylation sites. Clone DNA32290-1164 was deposited with the ATCC on Oct. 17, 1997 and assigned accession number 209384. The estimated molecular weight of the full-length PRO219 protein shown in Figure 8 is about 102,233 daltons and the estimated pI is about 6.02.

Analysis of the full-length PRO219 sequence shown in Figure 8 (SEQ ID NO: 15) showed that some of it was highly homologous to the mouse and human mattrelin-2 precursor polypeptides.

Example 6: Isolation of cDNA clones encoding human PRO221

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA28756. Based on the DNA28756 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO221.

PCR primer pairs (forward and reverse) were synthesized:

The forward PCR primer 5'-CCATGTGTCTCCTCCTACAAAG-3 '(SEQ ID NO: 21)

The reverse PCR primer 5'-GGGAATAGATGTGATCTGATTGG-3 '(SEQ ID NO: 22)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from consensus sequence DNA28756.

Hybridization probe

5'-CACCTGTAGCAATGCAAATCTCAAGGAAATACCTAGAGATCTTCCTCCTG-3 '(SEQ ID NO: 23)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO221 gene were then isolated with either the probe oligonucleotide and the PCR primers by using a positive library. RNA for cDNA library production was isolated from the lung tissue of human embryos.

DNA sequence analysis of the isolated clone as described above gave the full-length DNA sequence of DNA 33089-1132 (FIG. 9, SEQ ID NO: 19) and the protein sequence of PRO221 derived therefrom.

The entire coding sequence of DNA33089-1132 is shown in Figure 9 (SEQ ID NO: 19). Clone DNA33089-1132 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 179 to 181 and an apparent stop codon at nucleotide positions 956 to 958. The expected polypeptide precursor is 259 amino acids in length. Analysis of the sequence of the full-length PRO221 shown in Fig. 10 (SEQ ID NO: 20) demonstrates the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full length PRO221 polypeptide shown in Figure 10 showed that the signal peptide of about amino acid 1 to about amino acid 33, the transmembrane domain of about amino acid 204 to about amino acid 219, about amino acid 47 to about amino acid 51, and about amino acid 94 to about amino acid 98 Glycosylation sites of cAMP and cGMP dependent protein kinase phosphorylation sites of about 199 amino acids to about amino acid 203, about 37 to about amino acid 43, about amino acid 45 to about amino acid 51, and about amino acid 110 to about amino acid 116 of N- Demonstrating the presence of a misty stoichiometric site. Clone DNA33089-1132 was deposited with the ATCC on September 16, 1997 and deposited with accession number 209262. The estimated molecular weight of the full-length PRO221 protein shown in Figure 10 is about 29,275 daltons and the estimated pI is about 6.92.

Analysis of the full-length PRO221 sequence shown in Figure 10 (SEQ ID NO: 20) showed homology with members of the loop-rich repeated protein phase, including the SLIT protein.

Example 7: Isolation of cDNA clones encoding human PRO224

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA30845. Based on the DNA30845 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO224.

PCR primer pairs (forward and reverse) were synthesized:

The forward PCR primer 5'-AAGTTCCAGTGCCGCACCAGTGGC-3 '(SEQ ID NO: 26)

Reverse PCR primer 5'-TTGGTTCCACAGCCGAGCTCGTCG-3 '(SEQ ID NO: 27)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from consensus sequence DNA30845.

Hybridization probe

5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAATGCCCACC-3 '(SEQ ID NO: 28)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO224 gene were then isolated with either the probe oligonucleotide and the PCR primers by using a positive library. RNA for cDNA library production was isolated from human fetal liver tissue.

DNA sequence analysis of the isolated clone as described above gave the full-length DNA sequence of DNA33221-1133 (Fig. 11, SEQ ID NO: 24) and the protein sequence of PRO224 derived therefrom.

The entire coding sequence of DNA33221-1133 is shown in Figure 11 (SEQ ID NO: 24). Clone DNA33221-1133 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 33 to 35 and an apparent stop codon at nucleotide positions 879 to 881. The expected polypeptide precursor is 282 amino acids in length. Analysis of the sequence of the full-length PRO224 shown in Figure 12 (SEQ ID NO: 25) demonstrates the presence of several important polypeptide domains, the positions for which are important as described above. Analysis of the full length PRO224 polypeptide shown in Figure 12 showed that the signal peptide of about amino acid 1 to about amino acid 30, the transmembrane domain of about amino acid 231 to about amino acid 248, about amino acid 126 to about amino acid 130, about amino acid 195 to about amino acid 199 Glycosylation site of about amino acid 213 to about amino acid 217, about 3 to about amino acid 9, about 10 to about amino acid 16, about amino acid 26 to about amino acid 32, about amino acid 30 to about amino acid 36, about amino acid 112 From about amino acid 118 to about amino acid 166 to about amino acid 172, from about amino acid 212 to about amino acid 218, from about amino acid 224 to about amino acid 230, from about amino acid 230 to about amino acid 236, and from about amino acid 263 to about amino acid 269 Region, about 44 to about amino acid 55, of the prodrug lipoprotein lipid moiety Demonstrates the area, the presence of the leucine zipper form of about amino acid 17 to about amino acid 39. Clone DNA33221-1133 was deposited with the ATCC on September 16, 1997 and deposited with accession number 209263. The estimated molecular weight of the full-length PRO224 protein shown in Fig. 12 is about 28,991 daltons, and the estimated pI is about 4.62.

Analysis of the full-length PRO224 sequence shown in Figure 12 (SEQ ID NO: 25) showed that it was homologous to the lowest density lipoprotein receptor, the apolipoprotein E receptor and the chicken ovary receptor P95. Based on the BLAST and FastA sequence alignment analysis of the full-length sequence, PRO224 had sequence identity ranging from 28% to 45% with some of these proteins and had identity ranging from 33% to 39% with the entirety of these proteins.

Example 8: Isolation of cDNA clones encoding human PRO328

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA35615. Based on the DNA35615 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO328.

PCR primer pairs (forward and reverse) were synthesized:

The forward PCR primer 5'-TCCTGCAGTTTCCTGATGC-3 '(SEQ ID NO: 31)

Reverse PCR primer 5'-CTCATATTGCACACCAGTAATTCG-3 '(SEQ ID NO: 32)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus sequence DNA35165.

Hybridization probe

5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3 '(SEQ ID NO: 33)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO328 gene were then isolated by using either a probe oligonucleotide and PCR primers by using a positive library. RNA for cDNA library isolation was isolated from the human fetal kidney tissue.

The DNA sequence of the isolated clone as described above was used to obtain the full-length DNA sequence of DNA40587-1231 (Fig. 13, SEQ ID NO: 29) and the protein sequence of PRO328 derived therefrom.

The entire coding sequence of DNA40587-1231 is shown in Figure 13 (SEQ ID NO: 29). Clone DNA 40587-1231 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 15 to 17 and an apparent stop codon at nucleotide positions 1404 to 1406. The expected polypeptide precursor is 463 amino acids in length. The results of analyzing the sequence of the full-length PRO328 shown in Figure 14 (SEQ ID NO: 30) demonstrate the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full-length PRO328 polypeptide shown in Figure 14 showed that the signal peptide of about amino acid 1 to about amino acid 22, about amino acid 114 to about amino acid 118, about amino acid 403 to about amino acid 407, and about amino acid 409 to about amino acid 413, The glycosaminoglycan attachment site of about amino acid 439 to about amino acid 443, about amino acid 123 to about amino acid 129, about amino acid 143 to about amino acid 149, about amino acid 152 to about amino acid 158, about amino acid 169 to about amino acid 175 Amidation of the N-mistostoylation site of about amino acid 180 to about amino acid 186, about amino acid 231 to about amino acid 237 and about amino acid 250 to about amino acid 256, about amino acid 82 to about amino acid 88, and about amino acid 172 to about amino acid 176 Region, about 287 to about amino acid 298 of the peroxidase adjacent heme - extracellular protein SCP / Tpx-1 / Ag5 of the ligand signal, one extracellular protein SCP / Tpx-1 / Ag5 / PR-1 / Sc7 signal of about amino acid 127 to about amino acid 138, / PR-1 / Sc7 signal 2 domain. The clone DNA40587-1231 was deposited with the ATCC on Nov. 7, 1997 and assigned accession number 209438. The estimated molecular weight of the full-length PRO328 protein shown in Figure 14 is about 49,471 daltons and the estimated pI is about 5.36.

Analysis of the full-length PRO328 sequence shown in Figure 14 (SEQ ID NO: 30) showed that some of it showed considerable homology with human glioblastoma protein and cysteine-rich secretory proteins, thereby indicating that PRO328 is a novel glioblastoma protein or cysteine- Protein. ≪ / RTI >

Example 9: Isolation of cDNA clones encoding human PRO301

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA35936. Based on the DNA35936 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the sequence of interest by PCR, and 2) use as a probe to isolate clones of the full-length coding sequence of PRO301.

The oligonucleotides used in this procedure are as follows:

Omni-directional PCR primer 1 5'-TCGCGGAGCTGTGTTCTGTTTCCC-3 '(SEQ ID NO: 36)

Forward PCR primer 2 5'-ACACCTGGTTCAAAGATGGG-3 '(SEQ ID NO: 37)

Omni-directional PCR primer 3 5'-TTGCCTTACTCAGGTGCTAC-3 '(SEQ ID NO: 38)

Reverse PCR primer 1 5'-TAGGAAGAGTTGCTGAAGGCACGG-3 '(SEQ ID NO: 39)

Reverse PCR primer 2 5'-ACTCAGCAGTGGTAGGAAAG-3 '(SEQ ID NO: 40)

Hybridization probe 1

5'-TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT-3 '(SEQ ID NO: 41)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO301 gene were then isolated with either the probe oligonucleotide and the PCR primers by using positive libraries. The RNA for cDNA library preparation was isolated from the human fetal kidney tissue.

DNA sequence analysis of the isolated clone as described above gave the full-length DNA sequence of DNA 40628-1216 (Fig. 15, SEQ ID NO: 34) and the protein sequence of PRO301 derived therefrom.

The entire coding sequence of DNA40628-1216 is shown in Figure 15 (SEQ ID NO: 34). Clone DNA 40628-1216 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 52-54 and an apparent stop codon at nucleotide positions 949-951. The expected polypeptide precursor is 299 amino acids in length. Analysis of the sequence of the full-length PRO301 shown in Fig. 16 (SEQ ID NO: 35) demonstrates the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full length PRO301 polypeptide shown in Figure 16 showed that the signal peptide of about amino acid 1 to about amino acid 27, the transmembrane domain of about amino acid 235 to about amino acid 256, the N-glycosylation site of about amino acid 185 to about amino acid 189, CAMP and cGMP dependent protein kinase phosphorylation sites of amino acids 270 to about amino acid 274, about amino acid 105 to about amino acid 111, about amino acid 116 to about amino acid 122, about amino acid 158 to about amino acid 164, about amino acid 219 to about amino acid 225, 237 to about amino acid 243, and about 256 amino acids to about 262 amino acid residues. Clone DNA 40628-1216 was deposited with the ATCC on Nov. 7, 1997 and deposited with accession number 209432. The estimated molecular weight of the full-length PRO301 protein shown in Fig. 16 is about 32,583 daltons, and the estimated pI is about 8.29.

Based on the BLAST and FastA sequence alignment analysis of the full-length PRO301 sequence shown in Figure 16 (SEQ ID NO: 35), PRO301 showed amino acid sequence identity with the A33 antigen precursor (30%) and the Cockey and adenovirus receptor proteins (29% .

Example 10: Isolation of cDNA clones encoding human PRO526

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This initiation consensus sequence is referred to herein as DNA39626.init. In addition, a starting consensus DNA sequence was extended as long as the source of the EST sequence discussed above could be used, using a repeat cycle of BLAST and phrap. The assembled consensus sequence is referred to herein as < consen01 >. Based on the <consen01> consensus sequence, 1) the cDNA library containing the sequence of interest was confirmed by PCR and 2) oligonucleotides were synthesized for use as probes to isolate clones of the full-length coding sequence of PRO526.

PCR primer pairs (forward and reverse) were synthesized:

The forward PCR primer 5'-TGGCTGCCCTGCAGTACCTCTACC-3 '(SEQ ID NO: 44)

Reverse PCR primer 5'-CCCTGCAGGTCATTGGCAGCTAGG-3 '(SEQ ID NO: 45)

In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from <consen01> consensus sequences.

Hybridization probe

5'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCACAC-3 '(SEQ ID NO: 46)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO526 gene were then isolated with either the probe oligonucleotide and the PCR primers by using a positive library. RNA for cDNA library preparation was isolated from human fetal liver tissue (LIB228).

DNA sequence analysis of the isolated clone as described above gave the full-length DNA sequence of DNA44184-1319 (FIG. 17, SEQ ID NO: 42) and the protein sequence of PRO526 derived therefrom.

The entire coding sequence of DNA44184-1319 is shown in Figure 17 (SEQ ID NO: 42). Clone DNA44184-1319 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 514-516 and an apparent stop codon at nucleotide positions 1933-1935. The expected polypeptide precursor is 473 amino acids in length. The results of analyzing the sequence of the full-length PRO526 shown in Fig. 18 (SEQ ID NO: 43) demonstrate the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full-length PRO526 polypeptide shown in Figure 18 showed that the signal peptide of about amino acid 1 to about amino acid 26, the leucine zipper form of about amino acid 135 to about amino acid 157, the glycosaminoglycan attachment of about amino acid 436 to about amino acid 440 Glycosylation site of about amino acid 82 to about amino acid 86, about amino acid 179 to about amino acid 183, about amino acid 237 to about amino acid 241, about amino acid 372 to about amino acid 376, and about amino acid 423 to about amino acid 427, 411 &lt; / RTI &gt; to about amino acid 427 of the &lt; RTI ID = 0.0 &gt; Willebrand &lt; / RTI &gt; Clone DNA44184-1319 was deposited with the ATCC on Mar. 26, 1998 and assigned accession number 209704. The estimated molecular weight of the full-length PRO526 protein shown in Figure 18 is about 50,708 daltons and the estimated pI is about 9.28.

Analysis of the full-length PRO526 sequence shown in Fig. 18 (SEQ ID NO: 43) revealed that some of them showed considerable homology with proteins rich in lysine repeats, including human ALS, SLIT, carboxypeptidase and small- Indicating that PRO526 is a novel protein associated with protein-protein interactions.

Example 11: Isolation of cDNA clones encoding human PRO362

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA42257. Based on the DNA42257 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO362.

PCR primer pairs (forward and reverse) were synthesized:

Omni-directional PCR primer 1 5'-TATCCCTCCAATTGAGCACCCTGG-3 '(SEQ ID NO: 49)

Forward PCR primer 2 5'-GTCGGAAGACATCCCAACAAG-3 '(SEQ ID NO: 50)

Reverse PCR primer 1 5'-CTTCACAATGTCGCTGTGCTGCTC-3 '(SEQ ID NO: 51)

Reverse PCR primer 2 5'-AGCCAAATCCAGCAGCTGGCTTAC-3 '(SEQ ID NO: 52)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from consensus sequence DNA42257.

Hybridization probe

5'-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCTGAT-3 '(SEQ ID NO: 53)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO362 gene were then isolated with either the probe oligonucleotide and the PCR primers by using a positive library. RNA for cDNA library preparation was isolated from human fetal brain tissue (LIB153).

DNA sequence analysis of the isolated clone as described above gave the full-length DNA sequence of DNA45416-1251 (Fig. 19, SEQ ID NO: 47) and the protein sequence of PRO362 derived therefrom.

The entire coding sequence of DNA45416-1251 is shown in Figure 19 (SEQ ID NO: 47). Clone DNA45416-1251 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 119 to 121 and an apparent stop codon at nucleotide positions 1082 to 1084. The expected polypeptide precursor is 321 amino acids in length. Analysis of the sequence of the full-length PRO362 shown in Figure 20 (SEQ ID NO: 48) demonstrates the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full length PRO362 polypeptide shown in Figure 20 showed that the signal peptide of about amino acid 1 to about amino acid 19, the transmembrane domain of about amino acid 281 to about amino acid 300, the glycosaminoglycan attachment site of about amino acid 149 to about amino acid 153 About cAMP and cGMP dependent protein kinase phosphorylation sites from about amino acid 308 to about amino acid 312, from about amino acid 2 to about amino acid 8, from about amino acid 148 to about amino acid 154, from about amino acid 158 to about amino acid 164, from about amino acid 207 to about amino acid 213, Demonstrating the presence of the N-mistostoylation site of about 215 amino acids to about amino acid 221. Clone DNA45416-1251 was deposited with the ATCC on Feb. 5, 1998 and assigned accession number 209620. The estimated molecular weight of the full-length PRO362 protein shown in Figure 20 is about 35,544 daltons and the estimated pI is about 8.51.

Analysis of the full-length PRO362 sequence shown in Figure 20 (SEQ ID NO: 48) revealed a similarity with the A33 antigen protein and HCAR protein. More specifically, as a result of the analysis of the De Hoop database (version 35.45 Switzerland plot 35), significant phase differences between the amino acid sequences of PRO362 and the dehope sequences of AB002341_1, HSU55258_1, HSC7NRCAM_1, RNU81037_1, A33_HUMAN, P_W14158, NMNCAMRI_1, HSTITINN2_1, S71824_1 and HSU63041_1 Proved to have the same sex.

Example 12: Isolation of cDNA clones encoding human PRO356

PRO179 (DNA 16451-1078 identified in Example 2 above (Fig. 1, Fig. 1, Fig. 1) was identified by irradiating an expressed sequence tag (EST) DNA database (e.g., LIFESEQ (TM), Palo Alto, EST (# 2939340), which is homologous to EST (SEQ ID NO: 1), was identified. To clone PRO356, a human fetal lung library prepared from mRNA purchased from Clontech (Palo Alto, Calif.) Was used according to the manufacturer's instructions.

The cDNA used to isolate cDNA clones encoding human PRO356 was constructed by standard methods using commercially available reagents, such as, for example, Invitrogen's reagent from San Diego, CA. CDNA was primed with oligo dT having the NotI site, ligated to the hemizin-treated SalI adapter at the blunt end, digested with NotI, appropriately sorted by size by gel electrophoresis, and cloned into a suitable cloning vector , pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; see Holmes et al., Science , 253 : 1278-1280 (1991)) .

Then, based on the EST sequence described above, 1) a cDNA library containing the sequence of interest was confirmed by PCR, and 2) an oligonucleotide probe for use as a probe to isolate a clone of the full-length coding sequence of the PRO356 polypeptide Were synthesized. The forward and reverse PCR primers generally consist of 20 to 30 nucleotides and are often designed to provide PCR products of about 100-1000 bp in length. The probe sequence is usually 40-55 bp in length. To screen several libraries for full-length clones, DNA from these libraries was screened by PCR amplification according to the literature (Ausubel et al., Current Protocols in Molecular Biology, supra) using PCR primer pairs. Clones encoding the gene of interest were then isolated using a positive library using the probe oligonucleotide and one of the primer pairs.

The oligonucleotides used are as follows:

5'-TTCAGCACCAAGGACAAGGACAATGACAACT-3 '(SEQ ID NO: 56)

5'-TGTGCACACTTGTCCAAGCAGTTGTCATTGTC-3 '(SEQ ID NO: 57)

5'-GTAGTACACTCCATTGAGGTTGG-3 '(SEQ ID NO: 58)

The entire cDNA clone was identified and sequenced. The entire nucleotide sequence of DNA47470-1130-P1 is shown in Figure 21 (SEQ ID NO: 54). Clone DNA47470-1130-P1 contains a single open reading frame with an open translation initiation site at nucleotide positions 215 to 217 and a stop codon at nucleotide positions 1253 to 1255 (Figure 21, SEQ ID NO: 54). The predicted polypeptide precursor has a length of 346 amino acids, an estimated molecular weight of about 40,018 daltons, and an estimated pI of about 8.19. The full-length PRO356 protein is shown in Figure 22 (SEQ ID NO: 55).

Analysis of the full-length PRO356 sequence shown in Figure 22 (SEQ ID NO: 55) demonstrates the presence of several important polypeptide domains as shown in Figure 22, with the positions for the important polypeptide domains being approximate as described above. Analysis of the full-length PRO356 sequence revealed that the N-glycosylation site of the signal peptide of about amino acid 1 to about amino acid 26, about amino acid 58 to about amino acid 62, about amino acid 253 to about amino acid 257, and about amino acid 267 to about amino acid 271, The glycosaminoglycan attachment site of amino acids 167 to about amino acid 171, the cAMP and cGMP-dependent protein kinase phosphorylation sites of about amino acid 176 to about amino acid 180, about amino acid 168 to about amino acid 174, about amino acid 196 to about amino acid 202, 241 to about amino acid 247, about amino acid 252 to about amino acid 258, about amino acid 256 to about amino acid 262, and about amino acid 327 to about amino acid 333, about amino acid 199 to about amino acid 202 &Lt; / RTI &gt;

Clone DNA47470-1130-P1 was deposited with the ATCC on Oct. 28, 1997 and assigned accession number 209422. The deposited clone is understood to have a substantially correct sequence rather than a representative provided herein.

Analysis of the DEWOFF database (version 35.45 Swiss Prob 35) using the ALIGN-2 sequence alignment analysis of the full length sequence shown in Figure 22 (SEQ ID NO: 55) revealed that the PRO356 amino acid sequence and the two TIE-2L1 (32% 2L2 (34%). Abbreviation "TIE" is an acronym indicating a tyrosine kinase having a "Ig and EGF homology domains (t yrosine kinase containing I g and E GF homology domains), is coined to refer to a new family of receptor tyrosine kinases.

Example 13: Isolation of cDNA clones encoding human PRO509

To isolate the cDNA of DNA50148-1068, the bacteriophage library of human retinal cDNA (purchased from Clontech) was screened by hybridization with a synthetic oligonucleotide probe based on the EST sequence (Genbank locus AA021617) And showed some degree of homology with members. The oligonucleotide probe used for this screening was 60 bp in length. Five positive clones (containing a cDNA insert of 1.8-1.9 kb) were identified in the cDNA library and positive clones were confirmed to be specific by PCR using the hybridization probes as PCR primers. A single phage containing each of the five positive clones was isolated with a defined dilution and the DNA was purified using the Wizard Lambda Prep DNA purification kit (Promega).

CDNA inserts from three of the five bacteriophage clones were digested with EcoRI, excised from the vector arm, gel-purified, subcloned with pRK5, and sequenced in both strands. Sega's clone contained the same open reading frame (except introns found in one of the clones).

The entire nucleotide sequence of DNA 50148-1068 is shown in Figure 23 (SEQ ID NO: 59). The cDNA contained one open reading frame with the translation initiation site designated as the ATG codon at nucleotide positions 82-84. This open reading frame was terminated at the termination codon TGA of nucleotide positions 931-933.

The predicted amino acid sequence of the full-length PRO509 polypeptide sequence is 283 amino acids in length. The full-length PRO509 protein is shown in Figure 24 (SEQ ID NO: 60), with an estimated molecular weight of about 30,420 daltons and an estimated pI of about 7.34.

The analysis of the full-length PRO509 sequence shown in Figure 24 (SEQ ID NO: 60) demonstrates the presence of several important polypeptide domains, as shown in Figure 24, with the positions for the important polypeptide domains being approximate as described above. Analysis of the full-length PRO509 sequence (Figure 24, SEQ ID NO: 60) revealed that the signal peptide of about amino acid 1 to about amino acid 36, the transmembrane domain of about amino acid 205 to about amino acid 221, about amino acid 110 to about amino acid 114, Glycosylation site of about amino acid 177, about amino acid 81 to about amino acid 87, about amino acid 89 to about amino acid 95, about amino acid 104 to about amino acid 110, about amino acid 120 to about amino acid 126, about amino acid 153 to about amino acid 159, The N-myristoylation site of about amino acid 193 to about amino acid 199, about amino acid 195 to about amino acid 201, and about amino acid 220 to about amino acid 226, the presence of a cell attachment sequence of about amino acid 231 to about amino acid 234.

Some sequences were found to be similar by aligning the cytoplasmic region of the 58 amino acid length of PRO509 with the other known members of the human TNF receptor family (using the ALIGN ™ computer program), particularly CD40 (12 identical) and LT- Beta receptors (11 identical).

Example 14: Isolation of cDNA clones encoding human PRO866

The consensus DNA sequence was assembled against another EST sequence using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA42257. Based on the DNA44708 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full-length coding sequence of PRO866.

PCR primers (forward and reverse) were synthesized:

Omni-directional PCR primer 1 5'-CAGCACTGCCAGGGGAAGAGGG-3 '(SEQ ID NO: 63)

Forward PCR primer 2 5'-CAGGACTCGCTACGTCCG-3 '(SEQ ID NO: 64)

Forward PCR primer 3 5'-CAGCCCCTTCTCCTCCTTTCTCCC-3 '(SEQ ID NO: 65)

Reverse PCR primer 1 5'-GCAGTTATCAGGGACGCACTCAGCC-3 '(SEQ ID NO: 66)

Reverse PCR primer 2 5'-CCAGCGAGAGGCAGATAG-3 '(SEQ ID NO: 67)

Reverse PCR primer 3 5'-CGGTCACCGTGTCCTGCGGGATG-3 '(SEQ ID NO: 68)

In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus sequence DNA44708.

Hybridization probe

5'-CAGCCCCTTCTCCTCCTTTCTCCCACGTCCTATCTGCCTCTC-3 '(SEQ ID NO: 69)

In order to screen several libraries for the purpose of finding the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO866 gene were then isolated with either the probe oligonucleotide and the PCR primers by using a positive library. RNA for cDNA library construction was isolated from human fetal kidney tissue (LIB228).

DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence of DNA53971-1359 (Fig. 25, SEQ ID NO: 61) and the protein sequence of PRO866 derived therefrom.

The entire coding sequence of DNA53971-1359 is shown in Figure 25 (SEQ ID NO: 61). Clone DNA53971-1359 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 275 to 277 and an apparent stop codon at nucleotide positions 1268 to 1270. The expected polypeptide precursor is 331 amino acids in length. Analysis of the sequence of the full-length PRO866 shown in Figure 26 (SEQ ID NO: 62) demonstrates the presence of several important polypeptide domains, and the positions for the important polypeptide domains are approximate as described above. Analysis of the full-length PRO866 polypeptide shown in Fig. 26 revealed that the signal peptide of about amino acid 1 to about amino acid 26, the glycosaminoglycan attachment site of about 131 to about amino acid 135, the cAMP for about amino acid 144 to about amino acid 148, Dependent protein kinase phosphorylation site, about amino acid 26 to about amino acid 32, about amino acid 74 to about amino acid 80, about amino acid 132 to about amino acid 138, about amino acid 134 to about amino acid 140, about amino acid 190 to about amino acid 196, about amino acid 287 290 &lt; / RTI &gt; and about 290 to about 290 amino acids of the amino acid sequence of SEQ ID NO: 293. Clone DNA53971-1359 was deposited with the ATCC on Apr. 4, 1998 and assigned accession number 209750. The estimated molecular weight of the full-length PRO866 protein shown in Figure 26 is about 35,844 daltons and the estimated pI is about 5.45.

Analysis of the full-length PRO866 sequence shown in Figure 26 (SEQ ID NO: 62) showed that it was significantly similar to that of the mindin / spondin family, indicating that PRO866 could be a novel dindamine homolog. More specifically, the analysis of the De Hoff database (version 35.45 Swiss Probes 35) showed that significant differences between the amino acid sequences of PRO866 and the de Hoop sequences of AB006085_1, AB006084_1, AB006086_1, AF017267_1, CWU42213_1, AC004160_1, CPMICRP_1, S49108, A48569 and 146687 Proved to have the same sex.

Example 15: Hybridization in the system

In situ hybridization is a powerful versatile technology for the detection and localization of nucleic acid sequences in cells and tissue samples. For example, it may be useful in identifying gene expression sites, analyzing tissue distribution of transcription, identifying and locating virus infection, tracking changes in specific mRNA synthesis, and chromosomal mapping.

In situ hybridization was performed using the PCR-generated ³³ P-labeled riboprobe with the optimal version of the protocol according to the literature (Lu and Gillett, Cell Vision 1: 169-176 (1994)). Briefly, human tissues fixed with formalin and embedded in paraffin were cut and the paraffin removed and the proteins were removed with Proteinase K (20 g / ml) for 15 min at 37 ° C, Additional processing was performed for in-situ hybridization as described in the above article. Lt; RTI ID = 0.0 &gt; 55 C &lt; / RTI &gt; with the ( ³³ P) UTP-labeled antisense riboprobe generated from the PCR product. The slide was soaked in Kodak NTB2 (TM) nuclear track emulsion and exposed for 4 weeks.

³³ P-riboprobe synthesis

6.0 [mu] l (125 mCi) of ³³ P-UTP (Amersham BF 1002, SA <2000 Ci / mMol) was speed vac dried. For each tube containing dried ³³ P-UTP the following ingredients were added:

2.0 [mu] l 5x transcription buffer

1.0 [mu] l DTT (100 mM)

2.0 μl NTP mixture (2.5 mM: 10 mM GTP, 10 μl each of CTP and ATP + 10 μl H ₂ O)

1.0 [mu] l UTP (50 [mu] M)

1.0 [mu] l RNasin

1.0 [mu] l DNA template (l [mu] g)

1.0 ㎕ H ₂ 0

1.0 ㎕ RNA polymerase (usually T3 = AS, T7 = S for PCR products)

The tubes were incubated at 37 DEG C for 1 hour. 1.0 占 퐇 of RQ1 DNase was added, followed by incubation at 37 占 폚 for 15 minutes. 90 [mu] l of TE (10 mM Tris (pH 7.6) / lmM EDTA (pH 8.0)) was added and the mixture was pipetted onto DE81 paper. The residual solution was loaded into a Microcon-50 (trademark) ultrafiltration unit and spun for 6 minutes with program 10. The filtration unit was transferred to a second tube and spun for 3 minutes with program 2. After the final recovery spinning, a total of 100 [mu] l of TE was added followed by pipetting 1 [mu] l of the final product on DE81 paper and counting with 6 ml of Bioflour II (trademark).

Probes were electrophoresed on TBE / urea gel. A total of 1 to 3 [mu] l of probe or 5 [mu] l of RNA MrkII was added to 3 [mu] l of loading buffer. After heating for 3 minutes in a heat block at 95 ° C, the gel was immediately transferred to ice-cold. After washing the wells of the gel, the sample was loaded and electrophoresed at 180-250 volts for 45 minutes. The gel was wrapped in a Saran (trade name) lap and exposed to XAR film on a reinforcement screen for 1 hour to overnight in a -70 ° C freezer.

³³ P-hybridization

A. Preliminary treatment of frozen sections

The slide was taken out from the freezer, placed in an aluminum tray, and dissolved at room temperature for 5 minutes. To reduce condensation, the trays were placed in a 55 &lt; 0 &gt; C incubator for 5 minutes. Slides were fixed in ice-cold 4% paraformaldehyde for 10 minutes in a fume hood and washed with 0.5 x SSC (25 ml 20 x SSC + 975 ml SQ H ₂ O) for 5 minutes at room temperature. Proteinase K at 0.5 / / ml of Proteinase K for 10 minutes (12.5 10 of 10 mg / ml stock solution in 250 ml of pre-heated RNase buffer without RNase) was incubated at room temperature for 10 minutes with 0.5 x SSC The sections were washed. The sections were dehydrated with 70%, 95% and 100% ethanol for 2 minutes, respectively.

B. Pretreatment of sections embedded in paraffin

The paraffin on the slide was removed and placed in SQ H ₂ O and rinsed twice with 2 x SSC for 5 min at room temperature. (Human embryo), or 8x proteinase K (250 ml RNase buffer, 10 mg / ml, 500 ㎕, 37 캜, 15 min in 250 ml RNase buffer without RNase) At 37 캜 for 30 minutes) (formalin tissue). It was then rinsed with 0.5 x SSC as described above and dehydrated.

C. Prehybridization

Slides were placed in plastic boxes with filter paper saturated with Box buffer (4x SSC, 50% formamide). The tissue was heated with microwave for 2 to then vortexed covering (dextran sulfate + 6 ml SQ H ₂ O of the 3.75 g) 50 ㎕ hybridization buffer, a state of loose lid minutes. After ice cooling, 18.75 ml of formamide, 3.75 ml of 20 x SSC and 9 ml of SQ H ₂ O were added and the tissue was well vortexed and then incubated at 42 ° C for 1 to 4 hours.

D. Hybridization

1.0 x 10 &lt; ⁶ &gt; cpm of probe and 1.0 [mu] l of tRNA (50 mg / ml stock solution) were added to each slide and heated at 95 DEG C for 3 minutes. After ice-cooling, 48 [mu] l of hybridization buffer was added per slide. After vortexing, 50 μl of the ³³ P mixture was added to 50 μl of the prehybridization sample on the slide. The slides were incubated overnight at 55 ° C.

E. Wash

RNase A was treated for 30 minutes at 37 ° C (RNase buffer 250 (final volume = 4 L) after 2 washes of 2x SSC, EDTA at room temperature (400 ml of 20 x SSC + 16 ml of 0.25 M EDTA, final volume = 4 l) ml, 500 [mu] l = 20 [mu] g / ml). The slides were washed twice with 2 x SSC, EDTA for 10 min at room temperature. Strict washing conditions are as follows: 2 hours at 55 占 폚 with 0.1x SSC and EDTA (20 ml 20x SSC + 16 ml EDTA, final volume = 4 l).

F. oligonucleotides

In situ analysis was performed on five of the DNA sequences disclosed herein. The oligonucleotides used in the analysis are as follows.

(1) DNA30879-1152 (PRO207)

p1 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC TCC TGC GCC TTT CCT GAA CC-3 '(SEQ ID NO: 70)

p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GAC CCA TCC TTG CCC ACA GAG-3 '(SEQ ID NO: 71)

(3) DNA33089-1132 (PRO221)

p1 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC TGT GCT TTC ATT CTG CCA GTA-3 '(SEQ ID NO: 72)

p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GGG TAC AAT TAA GGG GTG GAT-3 '(SEQ ID NO: 73)

(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)

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

(5) DNA40628-1216 (PRO301)

p1 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GAG TCC TTC GGC GGC TGT T-3 '(SEQ ID NO: 76)

p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CGG GTG CTT TTG GGA TTC GTA-3 '(SEQ ID NO: 77)

(6) DNA45416-1251 (PRO362)

p1 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CTC CAA GCC CAC AGT GAC AA-3 '(SEQ ID NO: 78)

p2 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CCT CCA CAT TTC CTG CCA GTA-3 '(SEQ ID NO: 79)

G. Results

An in-situ analysis of the five DNA sequences disclosed herein was performed. The results of the analysis are as follows.

(1) DNA30879-1152 (PRO207) (Apo2L homolog)

Chondrosarcoma and other soft tissue sarcoma. All other tissues were negative.

The human fetal tissues examined (E12-E16) include placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, major blood vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, Body wall, pelvis and lower limb.

Adult tissues examined include kidney (normal kidney and end stage kidney), adrenal gland, myocardium, spleen, lymph node, pancreas, lung, skin, eye (including retina), bladder and liver (liver, liver, .

Inspected non-human primate tissues include:

Chimpanzee tissues: salivary glands, stomach, thyroid, parathyroid, tongue, thymus, ovary and lymph nodes,

Rhesus monkey tissue: cerebral cortex, hippocampal tissue, cerebellum, penis.

(2) DNA33089-1132 (PRO221) (1 TM receptor)

It was expressed specifically in the spinal cord neurons as well as in the fetal cerebral gray matter. The probe appears to cross-react with the rat. Mature rhesus monkeys were expressed at low levels in the cerebellum neurons of the brain. All other tissues were negative.

The fetal tissues examined (E12-E16) include placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, major blood vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, , Pelvis and lower limb.

The adult tissues examined included liver, kidney, adrenal gland, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cortex (rm), hippocampus (rm), cerebellum (rm), penis, , Stomach cancer, colon, colon cancer and chondrosarcoma, as well as liver damage and cirrhosis induced by acetaminophen.

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

Were expressed only in vascular endothelial tissues of fetal spleen, adult spleen, fetal liver, adult thyroid gland and adult lymph node (chimpanzee). Additional expression sites were observed in the growing spinal ganglia. All other tissues were negative.

The human fetal tissues examined (E12-E16) include placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, major blood vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, Body wall, pelvis and lower limb.

Adult tissues examined include kidney (normal kidney and end stage kidney), adrenal gland, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, eye (including retina), bladder and liver (liver, liver, ).

Inspected non-human primate tissues include:

Chimpanzee tissues: salivary glands, stomach, thyroid, parathyroid, skin, thymus, ovary and lymph nodes,

Rhesus monkey tissue: cerebral cortex, hippocampal tissue, cerebellum, penis.

(4) DNA40628-1216 (PRO301) (CD22 homologue (JAM hlog, A33 Ag hlog)

Expression in human inflammatory tissue (psoriasis, IBD, inflammatory kidney, inflammatory lung, hepatitis, normal tonsil, adult and chimpanzee multiblock)

Expression in human inflammatory tissues was evaluated to be superior to that of normal human and non-human primate tissues. Including epithelial cells of the colon, mucosal epithelium of the colon, airway epithelium of the bronchi, oral mucosa (tongue), tonsil and mucosa, placental mucosa, prostate mucosa, gastric mucosa, epithelial cells of the thymus Hassall body, hepatocyte, bile epithelium and placental epithelium , And was expressed in all epithelial structures. The only evidence for expression outside the epithelium is a weak, low level of irregular expression in the embryo center of the follicle in the tonsil with reactive hyperplasia.

Was observed in the following non-human primate tissues:

Chimpanzee tissue: Weak diffusion was observed in epidermis of tongue epithelium. In the thymus, weakly specific expression was observed in the thymus epithelium of the halal soma. At the top, a slight level of diffuse expression was observed in the epithelium of the gastric mucosa.

Human tissues: In the liver (multi-block including chronic cholangitis, lobular hyperplasia, acetaminophen toxicity), low to moderate diffusion expression was observed in hepatocytes and bile epithelium. It was most predominantly expressed in periportal / periportal hepatocytes. This was most prevalent in the bile epithelium of the liver site with chronic sclerosing cholangitis. It was not expressed in all samples and could reflect the quality of the sample rather than the expression variability.

In the case of psoriasis, weak expression was observed in the epidermis.

In lungs with chronic interstitial pneumonia and chronic bronchitis, low levels of diffusion were observed in the mucosal epithelium of the airway and weak diffusion was observed in the alveolar epithelium. But not in the epithelium of the submucosal gland of the bronchial (s).

In the placenta, median level of diffusion was expressed in the placental epithelium.

In the prostate gland, a low level of diffuse expression was observed in the prostate epithelium.

In the gallbladder, a medium level of diffuse expression was observed in the epithelium of the mucosa.

In the tonsil with reactive hyperplasia, highly diffuse expression was observed in the epithelium of the tonsil mucosa and the tonsil, and the signal was highest in the mucosal cells along the tonsil. Mild irregular spreading was observed in the embryo center of the cortical vesicle (B lymphocyte area), but not in other lymphocyte B lymphocytes in other tissues evaluated as having lymphoid structure or lymphocyte inflammation.

In the colon with inflammatory bowel disease and polyp / adenomatous changes, low levels of expression were observed in the mucosal epithelium and the highest expression was seen at the villus tip. There was no evidence of increased expression of polyps in polyps, as compared to adjacent mucosa. There was no apparent expression in the reactive mucosal lymphoid tissues present at various sites.

(5) DNA45416-1251 (PRO362) (Ig domain homologue)

Expression in human inflammatory tissues (psoriasis, IBD, inflammatory kidney, inflammatory lung, hepatitis, normal tonsils, adult and chimpanzee multi block)

Expression of new proteins in several human and non-human primate tissues was assessed and found to be highly restricted in this expression. It was expressed only in the Cooper cells of the alveolar macrophages and hepatocytes of the lungs. Expression in these cells was significantly increased when each of these cell populations was activated. Although two subgroups of these macrophages are located in different organs, they have similar biological functions. Both of these types of these phagocytic cells act as biological filters to remove substances from the bloodstream or airways, including pathogens, senile cells and proteins, and they can secrete a wide range of important pre-inflammatory cytokines.

In inflammatory lungs (7 patient samples), expression was predominant in reactive alveolar macrophage populations, defined as large, pale, often frightening cells present in the alveoli alone or in groups, and normal non-reactive macrophages Cells) were weakly expressed or negative. Expression in alveolar macrophages was increased during inflammatory reactions in which both the number and size of these cells were increased (activated). Despite the presence of hypertrophic sites of interstitial inflammatory sites and bronchial hyperplasia of these tissues, expression was restricted to alveolar macrophages. Several inflammatory lungs also had some degree of pyritic inflammation and were not expressed in neutrophilic granulocytes.

In liver, it was strongly expressed in reactive / activated Cooper cells in the liver with acute central nodule necrosis (acetaminophen toxicity) or markedly prominent periportal inflammation. However, it was weakly expressed or not expressed in liver Cooper cells with normal liver, mild inflammation or mild to moderate lobular hyperplasia / hypoplasia. Thus, in lungs, expression in activated / reactive cells was increased.

This molecule was not expressed in histiocytes / macrophages present in inflammatory bowel, hyperplastic / reactive tonsils or normal lymphocytes. Not all of these tissues, including those with tissue histiocytosis or resident macrophage populations, support that expression is restricted to a unique macrophage subpopulation defined as alveolar macrophages and liver Cooper cells.

Human tissues assessed as not detectable expression include inflammatory bowel disease tissues (seven patients with intermediate or severe disease), tonsil tissues with reactive hyperplasia, peripheral lymphocytes, psoriatic skin tissue (mild to moderate disease 2 patient samples), heart and peripheral nerve.

Chimpanzee tissues assessed as having no detectable expression include the tongue, stomach, and thymus.

Example 16: Use of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 as hybridization probes

The following method describes the use of a nucleotide sequence encoding a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 as a hybridization probe.

Encoding a naturally occurring variant of homologous DNA (e. G., PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 in human tissue cDNA library or human tissue genomic library) 6, 9, 14, 19, and 24 as probes for screening for the full-length or mature PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 as 29, 34, 42, 47, 54, 59 and 61, respectively) DNA or a fragment thereof is used.

Hybridization and washing of filters containing these library DNAs is performed under the following high stringency conditions. Hybridization of a radiolabeled probe derived from a gene encoding a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide into a filter was performed at 42 ° C in 50% , 5 × SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 × Denhardt's solution and 10% dextran sulfate solution for 20 hours. The filter is washed in an aqueous solution of 0.1 x SSC and 0.1% SDS at &lt; RTI ID = 0.0 &gt; 42 C. &lt; / RTI &gt;

Thereafter, DNA having the desired sequence identity with the DNA encoding the full-length native sequence is identified by standard methods known in the art.

Example 17: Expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 in E. coli

In this embodiment, Glycosylated forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with recombinant expression in E. coli.

DNA sequences coding for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were first amplified using selected PCR primers. The primer should contain a restriction enzyme site corresponding to the restriction enzyme site on the selected expression vector. Multiple expression vectors may be used. Examples of suitable vectors include pBR322 [which contains the ampicillin and tetracycline resistance gene. Derived from E. coli; Bolivar et al., Gene 2 : 95 (1977)]. The vector was digested with restriction enzymes and dephosphorylated. The PCR amplified sequence was then ligated to the vector. The vector preferably comprises an antibiotic resistance gene, a trp promoter, a poly-His leader (including the first six STII codons, a poly-His sequence and an enterokinase cleavage site), PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding regions, lambda transcription terminator and the argU gene.

The ligation mixture was then purified using the method described by Sambrook et al. (Supra). And used to transform E. coli strains. Transformants capable of growing in LB medium were identified and colonies with antibiotic resistance were selected. Plasmid DNA was isolated and identified using restriction analysis and DNA sequencing.

Selected clones were cultured overnight in a liquid culture medium such as LB broth supplemented with antibiotics. This culture was used for larger seed culture inoculation. The cells were then incubated to the desired optical density while the expression promoter was running.

After culturing the cells for several hours or more, cells can be recovered by centrifugation. The cell pellet obtained by centrifugation was dissolved with various reagents known in the art and dissolved in the solution containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 proteins can be purified.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, Coli was successfully expressed in a poly-His tagged form. The selected primers were used to initially amplify DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Primers included restriction sites corresponding to restriction sites on selected expression vectors and other useful sequences that provided efficient and reliable translation initiation, rapid purification on metal chelate columns, and proteolytic cleavage using enterokinase. The PCR-amplified, poly-His tagged sequence is then ligated to the expression vector and transformed into E. coli host according to strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq) were used. the first transformant was cultured in LB medium containing 50 ㎎ / ㎖ of carbenicillin (carbenicillin) until the OD ₆₀₀ reaches 3 to 5 with shaking at 30 ℃. then, CRAP the culture medium ( in 500 ㎖ water _{3.57 g (NH 4) 2 SO} 4, 0.71 g sodium citrate _{· 2H 2 O, 1.07 g KCl} , 5.36 g adipic nose (Difco) yeast extract, 5.36 g Sheffield Hi Carr claim (Sheffield hycase) SF, and 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO ₄ ) and incubated for about 20-30 hours at 30 ° C. The samples were separated Expression was confirmed by SDS-PAGE analysis and the bulk culture was centrifuged to pellet the cells (C) it was kept frozen until purification and refolding to the cell pellet.

0.5 to 1 L of this. The coli paste fermentation broth (6 to 10 g pellet) was resuspended in 10 volumes (w / v) of 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium bisulfite and sodium sodium sulfite were added to final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4 ° C. At this stage, a denatured protein with all cysteine residues blocked by sulfation was produced. The solution was centrifuged at 40,000 rpm for 30 minutes in a Beckman ultrafiltration centrifuge. The supernatant was diluted with a 3-5 fold volume of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through a 0.22 um filter to make it clear. The clear extract was loaded onto 5 ml of a Qiagen Ni ²⁺ -NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with additional buffer (pH 7.4) containing 50 mM imidazole (Calbiochem, Utrol grade). The protein was eluted with a buffer containing 250 mM imidazole and the fractions containing the desired protein were pooled and stored at 4 &lt; 0 &gt; C. The protein concentration was measured by absorbance at 280 nm using the extinction coefficient calculated according to the amino acid sequence.

The protein was refolded by slowly diluting the sample in a freshly prepared refolding buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. The refolding volume was determined such that the final protein concentration was 50-100 占 퐂 / ml. The refolding solution was slowly stirred at 4 캜 for about 12 to 36 hours. The refolding reaction was stopped by adding TFA to a final concentration of 0.4% (about pH 3). Before further protein purification, the solution was filtered through a 0.22 탆 filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was analyzed in Poros R1 / H reverse phase column chromatography using a mobile buffer of lysozyme 0.1% TFA with an acetonitrile concentration gradient of 10-80%. A fraction of the fraction exhibiting A ₂₈₀ absorbance was analyzed on SDS polyacrylamide gel to collect fractions containing homogeneously refolded proteins. Normally, properly refolded proteins elute at the lowest concentration of acetonitrile, with the appropriately refolded protein form in most proteins eluting from the lowest concentration of acetonitrile, since it most closely fits into the hydrophobic interior portion that is protected from interaction with the reverse phase resin. The aggregated proteins are usually eluted at high acetonitrile concentrations. The inverse phase step not only separates the protein of the wrongly folded form from the desired type of protein, but also removes the endotoxin from the sample.

Each fraction containing preferably the folded PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 proteins was recovered and the solution was loaded with acetonitrile . Proteins were formulated with 20 mM Hepes, pH 6.8, containing 0.14 M sodium chloride and 4% mannitol, by gel filtration using G25 superfine (Pharmacia) resin, equilibrated with dialysis or formulation buffer and sterile filtered.

PRO207, PRO224 and PRO301 were prepared by the procedure described above in the form of a poly-His tagged version. It was successfully expressed in E. coli.

Example 18 Expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 in Mammalian Cells

This example illustrates a method of producing a possible glycosylated form of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 by recombinant expression in mammalian cells.

The vector pRK5 (see EP 307,247, published May 5, 1989) was used as an expression vector. Optionally, the DNA may be amplified using the ligation method described by Sambrook et al. (Supra) using selected restriction enzymes, such as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, pRK5, and inserted into PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 DNA. PRK5-PRO356, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO301, pRK5-PRO526, pRK5-PRO362, pRK5-PRO356, pRK5- PRO509 or pRK5-PRO866.

In one embodiment, 293 cells may be selected as host cells. Human 293 cells (ATCC CCL 1573) were cultured in confluent tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally with nutrients and / or antibiotics. PRK5-PRO359, pRK5-PRO209, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO308, pRK5-PRO526, pRK5-PRO362, pRK5-PRO356, pRK5- Or pRK5-PRO866 DNA was mixed with about 1 쨉 g of DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31: 543 (1982)], and 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA and 0.227 M It was dissolved in CaCl _2. 500 쨉 l of 50 mM HEPES (pH 7.35), 280 mM NaCl, and 1.5 mM NaPO ₄ were added dropwise to the mixture to form a precipitate at 25 째 C for 10 minutes. The precipitate was suspended and added to 293 cells and allowed to stand at 37 DEG C for about 4 hours. The medium was aspirated off and 2 ml of 20% glycerol in PBS was added for 30 seconds. Then, 293 cells were washed with serum-free medium, fresh medium was added, and cells were cultured for about 5 days.

Approximately 24 hours after transfection, the medium was removed and replaced with medium (alone) or medium containing 200 μCi / ml ³⁵ S-cysteine and 200 μCi / μl ³⁵ S-methionine. After 12 hours incubation, the conditioned medium was collected, concentrated on a rotary filter and loaded onto 15% SDS gel. The treated gel was dried and exposed to film for a selected period of time to confirm the presence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. The culture containing the transfected cells was further cultured (in serum-free medium) and the medium was tested with the selected biochemical assay.

Alternatively, Somparyrac et al., Proc. Natl. Acad. Sci. To 7575 (1981)] dextran alcohol using the sulfate method PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or the PRO866 gene encoding the 293 cell as described in: USA, 12 It was possible to introduce it temporarily. 293 cells were cultured to the highest density in a spinning flask and 700 μg of pRK5-PRO179, pRK5-PRO207, pRK5-PRO209, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5- , pRK5-PRO362, pRK5-PRO356, pRK5-PRO509 or pRK5-PRO866 DNA. Cells were first centrifuged, concentrated from spin-flask and washed with PBS. The DNA-dextran precipitate was incubated on the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and then placed in a spinning flask containing tissue culture medium, 5 ug / ml bovine insulin, and 0.1 ug / ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and lysates. A sample containing the expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 was concentrated and analyzed by any selected method, e.g., dialysis and / or column chromatography &Lt; / RTI &gt;

In another embodiment, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding genes can be expressed in CHO cells. pRK5-PRO359, pRK5-PRO209, pRK5-PRO219, pRK5-PRO224, pRK5-PRO329, pRK5-PRO329, pRK5-PRO309, pRK5- It is known, for the PRO866 reagent, for using the CaPO ₄ or DEAE-dextran can be transfected into CHO cells. As mentioned above, the cell cultures were incubated and the medium was replaced with medium alone or with medium containing radioactive label such as ³⁵ S-methionine. After confirming the presence of the expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, the culture medium was replaced with serum-free medium. Preferably, the culture was incubated for about 6 days and the conditioned medium was recovered. The medium containing the expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides was concentrated and purified by any selected method.

In addition, epitope tagged PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be expressed in CHO host cells. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be subcloned from the pRK5 vector. Insertion of a subclone may be fused to a baculovirus expression vector in a form having a selected epitope tag such as a poly-His tag via PCR. The inserts of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 to which the poly-His tag was added are SV40 Can be subcloned into the derived vector. Finally, CHO cells can be transfected with an SV40 derived vector (as above). May be labeled in the same manner as described above to confirm expression. The culture medium containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with the expressed poly-His tag was then concentrated to give a Ni2 ⁺ And can be purified by a selected method such as chromatography.

In addition, it is also possible to express the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 in CHO and / or COS cells by a transient expression method, 0.0 &gt; CHO &lt; / RTI &gt;

And stably expressed in CHO cells using the following experimental procedure. The coding sequence of the soluble form of each protein (e. G., Extracellular domain) is expressed as an IgG construct (immunohedressin) fused to an IgGl constant region sequence comprising hinge, CH2 and CH2 domains and / The protein was expressed in an attached form.

Following PCR amplification, each DNA was subcloned into a CHO expression vector using standard methods described in Ausubel et al., Current Protocols in Molecular Biology, Unit 3.16, John Wiley and Sons (1997). The CHO expression vector is prepared to have restriction sites suitable for 5 'and 3' of the desired DNA so that the restriction sites of the cDNA can be conveniently shuttled. The vector used for expression in CHO cells is described in Lucas et al., Nucl. Acids Res. 24 : 9 1774-1779 (1996)), and the SV40 early promoter / enhancer was used to express the desired cDNA and dihydrofolate reductase (DHFR). DHFR expression allows selection of stably maintained cells of the plasmid after transfection.

Twelve micrograms of the desired plasmid DNA was mixed with about 50 μg of the plasmid DNA using commercially available transfection reagent (Superfect, Qiagen), Dosper (registered trademark) (Dosper) or Fugin (Fugene, Boehringer Mannheim) Were introduced into 10 million CHO cells. Cells were cultured according to the method described in Lucas et al., Supra. Approximately 3 x 10 &lt;&quot; ⁷ &gt; cells were frozen in ampoules according to the method described below for later culturing and production of the cells.

Plasmid DNA-containing ampoules were dissolved in a water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 ml of the medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated off and the cells resuspended in 10 ml of selective medium (PS20 filtered through 0.2 mu m filter paper containing 5% bovine serum of 0.2% hemodialyzed filtered). The cells were dispensed into a 100 ml round bottom flask containing 90 ml of selective medium. After 1-2 days, the cells were transferred to a 250 ml round-bottomed flask filled with 150 ml of selective medium and cultured at 37 ° C. After 2 to 3 days were inoculated with 250 ㎖, 500 ㎖ and 3 per 2000 ㎖ in ㎖ rotating flask x 10 ⁵ cells. The cell culture was centrifuged, replaced with fresh medium, and resuspended in the production medium. Any suitable CHO medium can be used, but the production medium described in U.S. Patent No. 5,122,469 (June 16, 1992) was actually used. A 3 L production spinning flask was inoculated with 1.2 x ¹⁰⁶ cells / ml. Cell number and pH were measured at day 0. On the first day, samples were taken in a rotating flask and the application of filtered air was started. On the second day, samples were taken in a rotating flask and the temperature was changed to 33 ° C and 30 ml of 500 g / L-glucose and 0.6 ml of 10% defoamer (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 milky emulsion ). During the entire batch, the pH was adjusted to maintain about 7.2 as needed. After 10 days or when the survival rate dropped to less than 70%, the cell culture was recovered by centrifugation and filtered through a 0.22 mu m filter. The filtrate was stored at 4 [deg.] C or immediately loaded onto the purification column.

For structures with poly-His tags, proteins were purified using a Ni ²⁺ -NTA column (Qiagen). Imidazole was added to conditioned medium at a concentration of 5 mM before purification. Conditioned medium at a flow rate of 4-5 ml / min at 4 [deg.] C was injected into 6 ml Ni2 ⁺ -NTA column equilibrated with 20 mM Hepes, pH 7.4 buffer containing 0.3 M NaCl and 5 mM imidazole. After loading, the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed through a 25 ml G25 suppine (Pharmacia) column into a storage buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol and stored at -80 ° C .

From the conditioned media, the immunoadhesin (including Fc) construct was purified as follows. The conditioned medium was injected into 5 ml of protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 6.8). After loading, the column was washed with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in 1 ml fractions in a tube containing 275 l of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then cleared of salts in the storage buffer as described above for the case of poly-His tagged proteins. SDS-polyacrylamide gel and N-terminal amino acid sequence analysis performed by Edman degradation method confirmed the homogeneity of the protein.

PRO179, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO356, PRO509 and PRO866 were stably expressed in CHO cells by the method described above. In addition, PRO224, PRO328, PRO301 and PRO356 were expressed in CHO cells by transient expression method.

Example 19 Expression in yeast of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866

The following method describes the recombinant expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding genes in yeast.

Yeast expression vectors for intracellular production or secretion of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were prepared from the ADH2 / GAPDH promoter. For direct intracellular expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO226, PRO329, PRO309, , PRO362, PRO356, PRO509 or PRO866 coding DNA and promoter were inserted into the appropriate restriction enzyme sites of the selected plasmid. In the case of secretion, PRO179, PRO209, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, or PRO306 were used for the expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, , PRO526, PRO366, PRO356, PRO509 or PRO866 signaling peptides or other nucleic acids encoding the ADH2 / GAPDH promoter, native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, May be cloned into a selected plasmid along with DNA encoding a mammalian signal peptide, e. G., A yeast alpha-factor or an invertase secretion signal / leader sequence and a linker sequence (if necessary).

Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatant can be precipitated with 10% trichloroacetic acid, separated by SDS-PAGE, and analyzed by staining the gel with Coomassie blue.

The yeast cells were then recovered from the fermentation broth by centrifugation and the culture was concentrated using the selected cartridge filter to produce recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO866 can be isolated and purified. Concentrates containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be further purified using the selected column chromatography resin.

Example 20 Expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 in Baculovirus-Infected Insect Cells

The following method describes recombinant expression in baculovirus-infected insect cells.

The coding sequences of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were fused upstream of the epitope tag included in the baculovirus expression vector. The epitope tag includes a poly-His tag and an immunoglobulin tag (such as the Fc region of an IgG). Several plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the coding sequences for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, , A PRO356, PRO509 or PRO866 coding sequence (for example, an extracellular domain of a transmembrane protein or a mature protein coding sequence in the case of an extracellular protein) is amplified by PCR using primers complementary to the 5 'and 3' Respectively. The 5 ' primer may comprise an adjacent (selected) restriction enzyme site. The product was then digested with the selected restriction enzymes and subcloned into an expression vector.

Recombinant baculovirus was prepared by using the above plasmid and BaculoGold (TM) virus DNA (Pharmingen) with Spodoptera frugiperda (" Sf9 &quot;) cells (ATCC CRL 1711 ). &Lt; / RTI &gt; After incubation at 28 ° C for 4 to 5 days, the released virus was recovered and used for subsequent amplification. Viral infections and protein expression were performed according to O'Reilley et al., Baculovirus Expression vectors: A laboratory Manual , Oxford: Oxford University Press (1994).

The PRO219, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 to which the expressed poly-his tag is attached are then analyzed by, for example, Ni ²⁺ -chelate affinity chromatography Can be purified as follows. Extracts are prepared from recombinant virus-infected Sf9 cells according to Rupert et al., Nature , 362 : 175-179 (1993). Briefly, washed Sf9 cells sonication buffer (25 ㎖ Hepes, pH 7.9; 12.5 mM MgCl 2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) was resuspended 20 seconds on ice in And then ultrasonicated twice. The supernatant was removed by centrifugation and the supernatant was diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 [mu] m filter. Ni ²⁺ -NTA agarose column (purchased from Qiagen) was prepared in 5 ml bed volume, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract was loaded onto the column at 0.5 ml per minute. At the start of point fraction recovery, the column was washed with loading buffer to the A ₂₈₀ baseline. Next, the column was washed with secondary washing buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 6.0) to elute nonspecifically bound proteins. When A ₂₈₀ reached the baseline again, the column was developed with a 0-500 mM imidazole gradient in the secondary wash buffer. 1 ml fractions were collected and analyzed by SDS-PAGE and Western blotting with Ni ²⁺ -NTA (Qiagen) coupled to silver stain or alkaline phosphatase. Fractions containing the eluted His ₁₀ tagged PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were pooled and dialyzed against loading buffer.

Alternatively, the purification of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with IgG tagged (or Fc tagged) For example, protein A or protein G column chromatography.

Followed by subcloning into baculovirus expression vectors (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins) after PCR amplification of the respective coding sequences, and Lipofectin Vector and BaculoGold ™ baculovirus DNA (Pharmingen) were simultaneously transfected into 105 Spodoptera frugiperda (Sf9) cells (ATCC CRL 1711) using a Gibco-BRL (GIBCO-BRL) pb.PH.IgG and pb.PH.His are variants of the commercially available baculovirus expression vector pVL1393 (Pharmingen) containing a modified polylinker region containing the His or Fc tag sequence. Cells were cultured in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). The cells were incubated at 28 DEG C for 5 days. The supernatants were harvested and subsequently Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS were infected with a MOI of approximately 10 and used for first viral amplification. The cells were incubated at 28 DEG C for 3 days. The supernatant was recovered to prepare 25 ml of Ni ²⁺ -NTA bead (QIAGEN) or IgG-added protein in the case of a histidine tagged protein. 1 ml of supernatant was placed in Protein A Sepharose CL-4B beads (Pharmacia) After binding, SDS-PAGE analysis was performed to determine the expression of the constructs in the baculovirus expression vector by comparing with standard proteins at known concentrations in Coomassie blue staining.

The first virus-amplified supernatant was used to infect spinner culture (500 ml) of Sf9 cells cultured to an MOI of about 0.1 in ESF-921 medium (Expression Systems LLC). The cells were incubated at 28 DEG C for 3 days. The supernatant was recovered and filtered. Batch binding and SDS-PAGE analysis were repeated as necessary until the expression of the spinner culture was confirmed.

The conditioned medium of transfected cells (0.5-3 L) was centrifuged to remove cells and filtered through a 0.22 mu m filter. The poly-His tagged structure was purified using a Ni ²⁺ -NTA column (Qiagen). Imidazole was added to the conditioned medium to a concentration of 5 mM before purification. The conditioned media was loaded onto a 6 ml Ni ²⁺ -NTA column equilibrated with 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole at 4 ° C at a flow rate of 4-5 ml / min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed from the salt in 25 ml of G25 Suppine (Pharmacia) column in Storage Buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol and stored at -80 ° C.

The protein immunoadhesin (Fc) construct was purified from conditioned media as follows. The conditioned media was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, it was extensively washed with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in 1 ml fractions in a tube containing 275 ml of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed from the storage buffer as in the case of the poly-His tagged protein described above. The homogeneity of the protein was confirmed by N-terminal amino acid sequence analysis performed by SDS-PAGE and Edman degradation.

PRO301, PRO362, PRO356, PRO509 and PRO866 were expressed in baculovirus-infected Sf9 insect cells.

Alternatively, modified baculovirus methods can be used in high-5 cells. In this method, the DNA encoding the desired sequence can be amplified using a suitable system, such as Pfu (Stratagene), or can be fused with the upstream (5 ') of the epitope tag contained in the baculovirus expression vector have. The epitope tag includes a poly-His tag and an immunoglobulin tag (such as the Fc region of an IgG). Several plasmids can be used, including commercially available plasmids, for example plasmids derived from pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors were designed to constitutively express the recombinant protein from the baculovirus ie1 promoter in the stably transformed insect cell (1). Both plasmids contain all the promoter sequences and the hr5 enhancer component known to be important for the ie1 mediated gene expression of non-infected insect cells, differing only in the direction of the multiple cloning site. pIE1-1 and pIE1-2 contain translation initiation sites and can be used to produce fusion proteins. Briefly, the desired sequence of the desired sequence or sequence, for example the sequence encoding the extracellular domain of the transmembrane protein, was amplified by PCR using primers complementary to the 5 'and 3' regions. The 5 ' primer may comprise an adjacent (selected) restriction enzyme site. The product was then digested with the selected restriction enzymes and subcloned into an expression vector. For example, a derivative of pEI1-1 may comprise the Fc region of human IgG (pb.PH.IgG) or 8 histidine (pb.PH.His) tag downstream (3 ') of the desired sequence. Preferably, the vector constructs are sequenced for identification.

High-5 cells were cultured at 50% confluency under conditions of CO ₂ , NO pen / strep member at 27 ° C. For each 150 mm plate, the PIE-based vector containing 30 μg of the sequence was mixed with 1 ml of Ex-Cell medium (Ex-Cell 401 + 1/100 L-Glu JRH Biosciences # 14401-78P, And mixed with 1 ml of Ex-Cell culture medium in a separate test tube with 100 占 퐇 of cell pectin (purchased from CellFECTIN, GIBCO-BRL # 10362-010, Vortexing). The two solutions were mixed and incubated at room temperature for 15 minutes. 8 ml of Ex-Cell medium was added to 2 ml of DNA / cell pectin mixture and the high-5 cell phase washed once with Ex-Cell medium was covered. Plates were then incubated in the dark for 1 hour at room temperature. The DNA / cell pectin mixture was then removed with a drawer and the cells were washed once with Ex-Cell to remove excess cell pectin. 30 ml of fresh Ex-Cell medium was added and the cells were incubated at 28 ° C for 3 days. The supernatant was recovered to prepare 25 ml of Ni ²⁺ -NTA bead (QIAGEN) or IgG-added protein in the case of a histidine tagged protein. 1 ml of supernatant was placed in Protein A Sepharose CL-4B beads (Pharmacia) After binding, SDS-PAGE analysis was performed and the expression of the sequence in the baculovirus expression vector was measured by coomassie blue staining compared to standard proteins at known concentrations.

The conditioned medium of transfected cells (0.5-3 L) was centrifuged to remove cells and filtered through a 0.22 mu m filter. The poly-His tagged structure was purified by using a Ni ²⁺ -NTA column (Qiagen). Imidazole was added to the conditioned medium to a concentration of 5 mM before purification. The conditioned media was loaded onto a 6 ml Ni ²⁺ -NTA column equilibrated with 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole at 48 ° C at a flow rate of 4-5 ml / min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed from the salt in 25 ml of G25 Suppine (Pharmacia) column in Storage Buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol and stored at -80 ° C.

The protein immunoadhesin (Fc) construct was purified from conditioned media as follows. The conditioned media was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 6.8). After loading, it was extensively washed with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in 1 ml fractions in a tube containing 275 ml of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed from the storage buffer as in the case of the poly-His tagged protein described above. The homogeneity of the protein was confirmed by SDS polyacrylamide gel and Edman degradation method and N-terminal amino acid sequence analysis performed if desired or other analytical methods as required.

PRO179, PRO221, PRO224, PRO328, PRO526, PRO362 and PRO356 were expressed by the baculovirus method described above using high-5 cells.

Example 21: Preparation of antibodies binding to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866

This embodiment is a method for producing a monoclonal antibody capable of specifically binding to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866.

Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that may be used include the purified PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO329, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, PRO179, PRO207, PRO320, PRO219, PRO221, , Fusion proteins comprising PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 and recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 Cells. Experts can choose an immunogen without unnecessary experimentation.

Mice were immunized subcutaneously or intraperitoneally with 1 to 100 占 퐂 immunized with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 emulsified in complete supplemental solution, I was injected and immunized. Alternatively, immunizations were emulsified in MPL-TDM supplement (Ribi Immunochemical Research, Hamilton, MT) and injected into the hindpaw of the animal to be immunized. The immunized mice were then boosted with additional immunogens emulsified in the selected adjuvant 10-12 days later. Thereafter, mice may be boosted with additional vaccines for several weeks. Anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti- PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibodies.

Once the appropriate antibody titer was detected, the animals that were " positive " to the antibody were finally intravenously injected with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. After 3 to 4 days, mice are sacrificed and spleen cells are recovered. The spleen cells were then fused (using 35% polyethylene glycol) with the selected mouse myeloma cell line, for example P3X63AgU.1, available from ATCC number CRL 1597. The fusions produced hybridoma cells which were then plated on 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin and thymidine) medium which inhibited the proliferation of non-fused cells, myeloma hybrids and spleen cell hybrids .

Hybridoma cells can be screened with an ELISA for reactivity to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Methods for determining " positive " hybridoma cells that secrete the desired monoclonal antibodies to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, Is well known to those skilled in the art.

The positive hybridoma cells were cultured in the same manner as in Example 1 except that the winter Balb / c mice were treated with anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti- -PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in tissue culture flasks or roller bottles. The monoclonal antibody produced in the plurality can be purified using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on protein A or protein G binding of the antibody may be used.

Example 22 Purification of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 Polypeptides with Specific Antibodies

The natural or recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be purified by a variety of standard techniques in the field of protein purification. For example, there can be mentioned, for example, pro-PRO179, pro-PRO207, pro-PRO320, pro-PRO219, pro-PRO221, pro-PRO224, pro-PRO328, , Pro-PRO509 or pro-PRO866 polypeptide, mature PRO179, mature PRO207, mature PRO320, mature PRO219, mature PRO221, mature PRO224, mature PRO328, mature PRO301, mature PRO526, mature PRO362, mature PRO356, mature PRO509 or mature PRO866 polypeptide Pre-PRO 369, pre-PRO 360, pre-PRO 320, pre-PRO 319, pre-PRO 212, pre-PRO 212, pre-PRO 328, pre- Or pre-PRO866 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides of interest. Generally, the immunoaffinity column is an anti-PR179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti- PRO328, anti- PRO301, anti- PRO356, anti-PRO509, or anti-PRO866 polypeptide antibody to the activated chromatographic resin by covalent coupling.

Polyclonal immunoglobulins were prepared from immune sera by precipitation with ammonium sulfate or by purification on immobilized protein A (Pharmacia LKB Biotechnology, Fitzgerald, FJ). Similarly, monoclonal antibodies were prepared from mouse remnants by ammonium sulfate precipitation or chromatography on immobilized protein A. Partially purified immunoglobulins were purified by chromatography on a CnBr-activated SEPHAROSE (TM) (Pharmacia LKB Biotechnology) . &Lt; / RTI &gt; The antibody was coupled to the resin, the resin was blocked, and the derived resin was washed according to the manufacturer's instructions.

Such immunoaffinity columns can be used for the purification of PRO polypeptides by preparing fractions from cells containing the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides in soluble form do. The agent is induced by solubilization of the subcellular fraction obtained by solubilization of the whole cell or differential centrifugation by detergent addition or by other methods well known in the art. Alternatively, soluble PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides comprising the signal sequence may be secreted into a medium in which the cells grow in a useful amount.

A formulation containing soluble PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide was passed over the immunoaffinity column and the column was washed with PRO179, PRO207, PRO320, PRO219, PRO221 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides (for example, a high ion concentration buffer in the presence of detergent). PRO356, an antibody / PRO216, an antibody / PRO221, an antibody / PRO224, an antibody / PRO328, an antibody / PRO301, an antibody / PRO526, an antibody / PRO362, an antibody / PRO356, an antibody / (E. G., A low pH buffer, such as about pH 2-3, or a high concentration of chaotrope such as urea or thiocyanate ions) to destroy the PRO509 or antibody / PRO866 polypeptide bond, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides were recovered.

Example 23: Drug screening

The present invention is particularly useful for screening compounds with any of a variety of drug screening techniques using PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or binding fragments thereof . The PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments used in these tests are free in solution, adhered to a solid support, Or may be located within the cell. One method of drug screening uses a recombinant nucleic acid expressing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments in a stably transformed eukaryotic or prokaryotic Host cells are used. Drugs were screened against such transformed cells by competitive binding assay. Such cells in viable or fixed form can be used for standard binding assays. For example, complex formation between PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments and the substance to be tested can be measured. Alternatively, the decrease in complex formation between the target cell or target receptor and the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide Can be inspected.

Accordingly, the present invention provides the use of a medicament or any other active substance capable of affecting PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide- Thereby providing a screening method. (I) contacting the active substance with a compound of the formula (I) or a pharmaceutically acceptable salt thereof, wherein the active substance is contacted with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments thereof, The presence of a complex between a substance and a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or fragment, or (ii) the presence of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224 , PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments and complexes between the cells. In this competitive binding assay, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments are usually labeled. After appropriate incubation, the free PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments are separated from the presence in the conjugate, The ability to bind to a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, or the ability of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, 0.0 &gt; PRO301, &lt; / RTI &gt; PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide / cell complexes.

Other drug screening techniques provide high throughput screening for compounds with binding affinities suitable for polypeptides and are described in detail in WO84 / 03564 (published Mar. 9, 1984). Briefly stated, a number of different small peptide test compounds are synthesized on a solid substrate, such as a plastic pin or some other surface. PRO279, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO306, PRO308, or PRO306 polypeptides by applying the peptide test compounds to the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides and washed. The combined PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are detected by methods well known in the art. The purified PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides may also be coated directly on the plate for use in the above-described drug screening techniques. In addition, non-digested antibodies can be used to capture the peptide and immobilize it on a solid support.

The present invention also relates to a method of treating or preventing a neuroprotective disorder wherein the neutralizing antibody capable of binding to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides is PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, Consider the use of competitive drug screening assays that specifically compete with test compounds for binding to PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments thereof. In this manner, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, .

Example 24: Rational drug design

The goal of the rational drug design is to identify the biological active polypeptides of interest (i.e., PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides) For example, to produce structural analogues of agonists, antagonists or inhibitors. Any of these examples may be combined with a more active or more stable form of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, Can be used to form drugs that enhance or inhibit the function of PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides (Hodgson, Bio / Technology , 9 : 1921 (1991)).

In one study, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, , PRO356, PRO509 or PRO866 polypeptide-inhibitor complex is determined by x-ray crystallography, computer modeling or most typically by a combination of the two methods. Both the morphology and charge of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides should be verified to reveal the structure of the molecule and determine the active site (s). Less frequently, useful information about the structure of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be obtained by modeling based on the structure of the homologous protein . In both cases, the relevant structural information is used to design or identify effective inhibitors of the cognate PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide-like molecules. Useful examples of rational drug design include those molecules that have enhanced activity or stability as shown in Braxton and Wells, Biochemistry, 31 : 7796-7801 (1992), or those that have been described in the literature [Athauda ), Et al. , J. Biochem. , &Lt; / RTI &gt; 113 : 742-746 (1993)], which act as inhibitors, agonists or antagonists of natural peptides.

It is also possible to isolate the target-specific antibody, select it by functional analysis as described above, and then analyze its crystal structure. In this way, in principle, a pharmacore is obtained in which subsequent drug design can serve as a basis. Protein Crystallization Method It is also possible to avoid all protein crystallization methods by producing an anti-genotype antibody (anti-id) against a functional pharmacologically active antibody. As a mirror image, the binding site of the anti-id will be expected to be the homolog of the original receptor. The peptides may then be identified and isolated from the chemically or biologically produced peptide banks using anti-id. The isolated peptide will then serve as a perming core.

By the present invention, sufficient amounts of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are available to perform analytical studies such as x- can do. The amino acid sequence knowledge of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO526, PRO362, PRO356, PRO509 or PRO866 provided herein may also be used in place of or in addition to the instructions for using computer modeling techniques .

Example 25: In vitro antitumor assay

The antiproliferative activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides is described in Skehan et al., J. Natl. In- vitro anti-cancer research for the National Cancer Institute (NCI) disease using the sulforfodamine B (SRB) dye binding assay, which is essentially described in Cancer Inst ., 82 : 1107-1112 . The 60 tumor cell lines (" NCI panel &quot;) and the maintenance and in vitro culture methods of these cell lines used in this study are described in Monks et al., J. Natl. Cancer Inst. , 83 : 757-766 (1991). The objective of this screening is to initially assess the cytotoxicity and / or cytotoxic activity of the test compound against different tumor types (Monks et al., Supra , Boyd, Cancer: Princ. Pract. Oncol. Update , 3 (10) : 1-12 (1989)].

Cells from approximately 60 human tumor cell lines were cultured in trypsin / EDTA (Gibco), washed once, resuspended in IMEM and viability measured. The cell suspension was pipetted into each 96 well microtiter plate (100 [mu] l volume). After 6 days of incubation, the cell density was inhibited to be lower than 2 days after incubation. Pre-incubation was carried out at 37 DEG C for 24 hours to stabilize the inoculum. A double dilution of the desired test concentration was added to the microtiter plate (1: 2 dilution) at 0 ° C with 100 μl aliquot of the well. Test compounds were evaluated at 5 1/2 log dilution concentrations (1000-100,000 times). Incubation was carried out for 2 days and 6 days at 5% CO ₂ 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 &lt; 0 &gt; C. The plates were washed 5 times with deionized water, dried and stained with 0.1 ml of 0.4% sulforfodamine B dye (Sigma) dissolved in 1% acetic acid and rinsed four times with 1% acetic acid to remove unbound dye, After drying, the dyed material was extracted with 0.1 ml of a 10 mM Tris base [tris (hydroxymethyl) aminomethane] (pH 10.5) for 5 minutes. The absorbance (OD) of sulforaphodamine B at 492 nm was measured by a computer- Using a microtiter platter reader.

The test sample was considered positive if it showed a growth inhibitory effect of more than 40% at more than one concentration. The results are shown in Table 4 below. In the abbreviation of tumor cell type, NSCL represents non-small cell lung cancer and CNS represents the central nervous system.

Deposit of a substance

The following substances were deposited with the American Type Culture Collection (ATCC), located at 10801 Boulevard, Manassas University, Virginia, USA, 20110-2209.

These depositions were made under the provisions of the Budapest Treaty on the International Recognition of Microorganism Deposit under the Patent Procedure and the Budapest Treaty (the Budapest Treaty). This ensures the maintenance of the survival culture of the deposit for 30 years from the date of deposit. The deposit will be sold under the agreement of the Budapest Treaty from the ATCC in accordance with an agreement between Genentech Inc. and the ATCC, which may be granted at the time of granting the relevant US patent or at the time of publication of the US or foreign patent application, (See 37 CFR §1.14, in particular 886 OG 638) by 35 USC § 122 and the subsequent US Patent and Trademark Attorney's Office regulations (see 37 CFR §1.14) by the US Patent and Trademark Attorneys office to ensure a permanent, To the person who has determined that it is entitled under the terms of this Agreement.

The assignee of the present application has agreed that if the culture of the deposit is killed, lost or destroyed when cultivated under suitable conditions, the notification will promptly replace the substance with another identical substance. The sale of the deposited material shall not be construed as permitting the implementation of the invention in violation of the rights granted by the national government under its patent law.

The foregoing specification is believed to be sufficient to enable those skilled in the art to practice the invention. The present invention is not intended to be limited to the scope of the deposited structure, and any functionally equivalent structure is within the scope of the present invention, as the deposited embodiments are intended as an illustration of certain aspects of the invention. The deposit of a substance here does not mean that the description contained herein is inappropriate to carry out any aspect including the best mode of the present invention and is intended to limit the scope of the claims to the specific description presented in the specification It should not be interpreted. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing detailed description, which is within the scope of the appended claims.

Claims (40)

Useful for inhibiting neoplastic cell growth comprising an effective amount of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof in combination with a pharmaceutically acceptable carrier. Composition. 4. The composition of claim 1, wherein the composition comprises growth inhibitory amounts of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof. The composition of claim 1, comprising a cytotoxic amount of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist thereof. The composition of claim 1, further comprising a growth inhibitor, a cytotoxic agent or a chemotherapeutic agent. A composition useful in tumor therapy in a mammal, comprising a therapeutically effective amount of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof. 6. The composition of claim 5, wherein the tumor is cancer. The composition according to claim 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, renal cancer, rectal cancer, uterine cancer, prostate cancer, lung cancer, bladder cancer, central nervous system cancer, melanoma and leukemia. Comprising the step of exposing an effective amount of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists thereof to tumor cells, How to. 9. The method of claim 8, wherein the agonist is selected from the group consisting of anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti- PRO224, anti- PRO328, anti- PRO301, anti- Anti-PRO356, anti-PRO509, or anti-PRO866 agonist antibody. 9. The method of claim 8, wherein the agonist is a small molecule that mimics the biological activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. 9. The method of claim 8, wherein the step of exposing is performed in a test tube. 9. The method according to claim 8, wherein the step of exposing is performed in vivo. (a) containers and (b) a composition comprising as an active substance a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or an agonist thereof in a container &Lt; / RTI &gt; 14. The article of claim 13, further comprising a label attached to the container, or a package insert contained within the container to refer to the use of the composition for inhibiting neoplastic cell growth. 14. The method of claim 13, wherein the agonist is selected from the group consisting of anti-PRO179, anti-PRO207, anti-PRO320, anti- PRO219, anti- PRO221, anti- PRO224, anti- PRO328, anti- PRO301, anti- Anti-PRO356, anti-PRO509, or anti-PRO866 agonist antibody. 14. The product of claim 13, wherein the agonist is a small molecule that mimics the biological activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. 14. The product of claim 13, wherein the active substance is present in a mammal in an amount effective to treat the tumor. 14. The article of claim 13, wherein said composition further comprises a growth inhibitor, a cytotoxic agent or a chemotherapeutic agent. (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 10), FIG. The amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: Nucleotide sequence identity with an amino acid sequence-encoding nucleic acid sequence identity of at least 80%. (SEQ ID NO: 1), 3 (SEQ ID NO: 6), 5 (SEQ ID NO: 9), 7 A nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 15 (SEQ ID NO: 34), 17 (SEQ ID NO: 42), 19 (SEQ ID NO: 47), 21 Nucleic acid sequence identity to a sequence. (SEQ ID NO: 1), 3 (SEQ ID NO: 6), 5 (SEQ ID NO: 9), 7 The full length coding sequence of the nucleotide sequence shown in SEQ ID NO: 15 (SEQ ID NO: 34), FIG. 17 (SEQ ID NO: 42), FIG. 19 (SEQ ID NO: 47), FIG. Lt; RTI ID = 0.0 &gt; 80% &lt; / RTI &gt; nucleic acid sequence identity with a selected nucleotide sequence in the group. Nucleic acid sequence identity with the full length coding sequence of DNA deposited with ATCC 209281, ATCC 209358, ATCC 209670, ATCC 209384, ATCC 209262, ATCC 209263, ATCC 209438, ATCC 209432, ATCC 209704, ATCC 209620, ATCC 209422 or ATCC 209750 is 80 % Of the isolated nucleic acid. 22. A vector comprising the nucleic acid of any one of claims 19 to 22. 24. The vector of claim 23, operably linked to a regulatory sequence recognized by a host cell transformed with said vector. 24. A host cell comprising the vector of claim 23. 26. The host cell of claim 25, wherein the cell is a CHO cell. 26. The method of claim 25, wherein the cell is a human. Host cells that are E. coli . 26. The host cell of claim 25, wherein the cell is a yeast cell. 26. The host cell of claim 25, wherein the cell is a Baculovirus-infected insect cell. 26. The method of claim 25, wherein the host cells of claim 25 are cultured under conditions suitable for expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides and recovering the polypeptide from the cell culture &Lt; / RTI &gt; (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 10), FIG. The amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. Wherein the amino acid sequence identity to the sequence is at least 80%. (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 10), FIG. The amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. Wherein the polypeptide is at least 80% positive compared to the sequence. The full length coding sequence of the DNA deposited with ATCC 209281, ATCC 209358, ATCC 209670, ATCC 209384, ATCC 209262, ATCC 209263, ATCC 209438, ATCC 209470, ATCC 209704, ATCC 209620, ATCC 209422 or ATCC 209750 Wherein the amino acid sequence identity is at least 80%. 33. A chimeric molecule comprising a polypeptide according to any one of claims 31 to 33 fused to a heterologous amino acid sequence. 35. The chimeric molecule of claim 34, wherein said heterologous amino acid sequence is an epitope tag sequence. 35. The chimeric molecule of claim 34, wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin. 33. An antibody which specifically binds to a polypeptide according to any one of claims 31 to 33. 38. The antibody of claim 37, wherein said antibody is a monoclonal antibody, humanized antibody or single chain antibody. (a) does not have a signal peptide associated therewith and does not have a signal peptide in the sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: (SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: A nucleotide sequence encoding the polypeptide shown in (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 10), FIG. 8, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: (SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: A nucleotide sequence encoding the extracellular domain of the polypeptide shown in (c) No signal peptide involved and no signal peptide exists in the sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, (SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: The nucleic acid sequence identity to the nucleotide sequence encoding the extracellular domain of the polypeptide set forth in SEQ ID NO. (a) does not have a signal peptide associated therewith and does not have a signal peptide in the sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: (SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: , &Lt; / RTI &gt; (SEQ ID NO: 2), 4 (SEQ ID NO: 7), 6 (SEQ ID NO: 15), 10 (SEQ ID NO: 42), FIG. 20, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: , Figure 32 (SEQ ID NO: 83) or Figure 34 (SEQ ID NO: 91), or (c) No signal peptide involved, and no signal peptide exists in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: (SEQ ID NO: 42), FIG. 20, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: , 32 (SEQ ID NO: 83), or FIG. 34 (SEQ ID NO: 91), wherein the amino acid sequence identity to the extracellular domain is 80% or more.