JP2005245451A - Composition and method for inhibiting growth of tumoral cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a composition for inhibiting the growth of tumoral cells, especially to provide an antitumor composition and a method for treating tumor, and to provide a screening method for identifying a growth inhibition composition, such as an antitumor composition. <P>SOLUTION: New polypeptides, and nucleic acid molecules encoding these polypeptides. A vector and a host cell each containing these nucleic acid molecules. A chimera polypeptide molecule containing the polypeptide fused with a different kind of a polypeptide sequence. An antibody bound to the polypeptide. And a method for producing the polypeptide. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

(Field of Invention)
The present invention relates to methods and compositions for inhibiting neoplastic cell growth. In particular, the present invention relates to anti-tumor compositions and methods for treating tumors. The invention further relates to screening methods for identifying growth inhibitory, eg, anti-tumor compounds.

(Background of the Invention)
Malignant tumors (cancers) are the second leading cause of death after heart disease in the United States (Boring et al., CA Cancel J. Clin., 43: 7 [1993]).
Cancer is derived from normal tissue and forms an abnormal or neoplastic cell that forms a tumor entity, invasion of adjacent tissue by these neoplastic tumor cells, and ultimately through the blood and lymphatic system Characterized by the generation of malignant cells that diffuse (metastasize) to local lymph nodes and distant sites. In a cancerous state, cells proliferate under conditions in which normal cells do not grow. Cancer itself manifests in a wide variety of forms characterized by different degrees of invasiveness and aggressiveness.

Despite recent developments 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 compounds that can inhibit the growth of tumor cells such as cancer cells.

(Summary of Invention)
A. Embodiments The present invention relates to methods and compositions for inhibiting tumor cell growth. More particularly, the present invention treats tumors, including breast cancer, prostate cancer, rectal cancer, lung cancer, ovarian cancer, kidney cancer and CNS cancer, leukemia, myeloma, etc. in mammalian patients, preferably humans. Relates to a method and a composition.
In one aspect, the present invention provides a pharmaceutical comprising a PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or an agonist thereof, as defined herein. The present invention relates to a composition of a substance useful for inhibiting neoplastic cell growth, which is mixed with an acceptable carrier. In a preferred embodiment, the composition of matter comprises a growth inhibitory amount of PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or an agonist thereof. Contains. In other preferred embodiments, the composition comprises a cytotoxic amount of PRO179, PRO297, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or an agonist thereof. Including. In some cases, the composition of matter may contain one or more growth inhibitory and / or cytotoxic and / or chemotherapeutic agents.
In a further aspect, the invention provides an effective amount of a PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or agonist thereof, as defined herein. Comprising a composition of matter useful for treating a tumor in a mammal. This tumor is preferably cancer.

In another aspect, the invention provides the tumor cell as defined herein with a PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, Or relates to a method of inhibiting tumor cell growth comprising exposing to an effective amount of an agonist thereof. In particular embodiments, the agonist is anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO362, -PRO356, anti-PRO509, or anti-PRO866 agonist antibody. In other embodiments, the agonist is a small molecule that mimics the biological activity of PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. . This method is performed in vitro or in vivo.
In yet a further embodiment, the present invention provides:
(A) a container; and (b) an article of manufacture comprising an active agent contained in the container, wherein the composition is useful for inhibiting neoplastic cell growth, eg, tumor cell growth. And the active agent in the composition is PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or an agonist thereof as defined herein; And (c) a container attached to the front container, or a packaging insert contained in the front container, the PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, Reference is made to the use of RO509, or PRO866 polypeptide, or an agonist thereof in the inhibition of growth of neoplastic cells, wherein the agonist is PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, It consists of an article of manufacture that would be an antibody that binds to PRO509 or PRO866 polypeptide. In particular embodiments, the agonist is anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO362, -PRO356, anti-PRO509, or anti-PRO866 agonist antibody. In other embodiments, the agonist is a small molecule that mimics the biological activity of PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. . Similar products containing PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide as defined herein in an amount effective for the treatment of tumors Included within the scope of the present invention. In addition, further growth inhibitors, cytotoxic agents, including PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides or agonists thereof as defined herein Alternatively, articles of manufacture that additionally contain chemotherapeutic agents are within the scope of the present invention.

B. Further Embodiments In other embodiments of the invention, the invention provides nucleotide sequences encoding PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides. An isolated nucleic acid comprising is provided.
In one aspect, the isolated nucleic acid molecule is (a) a PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide DNA molecule, wherein A full-length amino acid sequence disclosed herein, an amino acid sequence lacking a signal peptide disclosed herein, an extracellular domain of a transmembrane protein disclosed herein, with or without a signal peptide, or any of those disclosed herein Having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, with a fragment of a specifically defined full-length amino acid sequence, More preferably At least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% More preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity. Even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferred Have at least about 93% sequence identity, even more preferably less Also has about 94% sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity Nucleotide sequences having sequence identity, even more preferably at least about 98% sequence identity, even more preferably at least about 99% sequence identity, or (b) complement of the DNA molecule of (a) Have a body.

  In other aspects, the isolated nucleic acid molecule is (a) a PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide disclosed herein. A cDNA encoding the full-length sequence of PRO179, PRO207, PRO320, PRO219, or a sequence lacking the signal sequence of PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide disclosed herein, PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, A sequence comprising or excluding a signal sequence in the extracellular domain of a transmembrane protein of RO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or a fragment of any specifically defined full-length amino acid sequence disclosed herein; At least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity Even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more Preferably has at least about 87% sequence identity Even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably at least about 90% sequence identity, even more preferably Have at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity Have sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity Even more preferably having at least about 98% sequence identity. More preferably a nucleotide sequence having at least about 99% sequence identity, or the complement of the DNA molecule of (b) (a).

In a further aspect, the present invention provides (a) at least about 80% sequence identity with a DNA molecule encoding the same mature polypeptide encoded by any human cDNA deposited with the ATCC disclosed herein. Preferably having at least about 81% sequence identity, more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably Have at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity Having sequence identity, even more preferably having at least about 88% sequence identity, More preferably at least about 89% sequence identity, even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least About 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, even more preferably at least about 95% sequence Having identity, even more preferably having at least about 96% sequence identity, even more preferably having at least about 97% sequence identity, even more preferably having at least about 98% sequence identity, Even more preferably, a nucleo having at least about 99% sequence identity De sequence, or (b) an isolated nucleic acid molecule comprising the complement of the DNA molecule of (a).
In yet another aspect, the invention relates to PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides lacking or inactivating the transmembrane domain. Provided are isolated nucleic acid molecules comprising encoding nucleotide sequences, or the complements of such nucleotide sequences, and the transmembrane domains of these polypeptides are disclosed herein. Accordingly, soluble domains of PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides disclosed herein are contemplated.

  Other embodiments include, for example, anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO362, PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, optionally encoding a polypeptide comprising a binding site as a binding site for a PRO356, anti-PRO509, or anti-PRO866 antibody, or as an antisense oligonucleotide probe PRO179, PR that can find use as hybridization probes for coding fragments of PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides Indicates an O207, PRO320, PRO219, or a fragment of a sequence encoding a PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or a complement thereof. Such nucleic acid fragments are generally at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferably at least about 40 nucleotides in length, and even more preferably at least about 50 nucleotides in length. Nucleotide length, even more preferably at least about 60 nucleotides in length, even more preferably at least about 70 nucleotides in length, even more preferably at least about 80 nucleotides in length, even more preferably at least about 90 nucleotides in length A length, even more preferably a length of at least about 100 nucleotides, even more preferably a length of at least about 110 nucleotides, even more preferably a length of at least about 120 nucleotides, even more preferably at least about 130 nu. The length of the leotide, even more preferably at least about 140 nucleotides in length, even more preferably at least about 150 nucleotides in length, even more preferably at least about 160 nucleotides in length, even more preferably at least about 170 nucleotides in length. A length, even more preferably a length of at least about 180 nucleotides, even more preferably a length of at least about 190 nucleotides, even more preferably a length of at least about 200 nucleotides, even more preferably a length of at least about 250 nucleotides Even more preferably at least about 300 nucleotides in length, even more preferably at least about 350 nucleotides in length, even more preferably at least about 400 nucleotides in length, even more Preferably at least about 450 nucleotides in length, even more preferably at least about 500 nucleotides in length, even more preferably at least about 600 nucleotides in length, even more preferably at least about 700 nucleotides in length, even more preferably Is at least about 800 nucleotides in length, even more preferably at least about 900 nucleotides in length, even more preferably at least about 1000 nucleotides in length, and the term “about” in this context refers to the reference nucleotide sequence Means a reference length of plus or minus 10% of the length. Using any of the well-known sequence alignment programs, the PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide encoded nucleotide sequences and other Align nucleotide sequences to determine which PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide polypeptide-encoding nucleotide sequences are new PRO179, PRO207, PRO320, PRO219, or PRO221 by routine methods PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 novel fragments of a polypeptide-encoding nucleotide sequences can be determined are known. All such PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide coding sequences are discussed herein. Also contemplated are polypeptide fragments encoded by nucleotide fragments of PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, Preferably 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 PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO30, comprising a binding site for an anti-PRO866 antibody 1, a nucleotide fragment of a PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide.

In yet another embodiment, the invention relates to PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, encoded by any of the nucleic acid sequences identified above. PRO509, or PRO866 polypeptides are provided.
In one aspect, the invention provides a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal sequence as disclosed herein, a signal sequence in the extracellular domain of a transmembrane protein as disclosed herein. PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, with or without, or any of the well-defined full-length amino acid sequence fragments as disclosed herein , PRO356, PRO509, or PRO866 polypeptides having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity. Even more preferred Having at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity Having sequence identity, even more preferably having at least about 87% sequence identity, even more preferably having at least about 88% sequence identity, even more preferably having at least about 89% sequence identity Even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably Have at least about 93% sequence identity, even more preferably low Both about 94% sequence identity, even more preferably at least about 95% sequence identity, even more preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity An isolated PRO179, PRO207, PRO320, PRO219 comprising an amino acid sequence having sequence identity, even more preferably having at least about 98% sequence identity, and even more preferably having at least about 99% sequence identity Or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide.

  In a further aspect, the present invention has at least about 80% sequence identity, preferably at least about 81, to the amino acid sequence encoded herein by any human cDNA deposited with the ATCC. % Sequence identity, more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity. Even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferred Have at least about 88% sequence identity, even more preferably at least about 9% sequence identity, even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity Even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, even more preferably at least about 95% sequence identity, More preferably at least about 96% sequence identity, even more preferably at least about 97% sequence identity, even more preferably at least about 98% sequence identity, even more preferably at least about about Isolated PRO179 comprising an amino acid sequence with 99% sequence identity PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or to PRO866 polypeptide.

  In a further aspect, the invention provides a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal sequence as disclosed herein, a signal sequence in the extracellular domain of a transmembrane protein as disclosed herein. PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, with or without, or any of the well-defined full-length amino acid sequence fragments as disclosed herein , PRO356, PRO509, or PRO866 polypeptide, at least about 80% positive score, preferably at least about 81% positive score, more preferably at least about 82% positive score, even more preferred A positive score of at least about 83%, even more preferably at least about 84% positive score, even more preferably at least about 85% positive score, even more preferably at least about 86% positive score, Even more preferably at least about 87% positive score, even more preferably at least about 88% positive score, even more preferably at least about 89% positive score, even more preferably at least about 90% positive score Score, even more preferably at least about 91% positive score, even more preferably at least about 92% positive score, even more preferably at least about 93% positive score, even more preferably A positive score of at least about 94%, even more preferably at least about 95% positive score, even more preferably at least about 96% positive score, even more preferably at least about 97% positive score, More preferably an isolated PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301 having an amino acid sequence of at least about 98% positive score, and even more preferably at least about 99% positive score. , PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide.

In certain aspects, the invention relates to isolated PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or lacking an N-terminal signal sequence and / or the initiation methionine. PRO866 polypeptides are provided, which are further encoded by nucleotide sequences that encode the amino acid sequences set forth above. Processes for producing this same polypeptide are further illustrated herein, which include PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or Culturing a host cell comprising a vector comprising a suitable nucleic acid molecule under conditions suitable for expression of the PRO866 polypeptide, and PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, Including recovery of the PRO356, PRO509, or PRO866 polypeptide from the culture medium.
In another aspect, the invention relates to isolated PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, transmembrane domain deficient or transmembrane domain inactive, Alternatively, PRO866 polypeptides are provided. Processes for producing this same polypeptide are further illustrated herein, which include PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. Culturing a host cell comprising a vector containing a suitable nucleic acid molecule under conditions suitable for expression of the polypeptide, and PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, Including recovery of the PRO356, PRO509, or PRO866 polypeptide from the culture medium.

In yet another embodiment, the invention relates to an agonist of a native PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide as defined herein. In certain embodiments, the agonist is anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO362, -PRO356, anti-PRO509, or anti-PRO866 agonist antibody, or small molecule.
In a still further embodiment, the present invention relates to contacting the PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide with a candidate molecule, and said PRO179 , PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, or monitoring the biological activity mediated by PRO866 polypeptide, PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO36 , PRO356, PRO509, or to a method of identifying agonists to PRO866 polypeptides. Preferably, the PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides are native PRO179, PRO207, PRO320, PRO219, or PRO221, PRO3, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide.
And in a further embodiment, the invention relates to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or PRO179, PRO207, disclosed herein. PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO356, PRO509, or an agonist of PRO866 polypeptide, or in combination with a container, anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti -PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, It relates to a composition comprising an anti-PRO356, anti-PRO509, or anti-PRO866 agonist antibody. Optionally, the container is pharmaceutically acceptable.

Other embodiments of the invention include PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides, agonists thereof, or anti-PRO179, anti- Responds to PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, or anti-PRO866 agonist antibodies PRO179, PRO207, PRO320, PRO219, or PRO221, PRO224, PRO328, PRO301, PRO526, PR for adjustment of drugs useful in the treatment of sexual conditions O362, PRO356, PRO509, or PRO866 polypeptide, or agonists thereof as indicated above, or 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 agonist antibodies.
In another embodiment of the invention, a vector comprising DNA encoding any of the polypeptides set forth above is provided. Host cells comprising any of the vectors are also provided. By way of example, the host cell is a CHO cell, E. coli, yeast, or baculovirus infected insect cell. Methods for producing any of the polypeptides set forth herein are further provided, including culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the culture medium.

In another embodiment, the invention provides a chimeric molecule comprising any of the polypeptides shown herein fused to a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules include any of the polypeptides shown herein fused to an epitope tag sequence or an Fc region of an immunoglobulin.
In still other embodiments, the present invention provides antibodies that specifically bind to the polypeptides set forth above and below. Optionally, the antibody is a monoclonal antibody, a humanized antibody, an antibody fragment, or a single chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful as isolation of genomic or cDNA nucleotide sequences, or as antisense probes, which probes are derived from any of the nucleotide sequences shown above or below. can get.

(Detailed Description of the Invention)
“PRO179”, “PRO207”, “PRO320”, “PRO219”, “PRO221”, “PRO224”, “PRO328”, “PRO301”, “PRO526”, “PRO362”, “PRO356”, The term “PRO509” or “PRO866” polypeptide or protein refers to the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides, and further to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO 09 encompasses and PRO866 polypeptide variants (wherein, further defined) a. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can be isolated from a variety of sources, such as human tissue and other sources, and recombinant methods And / or adjustable by synthesis

  “Native Sequence 179”, “Natural Sequence 207”, “Natural Sequence 320”, “Natural Sequence 219”, “Native Sequence 221”, “Native Sequence 224”, “Native Sequence 328”, “Native Sequence 301”, “Natural Sequence 301” The “sequence 526”, “native sequence 362”, “native sequence 356”, “native sequence 509”, or “native sequence 866” is derived from naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526. , PRO362, PRO356, PRO509, and PRO866 polypeptides having the same amino acid sequence. Such native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide can be isolated from nature, or recombinant / or synthetic. Can be produced by. The term “native sequence” PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide is a naturally cleaved or selected type (eg, extracellular domain). Sequence), naturally occurring variants (eg, alternative splice forms), and naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides In particular. In one embodiment of the invention, the native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides are each shown in FIG. 2 (SEQ ID NO: 2). ), FIG. 4 (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 10), FIG. 8 (SEQ ID NO: 15), FIG. 10 (SEQ ID NO: 20), FIG. 12 (SEQ ID NO: 25), FIG. SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. 22 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 60) Or the mature or full-length native sequences PRO179, PRO207, PRO320, PRO219 shown in FIG. 26 (SEQ ID NO: 62) PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. Furthermore, FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 10), FIG. 8 (SEQ ID NO: 15), FIG. 10 (SEQ ID NO: 20), FIG. 12 (SEQ ID NO: 25), FIG. 14 (SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. 55), FIG. 24 (SEQ ID NO: 60), or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, disclosed in FIG. 26 (SEQ ID NO: 62). And PRO866 polypeptides start with the methionine residue designated as the first amino acid among them, while FIG. 2 (SEQ ID NO: 2), FIG. No. 7), FIG. 6 (SEQ ID NO: 10), FIG. 8 (SEQ ID NO: 15), FIG. 10 (SEQ ID NO: 20), FIG. 12 (SEQ ID NO: 25), FIG. 14 (SEQ ID NO: 30), FIG. 16 (SEQ ID NO: 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. 22 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 60), or FIG. Number: 62) Other methionine residues located upstream or downstream of the first amino acid are PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. It is conceivable and possible that it could be used as the starting amino acid of a polypeptide.

  The “extracellular domain” or “ECD” of the polypeptides disclosed herein corresponds to a form that has essentially no transmembrane and cytoplasmic domains. Usually, the polypeptide ECD has less than about 1% of such transmembrane and / or cytoplasmic domains, preferably less than 0.5% of such domains. Any transmembrane domain identified as that of the polypeptide of the present invention has been confirmed to conform to the criteria routinely used to identify that type of hydrophobic domain. The exact transmembrane domain boundary may vary, but it is most likely within as little as about 5 on either side of the domain, as initially identified and shown in the accompanying drawings Limited to amino acids. Thus, in one embodiment of the invention, the extracellular domain of the polypeptide of the invention comprises amino acids 1 to X of the mature amino acid sequence, where X is the extracellular domain / transmembrane domain. Corresponds to any of the 5 amino acids on either side of the boundary.

  The approximate locations of the “signal peptides” of the various PRO polypeptides disclosed herein are shown in the accompanying figures. However, although the C-terminal boundary of the signal sequence can vary, the most likely is no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as first identified herein. Here, the C-terminal boundary of the signal peptide can be identified according to criteria for identifying amino acid sequences routinely used in the art (eg, Nielsen). Et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). Furthermore, in some cases, it is also observed that cleavage of the signal peptide from the secreted polypeptide is not completely uniform, resulting in one or more secreted species. These mature polypeptides, and polynucleotides encoding them, which are cleaved within less than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as defined herein, are contemplated in the present invention. The

“PRO179 variant polypeptide” means an active PRO179 polypeptide (other than the native sequence PRO179 polypeptide) as defined below, (a) of the PRO179 polypeptide shown in FIG. 2 (SEQ ID NO: 2). Residue 1 or about 17-460, (b) X of PRO179 shown in FIG. 2 (SEQ ID NO: 2) when X is any amino acid residue 12-21 of FIG. 2 (SEQ ID NO: 2) 460, or at least about 80% amino acid sequence identity to the amino acid sequence of a specific derivative fragment of the amino acid sequence shown in (c).
“PRO207 variant polypeptide” means an active PRO207 polypeptide (other than the native sequence PRO207 polypeptide) as defined below, (a) of the PRO207 polypeptide shown in FIG. 4 (SEQ ID NO: 7). Residue 1 or about 41-249, or (b) X of PRO207 shown in FIG. 4 (SEQ ID NO: 7) when X is any amino acid residue from 36-45 of FIG. 4 (SEQ ID NO: 7) ˜249, or (c) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 4 (SEQ ID NO: 7).
“PRO320 variant polypeptide” means an active PRO320 polypeptide (other than the native sequence PRO320 polypeptide) as defined below, (a) of the PRO320 polypeptide shown in FIG. 6 (SEQ ID NO: 10). X of PRO207 shown in FIG. 6 (SEQ ID NO: 10) when residue 1 or about 22-338, or (b) X is any amino acid residue 17-26 of FIG. 6 (SEQ ID NO: 10) ˜338, or (c) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 6 (SEQ ID NO: 10).

“PRO219 variant polypeptide” means an active PRO219 polypeptide (other than the native sequence PRO219 polypeptide) as defined below, (a) of the PRO219 polypeptide shown in FIG. 8 (SEQ ID NO: 15). Residue 1 or about 24 to 1005, or (b) X of PRO219 shown in FIG. 8 (SEQ ID NO: 15) when X is any amino acid residue from 19 to 28 of FIG. 8 (SEQ ID NO: 15) Or (c) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 8 (SEQ ID NO: 15).
“PRO221 variant polypeptide” means an active PRO221 polypeptide (other than the native sequence PRO221 polypeptide) as defined below, (a) of the PRO221 polypeptide shown in FIG. 10 (SEQ ID NO: 20). PRO221 X shown in FIG. 10 (SEQ ID NO: 20) when residue 1 or about 34-259, or (b) X is any amino acid residue 29-38 of FIG. 10 (SEQ ID NO: 20) ~ 259, (c) 1 or about 34-X of PRO221 in Figure 10 (SEQ ID NO: 20), where X is any amino acid residue from 199 to 208 in Figure 10 (SEQ ID NO: 20), or ( d) At least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 10 (SEQ ID NO: 20).
“PRO224 variant polypeptide” means an active PRO224 polypeptide (other than the native sequence PRO224 polypeptide) as defined below, (a) of the PRO224 polypeptide shown in FIG. 12 (SEQ ID NO: 25). Residue 1 or about 1-31 or (b) X of PRO224 shown in FIG. 12 (SEQ ID NO: 25) when X is any amino acid residue of 26-35 of FIG. 12 (SEQ ID NO: 25) ~ 282, (c) 1 or about 31-X of PRO224 in Figure 12 (SEQ ID NO: 25) when X is any amino acid residue from 226 to 235 of Figure 12 (SEQ ID NO: 25), or ( d) At least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25).

“PRO328 variant polypeptide” means an active PRO328 polypeptide (other than the native sequence PRO328 polypeptide) as defined below, (a) of the PRO328 polypeptide shown in FIG. 14 (SEQ ID NO: 30). Residue 1 or about 23-463, or (b) X of PRO221 shown in FIG. 14 (SEQ ID NO: 30) when X is any amino acid residue 18-27 of FIG. 14 (SEQ ID NO: 30) ~ 463, (c) or (d) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in Figure 14 (SEQ ID NO: 30).
“PRO301 variant polypeptide” means an active PRO301 polypeptide (other than the native sequence PRO301 polypeptide) as defined below, (a) of the PRO301 polypeptide shown in FIG. 16 (SEQ ID NO: 35). Residue 1 or about 28-229, or (b) X of PRO301 shown in FIG. 16 (SEQ ID NO: 35) when X is any amino acid residue 23-32 of FIG. 16 (SEQ ID NO: 35) 299, (c) 1 or about 28-X of PRO301 in FIG. 16 (SEQ ID NO: 35) when X is any amino acid residue from 230 to 239 in FIG. 16 (SEQ ID NO: 35), or ( d) At least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 16 (SEQ ID NO: 35).
“PRO526 variant polypeptide” means an active PRO526 polypeptide (other than the native sequence PRO526 polypeptide) as defined below, (a) of the PRO526 polypeptide shown in FIG. 18 (SEQ ID NO: 43). Residue 1 or about 27-473, or (b) X of PRO526 shown in FIG. 18 (SEQ ID NO: 43) when X is any amino acid residue 22-31 of FIG. 18 (SEQ ID NO: 43) ˜473, or (d) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 18 (SEQ ID NO: 43).

“PRO362 variant polypeptide” means an active PRO362 polypeptide (other than the native sequence PRO362 polypeptide) as defined below, (a) of the PRO362 polypeptide shown in FIG. 20 (SEQ ID NO: 48). Residue 1 or about 20-321, or (b) X of PRO362 shown in FIG. 20 (SEQ ID NO: 48) when X is any amino acid residue 15-24 of FIG. 20 (SEQ ID NO: 48) ~ 321, (c) 1 or about 20-X of PRO362 in Figure 20 (SEQ ID NO: 48) where X is any amino acid residue from 276 to 285 in Figure 20 (SEQ ID NO: 48), or ( c) At least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 20 (SEQ ID NO: 48).
“PRO356 variant polypeptide” means an active PRO356 polypeptide (other than the native sequence PRO356 polypeptide) as defined below, (a) of the PRO356 polypeptide shown in FIG. 22 (SEQ ID NO: 55). Residue 1 or about 27-346, or (b) X of PRO362 shown in FIG. 22 (SEQ ID NO: 55) when X is any amino acid residue 22-31 of FIG. 22 (SEQ ID NO: 55) ˜346, or (c) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in FIG. 22 (SEQ ID NO: 55).
“PRO509 variant polypeptide” means an active PRO509 polypeptide (other than the native sequence PRO509 polypeptide) as defined below, (a) of the PRO509 polypeptide shown in FIG. 24 (SEQ ID NO: 60). Residue 1 or about 37-283, (b) X of PRO509 shown in FIG. 24 (SEQ ID NO: 60) when X is any amino acid residue 32-41 of FIG. 24 (SEQ ID NO: 60) 283, (c) 1 or about 37 to X of FIG. 24 (SEQ ID NO: 60), or X (d) when X is any amino acid from amino acid 200 to amino acid 209 of FIG. 24 (SEQ ID NO: 60) It has at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in 24 (SEQ ID NO: 60).
“PRO866 variant polypeptide” means an active PRO866 polypeptide (other than the native sequence PRO866 polypeptide) as defined below, (a) of the PRO866 polypeptide shown in FIG. 26 (SEQ ID NO: 62). Residue 1 or about 27 to 331, or (b) X of PRO866 as shown in FIG. 26 (SEQ ID NO: 62) when X is any amino acid residue from 22 to 31 of FIG. 26 (SEQ ID NO: 62) ~ 331, or (c) having at least about 80% amino acid sequence identity to the amino acid sequence of the specific derivative fragment of the amino acid sequence shown in Figure 26 (SEQ ID NO: 62).
These PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 variants, for example, in the N- and / or C-terminus and in one or more internal domains It includes PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides with one or more amino acid residues added or deleted.

Typically, the PRO179 variant is (a) residues 1 or about 17-460 of the PRO179 polypeptide shown in FIG. 2 (SEQ ID NO: 2), and (b) X is 12-21 of FIG. 2 (SEQ ID NO: 2). PRO-179 X-460 as shown in FIG. 2 (SEQ ID NO: 2) when it is any amino acid residue, or (c) a specific derivative fragment of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), and at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity; Preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity More preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence. Identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98%. Amino acid sequence identity, and more preferably At least about 99% amino acid sequence identity.
Typically, the PRO207 variant is (a) residues 1 or about 41-249 of the PRO207 polypeptide shown in FIG. 4 (SEQ ID NO: 7), and (b) X is 36-45 of FIG. 4 (SEQ ID NO: 7). 4 to 249 of PRO207 shown in FIG. 4 (SEQ ID NO: 7) in the case of any amino acid residue, or (c) a specific derivative fragment of the amino acid sequence shown in FIG. 4 (SEQ ID NO: 7), and at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity; Preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity More preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence. Identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98%. Amino acid sequence identity, and more preferably At least about 99% amino acid sequence identity.

Typically, the PRO320 variant is (a) residues 1 or about 22-338 of the PRO320 polypeptide shown in FIG. 6 (SEQ ID NO: 10), and (b) X is 17-26 of FIG. 6 (SEQ ID NO: 10). X-338 of PRO320 shown in FIG. 6 (SEQ ID NO: 10) when it is any amino acid residue, or (c) a specific derivative fragment of the amino acid sequence shown in FIG. 6 (SEQ ID NO: 10), and at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity More preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid Sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% Amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about about 98% amino acid sequence identity, and more Mashiku has at least about 99% amino acid sequence identity.
Typically, the PRO219 variant is (a) residues 1 or about 24 to 1005 of the PRO219 polypeptide shown in FIG. 8 (SEQ ID NO: 15), and (b) X is 19 to 28 of FIG. 8 (SEQ ID NO: 15). At least about a specific derivative fragment of PRO219 X-10005 as shown in FIG. 8 (SEQ ID NO: 15), or (c) the amino acid sequence shown in FIG. 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity Identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90%. Amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94 % Amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least About 98% amino acid sequence identity, and Ri preferably at least about 99% amino acid sequence identity.

Typically, the PRO221 variant is (a) residues 1 or about 34-259 of the PRO221 polypeptide shown in FIG. 10 (SEQ ID NO: 20), and (b) X is 29-38 of FIG. 10 (SEQ ID NO: 20). When it is an arbitrary amino acid residue, X to 259 of PRO221 shown in FIG. 10 (SEQ ID NO: 20), (c) X is an arbitrary amino acid residue of 199 to 208 in FIG. 10 (SEQ ID NO: 20) 1 or about 34-X of PRO221 shown in FIG. 10 (SEQ ID NO: 20), or (d) a specific derivative fragment of the amino acid sequence shown in FIG. 10 (SEQ ID NO: 20) and at least about 80% amino acids Sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more Preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity. More preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid Sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% Amino acid sequence identity, more preferably at least About 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, and more preferably at least about 99% amino acid sequence identity. Have.
Typically, the PRO224 variant is (a) residues 1 or about 31-282 of the PRO224 polypeptide shown in FIG. 12 (SEQ ID NO: 25), and (b) X is 26-35 of FIG. 12 (SEQ ID NO: 25). In the case of any amino acid residue, X-282 of PRO221 shown in FIG. 12 (SEQ ID NO: 25), (c) X is any amino acid residue of 226-235 in FIG. 12 (SEQ ID NO: 25) 1 or about 31-X of PRO224 shown in FIG. 12 (SEQ ID NO: 25), or (d) a specific derivative fragment of the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25) and at least about 80% amino acids Sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more Preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about 87% amino acid sequence identity. More preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91% amino acid Sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95% Amino acid sequence identity, more preferably at least About 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, and more preferably at least about 99% amino acid sequence identity. Have.

Typically, the PRO328 variant is (a) residues 1 or about 23-463 of the PRO328 polypeptide shown in FIG. 14 (SEQ ID NO: 30), (b) X is 18-27 of FIG. 14 (SEQ ID NO: 30). X-463 of PRO328 shown in FIG. 14 (SEQ ID NO: 30), or (c) a specific derivative fragment of the amino acid sequence shown in FIG. 14 (SEQ ID NO: 30), which is any amino acid residue, and at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence Sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% Amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably At least about 98% amino acid sequence identity, and , More preferably at least about 99% amino acid sequence identity.
Typically, the PRO301 variant is (a) residues 1 or about 28-299 of the PRO301 polypeptide shown in FIG. 16 (SEQ ID NO: 35), and (b) X is 23-32 of FIG. 16 (SEQ ID NO: 35). X-299 of PRO301 shown in FIG. 16 (SEQ ID NO: 35) in the case of any amino acid residue, (c) FIG. 16 (SEQ ID NO: 35) X is 230-239 in FIG. 16 (SEQ ID NO: 35) Or about 28 to X of PRO301 shown in FIG. 16 (SEQ ID NO: 35), or (d) a specific derivative of the amino acid sequence shown in FIG. 16 (SEQ ID NO: 35). And at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83%. Mino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably At least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, and more Preferably at least about 95% amino acid sequence identity More preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, and more preferably at least about 99% amino acid sequence Has sequence identity.

Usually, the PRO526 variant is (a) residues 1 or about 27 to 473 of the PRO526 polypeptide shown in FIG. 18 (SEQ ID NO: 43), and (b) X is 22 to 31 in FIG. 18 (SEQ ID NO: 43). X-473 of PRO526 as shown in FIG. 18 (SEQ ID NO: 43) in the case of any amino acid residue, or (d) a specific derivative fragment of the amino acid sequence shown in FIG. 18 (SEQ ID NO: 43), and at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence Sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% Amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably At least about 98% amino acid sequence identity, and , More preferably at least about 99% amino acid sequence identity.
Typically, the PRO362 variant is (a) residues 1 or about 20-321 of the PRO362 polypeptide shown in FIG. 20 (SEQ ID NO: 48), and (b) X is 15-24 of FIG. 20 (SEQ ID NO: 48). X-321 of PRO362 shown in FIG. 20 (SEQ ID NO: 48) in the case of any amino acid residue, (c) FIG. 20 (SEQ ID NO: 48) X is 276-285 in FIG. 20 (SEQ ID NO: 48). 1 or about 20 to X of PRO362 shown in FIG. 20 (SEQ ID NO: 48), or (d) a specific derivative fragment of the amino acid sequence shown in FIG. 20 (SEQ ID NO: 48) And at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83%. Mino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably At least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, and more Preferably at least about 95% amino acid sequence identity More preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, and more preferably at least about 99% amino acid sequence Has sequence identity.

Typically, the PRO356 variant is (a) residues 1 or about 27-346 of the PRO356 polypeptide shown in FIG. 22 (SEQ ID NO: 55), and (b) X is 22-31 of FIG. 22 (SEQ ID NO: 55). X-346 of PRO356 shown in FIG. 22 (SEQ ID NO: 55) when it is an arbitrary amino acid residue, or (c) a specific derivative fragment of the amino acid sequence shown in FIG. 22 (SEQ ID NO: 55), and at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least About 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence Sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% Amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about about 94% amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably At least about 98% amino acid sequence identity, and , More preferably at least about 99% amino acid sequence identity.
Usually, the PRO509 variant is (a) residues 1 or about 37-283 of the PRO509 polypeptide shown in FIG. 24 (SEQ ID NO: 60), and (b) X is 32-41 of FIG. 24 (SEQ ID NO: 60). X-283 of PRO509 shown in FIG. 24 (SEQ ID NO: 60) in the case of any amino acid residue, (c) FIG. 24 (SEQ ID NO: 60) X is 200-209 in FIG. 24 (SEQ ID NO: 60) 1 or about 34 to X of PRO507 shown in FIG. 24 (SEQ ID NO: 60), or (d) a specific derivative fragment of the amino acid sequence shown in FIG. 24 (SEQ ID NO: 60) And at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83%. Mino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity, more preferably at least about about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably At least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, and more Preferably at least about 95% amino acid sequence identity More preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, and more preferably at least about 99% amino acid sequence Has sequence identity.

Typically, the PRO866 variant is (a) residues 1 or about 27-331 of the PRO866 polypeptide shown in FIG. 26 (SEQ ID NO: 62), and (b) X is 22-31 of FIG. 26 (SEQ ID NO: 62). At least about 80 from the X-331 of PRO866 shown in FIG. 26 (SEQ ID NO: 62), and (c) the amino acid sequence shown in FIG. % Amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% amino acid sequence identity Identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about 90%. Amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94 % Amino acid sequence identity, more preferably at least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least About 98% amino acid sequence identity, and Ri preferably at least about 99% amino acid sequence identity.
PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 mutant peptides are native PRO179, PRO207, PRO320, PRO219, PRO221, PRO226, PRO226, PRO226 Does not include PRO362, PRO356, PRO509, and PRO866 peptide sequences. Typically, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variant polypeptides are at least about 10 amino acids long, more at least about 20 amino acids long, More are at least about 30 amino acids long, more are at least about 40 amino acids long, more are at least about 50 amino acids long, more are at least about 60 amino acids long, more are at least about 70 amino acids long, more are at least about at least about 80 amino acids long, more at least about 90 amino acids long, more at least about 100 amino acids long, more at least about 150 amino acids long, more at least about 200 amino acids long, Ri Many least about 250 amino acids in length, more often is at least about 300 amino acids in length, or more.

As shown below, Table 1 provides the complete source code for the ALIGN-2 sequence comparison computer program. This source code is routinely compiled for use on the UNIX operating system and provides an ALIGN-2 sequence comparison computer program.
In addition, Tables 2A-2D use the following method to determine% amino acid sequence identity (Table 2A-2B) and% nucleic acid sequence identity (Table 2C-2D) using the ALIGN-2 sequence comparison computer program. Shows hypothetical examples for use, where “PRO” is the target hypothetical PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 poly The amino acid sequence of the peptide, “comparison protein” is the amino acid sequence of the polypeptide to be compared with the target “PRO” polypeptide, and “PRO-DNA” is the target hypothetical PRO179-, PRO207-, PRO320-, PRO219- , PRO221-, PRO224 , PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509-, or PRO866-encoding nucleic acid sequence, "Comparative DNA" is the nucleotide of the nucleic acid molecule to which the "PRO-DNA" nucleic acid molecule of interest is compared The sequences “X”, “Y” and “Z” each represent a different hypothetical amino acid residue, and “N”, “L” and “V” each represent a different hypothetical nucleotide.

  “Percent amino acid sequence identity” with respect to the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide sequences defined herein is the largest sequence alignment. Introducing gaps if necessary to obtain percent sequence identity and not considering any conservative substitutions as part of sequence identity, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, Defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues of a PRO362, PRO356, PRO509, or PRO866 polypeptide sequenceAlignments for the purpose of determining percent amino acid sequence identity can be obtained by various methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. This can be achieved by using available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are derived from the sequence comparison program ALIGN-2, whose complete source code for the ALIGN-2 program is provided in Table 1, as described below. Is obtained. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code shown in Table 1 was submitted to the US Copyright Office, Washington DC, 20559 with user documentation and under US Copyright Registration Number TXU510087 It is registered. ALIGN-2 is publicly available through Genentech, South San Francisco, California, and may be compiled from the source code given in Table 1. The ALIGN-2 program is compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the% amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (or with or against a given amino acid sequence B or A given amino acid sequence A that has or contains a certain% amino acid sequence identity) is calculated as follows:
100 times the fraction X / Y, where X is the number of amino acid residues recorded as the same pair by alignment of A and B of the sequence alignment program ALIGN-2, and Y is the total amino acid residue of B Is a number. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, then the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. As an example of calculating% amino acid sequence identity using this method, Tables 2A-2B calculate% amino acid sequence identity to the amino acid sequence designated “PRO” of the amino acid sequence designated “Comparative Protein”. The method is shown.

  Unless otherwise indicated, all% amino acid sequence identity values herein are obtained using the ALIGN-2 sequence comparison computer program as described above. However,% amino acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://ww.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, all of which are set to initial values, eg unmask = allowable, chain = all, predicted occurrence = 10, minimum low composite length = 15/5 Multipath e-value = 0.01, multipath constant = 25, final gap alignment dropoff = 25, and scoring matrix = BLOSUM62.

In situations where NCBI-BLAST2 is used for amino acid sequence comparison,% amino acid sequence identity of a given amino acid sequence A with or against a given amino acid sequence B (or with a given amino acid sequence B, or A given amino acid sequence A, which has or contains some% amino acid sequence identity, on the other hand) is calculated as follows:
100 times the fraction X / Y, where X is the number of amino acid residues recorded as the same pair by alignment of A and B of the sequence alignment program NCBI-BLAST2, and Y is the total amino acid residue of B Is a number. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, then the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A.

  Furthermore,% amino acid sequence identity values may be determined using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). Most WU-BLAST-2 search parameters are set to initial values. Parameters that are not set to initial values, ie adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein,% amino acid sequence identity values are: (a) the amino acid sequence of the subject PRO polypeptide having a sequence derived from a native PRO polypeptide and the subject comparative amino acid sequence (ie, The number of identical identical amino acid residues determined by WU-BLAST-2 between (the sequence that may be a PRO polypeptide variant to which the subject PRO polypeptide is compared), and (b) subject Determined by the quotient divided by the total number of residues in the PRO polypeptide. For example, in the expression “polypeptide comprising amino acid sequence A having at least 80% amino acid sequence identity to amino acid sequence B”, amino acid sequence A is a comparative amino acid sequence of interest. Amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.

  “PRO179 variant polynucleotide” or “PRO179 variant nucleic acid sequence” refers to a nucleic acid molecule that encodes an active PRO179 polypeptide as defined below, and is shown in (a) FIG. 2 (SEQ ID NO: 2). A nucleic acid sequence encoding residue 1 or about 17-460 of the PRO179 polypeptide, (b) Figure 2 (SEQ ID NO: when X is any amino acid residue 12-12 of Figure 2 (SEQ ID NO: 2)) 2) at least about the nucleic acid sequence encoding amino acids X to 460 of PRO179, or (c) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) Has 80% nucleic acid sequence identity. Typically, a PRO179 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 17-460 of the PRO179 polypeptide shown in FIG. 2 (SEQ ID NO: 2), (b) X is FIG. 2) the nucleic acid sequence encoding amino acids X to 460 of PRO179 shown in FIG. 2 (SEQ ID NO: 2) in the case of any amino acid residue of 12 to 21, or (c) FIG. 2 (SEQ ID NO: 2) ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% Nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89 % Nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least About 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably Is at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity And, more preferably at least about 99% nucleic acid sequence identity. PRO179 polynucleotide variants do not include the native PRO179 nucleotide sequence.

  “PRO207 variant polynucleotide” or “PRO207 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO207 polypeptide as defined below, (a) shown in FIG. 4 (SEQ ID NO: 7). A nucleic acid sequence encoding residue 1 or about 41-249 of the PRO207 polypeptide, (b) Figure 4 (SEQ ID NO: when X is any amino acid residue 36-45 of Figure 4 (SEQ ID NO: 7)) 7) at least about a nucleic acid sequence encoding amino acids X to 249 of PRO179, or (c) a nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 4 (SEQ ID NO: 7) Has 80% nucleic acid sequence identity. Usually, the PRO207 variant polynucleotide is (a) a nucleic acid sequence encoding residues 1 or about 41 to 249 of the PRO207 polypeptide shown in FIG. 4 (SEQ ID NO: 7), and (b) X is FIG. 7), the nucleic acid sequence encoding amino acids X to 249 of PRO207 shown in FIG. 4 (SEQ ID NO: 7) in the case of any amino acid residue of 36 to 45, or (c) FIG. 4 (SEQ ID NO: 7 ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specifically derived fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% Nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89 % Nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least About 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably Is at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity And, more preferably at least about 99% nucleic acid sequence identity. PRO207 polynucleotide variants do not include the native PRO207 nucleotide sequence.

  “PRO320 variant polynucleotide” or “PRO320 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO320 polypeptide as defined below, and is (a) shown in FIG. 6 (SEQ ID NO: 10). A nucleic acid sequence encoding residue 1 or about 22-338 of the PRO320 polypeptide, (b) Figure 6 (SEQ ID NO: when X is any amino acid residue 17-26 of Figure 6 (SEQ ID NO: 10) 10) at least about the nucleic acid sequence encoding amino acids X to 338 of PRO320, or (c) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 6 (SEQ ID NO: 10) Has 80% nucleic acid sequence identity. In general, a PRO320 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 17-26 of the PRO320 polypeptide shown in FIG. 6 (SEQ ID NO: 10), (b) X is FIG. 10) of a nucleic acid sequence encoding amino acids X to 338 of PRO320 shown in FIG. 6 (SEQ ID NO: 10) in the case of any amino acid residue of 17 to 26, or (c) FIG. ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 5% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably At least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, and more Preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity. More preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence. One of, and more preferably at least about 99% nucleic acid sequence identity. PRO320 polynucleotide variants do not include the native PRO320 nucleotide sequence.

  “PRO219 variant polynucleotide” or “PRO219 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO219 polypeptide as defined below, (a) shown in FIG. 8 (SEQ ID NO: 15). A nucleic acid sequence encoding residue 1 or about 17-460 of the PRO219 polypeptide, (b) Figure 8 (SEQ ID NO: when X is any amino acid residue from 24 to 1005 of Figure 2 (SEQ ID NO: 2)) 15) at least about the nucleic acid sequence encoding amino acids X-10005 of PRO219, or (c) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 8 (SEQ ID NO: 15) Has 80% nucleic acid sequence identity. Usually, a PRO219 mutant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 24 to 1005 of the PRO219 polypeptide shown in FIG. 8 (SEQ ID NO: 15), (b) X is FIG. 15), the nucleic acid sequence encoding amino acids X to 1005 of PRO219 shown in FIG. 8 (SEQ ID NO: 15), or (c) FIG. 8 (SEQ ID NO: 15). ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably less About 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, and more Preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity More preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity. Identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleus Sequence identity, and more preferably at least about 99% nucleic acid sequence identity. PRO219 polynucleotide variants do not include the native PRO219 nucleotide sequence.

  “PRO221 variant polynucleotide” or “PRO221 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO221 polypeptide as defined below, and is (a) shown in FIG. 10 (SEQ ID NO: 20). A nucleic acid sequence encoding residue 1 of PRO221 polypeptide or about 34-259, (b) Figure 10 (SEQ ID NO: when X is any amino acid residue 29-38 of Figure 10 (SEQ ID NO: 20)) 20) nucleic acid sequence encoding amino acids X to 259 of PRO221, (c) FIG. 10 (SEQ ID NO :) when X is any amino acid from amino acid 199 to amino acid 208 of FIG. 10 (SEQ ID NO: 20) 20) a nucleic acid sequence encoding amino acid 1 or about 34-X, or (d) another specific derivative of the amino acid sequence shown in FIG. 10 (SEQ ID NO: 20) Having at least about 80% nucleic acid sequence identity to the nucleic acid sequence over de. Usually, a PRO221 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 34-259 of the PRO221 polypeptide shown in FIG. 10 (SEQ ID NO: 20), (b) X is FIG. 20), the nucleic acid sequence encoding amino acids X to 259 of PRO221 shown in FIG. 10 (SEQ ID NO: 20) in the case of any amino acid residue of 29 to 38, (c) X is FIG. 10 (SEQ ID NO: 20) a nucleic acid sequence encoding amino acid 1 of FIG. 10 (SEQ ID NO: 20) or about 34-X when it is any amino acid of amino acids 199 to 208, or (c) FIG. 10 (SEQ ID NO: 20) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity with any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequences shown in More preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity. More preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid. Sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% Nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably low At least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity; And more preferably has at least about 99% nucleic acid sequence identity. PRO221 polynucleotide variants do not include the native PRO221 nucleotide sequence.

  “PRO224 variant polynucleotide” or “PRO224 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO224 polypeptide as defined below, and is (a) shown in FIG. 12 (SEQ ID NO: 25). A nucleic acid sequence encoding residue 1 or about 31-282 of PRO224 polypeptide, (b) Figure 12 (SEQ ID NO: when X is any amino acid residue from 26-35 of Figure 12 (SEQ ID NO: 25)) 25) nucleic acid sequence encoding amino acids X to 282 of PRO224 shown in FIG. 25, (c) FIG. 12 (SEQ ID NO :) when X is any amino acid from amino acids 226 to 235 of FIG. 12 (SEQ ID NO: 25) 25) a nucleic acid sequence encoding amino acid 1 or about 31-X, or (d) another specific derivative of the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25) Having at least about 80% nucleic acid sequence identity to the nucleic acid sequence over de. Typically, the PRO224 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 31-282 of the PRO224 polypeptide shown in FIG. 12 (SEQ ID NO: 25), (b) X is FIG. 25), the nucleic acid sequence encoding amino acids X to 282 of PRO224 shown in FIG. 12 (SEQ ID NO: 25) in the case of any amino acid residue of 26 to 35, (c) X is FIG. 12 (SEQ ID NO: 25) a nucleic acid sequence encoding amino acid 1 or about 31-X in FIG. 12 (SEQ ID NO: 25) when it is any amino acid from amino acid 226 to amino acid 235, or (d) FIG. 10 (SEQ ID NO: 25) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity with any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequences shown in More preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity. More preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid. Sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% Nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably low At least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity; And more preferably has at least about 99% nucleic acid sequence identity. PRO224 polynucleotide variants do not include the native PRO224 nucleotide sequence.

  “PRO328 variant polynucleotide” or “PRO328 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO328 polypeptide as defined below, and is (a) shown in FIG. 14 (SEQ ID NO: 30). A nucleic acid sequence encoding residue 1 of PRO328 polypeptide or about 23-463, (b) Figure 14 (SEQ ID NO: when X is any amino acid residue 18-27 of Figure 14 (SEQ ID NO: 30)) 30) at least about the nucleic acid sequence encoding amino acids X-463 of PRO328, or (c) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 14 (SEQ ID NO: 30) Has 80% nucleic acid sequence identity. Typically, the PRO328 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 23-463 of the PRO328 polypeptide shown in FIG. 14 (SEQ ID NO: 30), (b) X is FIG. : 30) a nucleic acid sequence encoding amino acids X to 463 of PRO328 shown in Fig. 14 (SEQ ID NO: 30) in the case of any amino acid residue of 18 to 27, or (c) Fig. 14 (SEQ ID NO: 30) ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably low At least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity; More preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity. More preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid. Sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% Nucleic acid sequence identity and more preferably at least about 99% nucleic acid sequence identity. PRO328 polynucleotide variants do not include the native PRO328 nucleotide sequence.

  “PRO301 variant polynucleotide” or “PRO301 variant nucleic acid sequence” refers to a nucleic acid molecule that encodes an active PRO301 polypeptide as defined below, (a) shown in FIG. 16 (SEQ ID NO: 35). A nucleic acid sequence encoding residue 1 or about 28-299 of a PRO301 polypeptide, (b) Figure 16 (SEQ ID NO: when X is any amino acid residue 23-32 of Figure 16 (SEQ ID NO: 35)) FIG. 16 (SEQ ID NO :) when the nucleic acid sequence encoding amino acids X to 299 of PR301 shown in FIG. 35) and (c) X is any amino acid from amino acids 230 to 239 of FIG. 16 (SEQ ID NO: 35) 35) a nucleic acid sequence encoding amino acid 1 or about 28-X, or (d) another specific derivative of the amino acid sequence shown in FIG. 16 (SEQ ID NO: 35). Having at least about 80% nucleic acid sequence identity to nucleic acid encoding sequences. Usually, a PRO301 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 28-299 of PRO301 polypeptide shown in FIG. 16 (SEQ ID NO: 35), (b) X is FIG. 16 (SEQ ID NO: : Nucleic acid sequence encoding amino acids X to 299 of PRO301 shown in FIG. 16 (SEQ ID NO: 35) in the case of any amino acid residue of 23 to 299 of (35), (c) X is FIG. 16 (SEQ ID NO: 35) a nucleic acid sequence encoding amino acid 1 or about 28 to X in FIG. 16 (SEQ ID NO: 35) when any amino acid from amino acid 230 to amino acid 239, or (c) F6g10 (SEQ ID NO: 35) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity with any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequences shown More preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid. Sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% Nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferred Is at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity; And more preferably has at least about 99% nucleic acid sequence identity. PRO301 polynucleotide variants do not include the native PRO301 nucleotide sequence.

  “PRO526 variant polynucleotide” or “PRO526 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO526 polypeptide as defined below, and is (a) shown in FIG. 18 (SEQ ID NO: 43). A nucleic acid sequence encoding residue 1 or about 27-473 of the PRO526 polypeptide, (b) Figure 18 (SEQ ID NO: when X is any amino acid residue 22-31 of Figure 18 (SEQ ID NO: 43)) 43) at least about the nucleic acid sequence encoding amino acids X to 473 of PRO526, or (c) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 18 (SEQ ID NO: 43) Has 80% nucleic acid sequence identity. Typically, the PRO526 mutant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 27-473 of the PRO526 polypeptide shown in FIG. 18 (SEQ ID NO: 43), (b) X is FIG. : 43) a nucleic acid sequence encoding amino acids X to 473 of PRO526 shown in Fig. 18 (SEQ ID NO: 43) in the case of any amino acid residue of 22 to 31, or (c) Fig. 18 (SEQ ID NO: 43) ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably low At least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity; More preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity. More preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid. Sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% Nucleic acid sequence identity and more preferably at least about 99% nucleic acid sequence identity. PRO526 polynucleotide variants do not include the native PRO526 nucleotide sequence.

  “PRO362 variant polynucleotide” or “PRO362 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO362 polypeptide as defined below and is (a) shown in FIG. 20 (SEQ ID NO: 48). A nucleic acid sequence encoding residue 1 or about 20-321 of the PRO362 polypeptide, (b) Figure 20 (SEQ ID NO: when X is any amino acid residue 15-24 of Figure 20 (SEQ ID NO: 48)) 48) nucleic acid sequence encoding amino acids X to 321 of PRO362 shown in FIG. 48, (c) FIG. 20 (SEQ ID NO :) when X is any amino acid from amino acids 276 to 285 of FIG. 20 (SEQ ID NO: 48) 48) a nucleic acid sequence encoding amino acid 1 or about 20-X, or (d) another specific derivative of the amino acid sequence shown in FIG. 20 (SEQ ID NO: 48). Having at least about 80% nucleic acid sequence identity to the nucleic acid sequence over de. Typically, a PRO362 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 20-321 of the PRO362 polypeptide shown in FIG. 20 (SEQ ID NO: 48), (b) X is FIG. 48) is a nucleic acid sequence encoding amino acids X to 321 of PRO362 shown in FIG. 20 (SEQ ID NO: 48) when it is any amino acid residue of 15 to 24, (c) X is FIG. 20 (SEQ ID NO: 48) the nucleic acid sequence encoding amino acid 1 or about 20-X in FIG. 20 (SEQ ID NO: 48) when it is any amino acid from amino acid 276 to amino acid 285, or (d) FIG. 20 (SEQ ID NO: 48) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity with any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequences shown in More preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity. More preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid. Sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% Nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably low At least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity; And more preferably has at least about 99% nucleic acid sequence identity. PRO2362 polynucleotide variants do not include the native PRO362 nucleotide sequence.

  “PRO356 variant polynucleotide” or “PRO356 variant nucleic acid sequence” refers to a nucleic acid molecule that encodes an active PRO356 polypeptide as defined below, (a) shown in FIG. 22 (SEQ ID NO: 55). Nucleic acid sequence encoding residue 1 or about 27-346 of PRO356 polypeptide, (b) Figure 22 (SEQ ID NO: when X is any amino acid residue 22-31 of Figure 22 (SEQ ID NO: 55)) 55) at least about the nucleic acid sequence encoding amino acids X to 346 of PRO356, or (c) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 22 (SEQ ID NO: 55) Has 80% nucleic acid sequence identity. Usually, the PRO356 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 27 to 346 of the PRO356 polypeptide shown in FIG. 22 (SEQ ID NO: 55), (b) X is FIG. 22 (SEQ ID NO: : 55) in the case of any amino acid residue of 22-31, the nucleic acid sequence encoding amino acids X-346 of PRO356 shown in FIG. 22 (SEQ ID NO: 55), or (c) FIG. 22 (SEQ ID NO: 55) ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably low At least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity; More preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity. More preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid. Sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% Nucleic acid sequence identity and more preferably at least about 99% nucleic acid sequence identity. PRO356 polynucleotide variants do not include the native PRO356 nucleotide sequence.

  “PRO509 variant polynucleotide” or “PRO509 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO509 polypeptide as defined below, and is (a) shown in FIG. 24 (SEQ ID NO: 60). A nucleic acid sequence encoding residue 1 or about 37-283 of the PRO509 polypeptide, (b) Figure 24 (SEQ ID NO: when X is any amino acid residue 32-32 of Figure 24 (SEQ ID NO: 60)) 60), a nucleic acid sequence encoding amino acids X to 283 of PRO509, (c) FIG. 24 (SEQ ID NO :) when X is any amino acid from amino acids 200 to 209 of FIG. 24 (SEQ ID NO: 60) 60) a nucleic acid sequence encoding amino acid 1 or about 37-X, or (d) another specific derived fragment of the amino acid sequence shown in FIG. 24 (SEQ ID NO: 60) Having at least about 80% nucleic acid sequence identity to the nucleic acid sequence over de. Typically, the PRO221 variant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 37-283 of the PRO509 polypeptide shown in FIG. 24 (SEQ ID NO: 60), (b) X is FIG. : 60) the nucleic acid sequence encoding amino acids X to 283 of PRO509 shown in Fig. 24 (SEQ ID NO: 60) when it is any amino acid residue of 32-41, (c) X is Fig. 24 (SEQ ID NO :) 60) a nucleic acid sequence encoding amino acid 1 or about 37-X in FIG. 24 (SEQ ID NO: 60) when it is any amino acid from amino acid 200 to amino acid 209, or (d) FIG. 24 (SEQ ID NO: 60) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity with any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequences shown in More preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity. More preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid. Sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% Nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably low At least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity; And more preferably has at least about 99% nucleic acid sequence identity. PRO509 polynucleotide variants do not include the native PRO509 nucleotide sequence.

  “PRO866 variant polynucleotide” or “PRO866 variant nucleic acid sequence” means a nucleic acid molecule that encodes an active PRO866 polypeptide as defined below, and is (a) shown in FIG. 26 (SEQ ID NO: 62). Nucleic acid sequence encoding residue 1 or about 27-331 of PRO866 polypeptide, (b) Figure 26 (SEQ ID NO: when X is any amino acid residue 22-31 of Figure 26 (SEQ ID NO: 62)) 62) to the nucleic acid sequence encoding amino acids X to 331 of PRO866, or (c) (d) the nucleic acid sequence encoding another specific derivative of the amino acid sequence shown in FIG. 26 (SEQ ID NO: 62) Having at least about 80% nucleic acid sequence identity. Typically, a PRO866 mutant polynucleotide is (a) a nucleic acid sequence encoding residue 1 or about 22-31 of the PRO866 polypeptide shown in FIG. 26 (SEQ ID NO: 62), (b) X is FIG. : 62) a nucleic acid sequence encoding amino acids X to 331 of PRO866 shown in FIG. 26 (SEQ ID NO: 62) in the case of any amino acid residue of 22 to 31, or (c) FIG. 26 (SEQ ID NO: 62) ) At least about 80% nucleic acid sequence identity, preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% of any of the nucleic acid sequences encoding other specific derivative fragments of the amino acid sequence shown in Nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably less Both about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more Preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity More preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity. Identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98%. Acid sequence identity, and more preferably at least about 99% nucleic acid sequence identity. PRO866 polynucleotide variants do not include the native PRO866 nucleotide sequence.

  Typically, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variant polynucleotides are at least about 30 nucleotides in length, and more at least about 60 nucleotides in length, More at least about 90 nucleotides long, more at least about 120 nucleotides long, more at least about 150 nucleotides long, more at least about 180 nucleotides long, more at least about 210 nucleotides long, more at least about at least about 240 nucleotides long, more at least about 270 nucleotides long, more at least about 300 nucleotides long, more at least about 4 0 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.

"Percent (%)" nucleic acids identified relative to the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide-encoding nucleic acid sequences defined herein "Sequence identity" refers to PRO179, PRO207, PRO179, PRO207, which introduced the gap if necessary to align the sequences and obtain maximum percent sequence identity and did not consider any conservative substitutions as part of the sequence identity. Nucleotide in the candidate sequence that is identical to the nucleotide of the PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide coding sequence. It is defined as the percent of Reochido. Alignments for the purpose of determining percent nucleic acid sequence identity can be obtained by various methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Can be achieved by using available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% nucleic acid sequence identity values are derived from the sequence comparison program ALIGN-2, whose complete source code for the ALIGN-2 program is provided in Table 1, as described below. Is obtained. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code shown in Table 1 was submitted to the US Copyright Office, Washington DC, 20559 with user documentation and under US Copyright Registration Number TXU510087 It is registered. ALIGN-2 is publicly available through Genentech, South San Francisco, California, and may be compiled from the source code given in FIGS. 2A-P. The ALIGN-2 program is compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the% nucleic acid sequence identity of a given nucleic acid sequence C with, or relative to, a given nucleic acid sequence D (or with or against a given nucleic acid sequence D or A given amino acid sequence C, which has or contains a certain percentage of nucleic acid sequence identity) is calculated as follows:
100 times the fraction W / Z where W is the number of nucleotides recorded as identical pairs by C and D alignment of the sequence alignment program ALIGN-2, and Z is the total number of nucleotides in D. It will be appreciated that if the length of the nucleic acid sequence C is different from the length of the nucleic acid sequence D, then the% nucleic acid sequence identity of C to D is different from the% nucleic acid sequence identity of D to C. As an example of calculating% nucleic acid sequence identity using this method, Tables 2C-2D show the% nucleic acid sequence identity of the nucleic acid sequence designated “Comparative DNA” to the nucleic acid sequence designated “PRO-DNA”. The calculation method of is shown.

Unless otherwise indicated, all% nucleic acid sequence identity values herein are obtained using the ALIGN-2 sequence comparison computer program as described above. However,% nucleic acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http://ww.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, all of which are set to initial values, for example, unmask = possible, chain = all, predicted occurrence = 10, minimum low composite length = 15/5 Multipath e-value = 0.01, multipath constant = 25, final gap alignment dropoff = 25, and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is used for sequence comparison, the% nucleic acid sequence identity of, or relative to, a given nucleic acid sequence C (or a given nucleic acid sequence D, or to it) A given nucleic acid sequence C, which has or contains a certain percentage of nucleic acid sequence identity), is calculated as follows:
100 times the fraction W / Z, where W is the number of nucleotides recorded as identical pairs by C and D alignment of the sequence alignment program NCBI-BLAST2, and Z is the total number of nucleotides in D . It will be appreciated that if the length of the nucleic acid sequence C is different from the length of the nucleic acid sequence D, then the% nucleic acid sequence identity of C to D is different from the% nucleic acid sequence identity of D to C.
Furthermore,% nucleic acid sequence identity values may be determined using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). Most WU-BLAST-2 search parameters are set to initial values. Parameters that are not set to initial values, ie adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, the% nucleic acid sequence identity value is: (a) a nucleic acid sequence of a subject PRO polypeptide-encoding sequence having a sequence derived from a native sequence PRO polypeptide-encoding nucleic acid; The identical identity determined by WU-BLAST-2 with the comparison nucleic acid molecule (ie, the sequence of the subject PRO polypeptide-encoding nucleic acid molecule, which may be a variant polynucleotide to be compared) The number of nucleotides is determined by the quotient divided by (b) the total number of nucleotides of the subject PRO polypeptide-encoding nucleic acid. For example, in the expression “an isolated nucleic acid molecule comprising a nucleic acid sequence A having or having at least 80% amino acid sequence identity to the nucleic acid sequence B”, the reference nucleic acid to which the nucleic acid sequence A is directed The nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest.

  In another embodiment, PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variant polynucleotides are active PRO179, PRO207, PRO320, PRO219, PRO22, PRO22 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, preferably as shown in FIG. 2 (SEQ ID NO: 2), respectively, under stringent hybridization and wash conditions Nucleotide sequence encoding full length PRO179 polypeptide, encoding full length PRO207 polypeptide shown in FIG. 4 (SEQ ID NO: 7) Nucleotide sequence encoding the full-length PRO320 polypeptide shown in FIG. 6 (SEQ ID NO: 10), nucleotide sequence encoding the full-length PRO219 polypeptide shown in FIG. 8 (SEQ ID NO: 15), FIG. 10 (SEQ ID NO: 20) ), A nucleotide sequence encoding the full-length PRO224 polypeptide shown in FIG. 12 (SEQ ID NO: 25), and a full-length PRO328 polypeptide shown in FIG. 14 (SEQ ID NO: 30). Nucleotide sequence, nucleotide sequence encoding full-length PRO301 polypeptide shown in FIG. 16 (SEQ ID NO: 35), nucleotide sequence encoding full-length PRO526 polypeptide shown in FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48) Total length P shown in Nucleotide sequence encoding O362 polypeptide, nucleotide sequence encoding full-length PRO356 polypeptide shown in FIG. 22 (SEQ ID NO: 55), nucleotide sequence encoding full-length PRO509 polypeptide shown in FIG. 24 (SEQ ID NO: 60), It can hybridize to the nucleotide sequence encoding the full-length PRO866 polypeptide shown in Figure 26 (SEQ ID NO: 62). PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variant polypeptides are PRO179, PRO207, PRO320, PRO219, PRO221, PRO226, PRO226, PRO226 It may be encoded by a PRO362, PRO356, PRO509, or PRO866 variant polynucleotide.

The term “positive” in the context of amino acid sequence identity comparisons performed as described above includes not only amino acid residues that are identical in the compared sequences, but also those that have similar properties. The amino acid residue recorded with a positive value for the target amino acid residue is the same as the target amino acid residue, or the target amino acid residue (as specified in Table 3 below). ) Preferred substituents.
For purposes herein, a given amino acid sequence A, or a given amino acid sequence B, or a% positive value relative thereto (or a given amino acid sequence B, or to some extent relative thereto). A given amino acid sequence A having or containing% positive) is calculated as follows:
100 times the fraction X / Y, where X is the number of amino acid residues that recorded positive values by alignment of A and B of the sequence alignment program ALIGN-2, 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 different from the length of amino acid sequence B, then% positive for A to B is different from% positive for B to A.

  “Isolated”, when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Preferably, an isolated polypeptide does not have any of the attendants that naturally accompany it. Contaminant components of the natural environment are substances that typically 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) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequenator, or (2) Coomassie blue or It is preferably purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using silver staining. Since the isolated polypeptide is free of at least one component of the natural environment of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, In situ proteins in recombinant cells are included. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

  An “isolated” nucleic acid molecule encoding a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or anti-PRO179, anti-PRO207, anti -Isolation encoding -PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, or anti-PRO866 antibody The identified nucleic acid molecule was identified as PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO35. -, Nucleic acid encoding PRO509- or PRO866- or anti-PRO179-, anti-PRO207-, anti-PRO320-, anti-PRO219-, anti-PRO221-, anti-PRO224-, anti-PRO328-, anti From at least one contaminating nucleic acid molecule normally associated with a natural source of nucleic acid encoding -PRO301-, anti-PRO526-, anti-PRO362-, anti-PRO356-, anti-PRO509-, or anti-PRO866- antibody An isolated nucleic acid molecule. Preferably, an isolated nucleic acid is not associated with all components that naturally accompany it. An isolated nucleic acid molecule encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, or anti-PRO179, anti-PRO207, anti-PRO320, anti Nucleic acid molecules encoding -PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, or anti-PRO866 are found in nature It is something other than form or setting. Thus, an isolated nucleic acid molecule is a nucleic acid molecule that encodes PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 present in a natural cell, or 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 -Differentiated from nucleic acid molecules encoding PRO866. However, an isolated nucleic acid molecule that encodes a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or anti-PRO179, anti-PRO207, anti Isolated encoding -PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, or anti-PRO866 antibodies The nucleic acid molecule is, for example, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PR when the nucleic acid molecule is at a chromosomal location different from that of natural cells. O301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or 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 PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, which are normally expressed in cells expressing anti-PRO866 antibody Or a PRO866 nucleic acid molecule or anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, -PRO301, anti -PRO526, anti -PRO362, including anti -PRO356, anti -PRO509, or anti -PRO866 nucleic acid molecule.

The expression “control sequence” means a DNA sequence necessary for the expression of a coding sequence operably linked in a particular host organism. For example, suitable control sequences for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
A nucleic acid is “operably linked” when it is in a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA, if expressed as a preprotein that participates in polypeptide secretion, is operably linked to the polypeptide DNA; a promoter or enhancer is responsible for transcription of the sequence. If it affects, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is in a position that facilitates translation. . In general, “operably linked” means that the bound DNA sequences are in close proximity and, in the case of a secretory leader, in close proximity and in the reading phase. However, enhancers do not necessarily have to be close together. Binding is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional techniques.

  The term “antibody” is used in the broadest sense, in particular for example a single anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301. Anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, and anti-PRO866 monoclonal antibodies (including agonist antibodies), poly-epitope specific anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO320, PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, and anti-PRO866 antibody compositions, single chain anti-PRO179, Anti-PRO207, anti-PRO320, anti-PRO219, anti-PR 221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, and anti-PRO866 antibody, anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO320 Includes PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509, and anti-PRO866 antibody fragments (see below). The term “monoclonal antibody” as used herein is identical except for a substantially homogeneous population of antibodies, ie, naturally occurring mutations in which the constituent individual antibodies may be present in minor amounts. Refers to an antibody obtained from a population.

The “stringency” of a hybridization reaction is readily determined by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment close to but below its melting point. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures make the reaction conditions more stringent, while lower temperatures reduce stringency. Furthermore, stringency is inversely proportional to salt concentration. For further details and explanation of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
As defined herein, “stringent conditions” or “highly stringent conditions” are: (1) 0.015 M sodium chloride / 0.0015 M citric acid at low ionic strength and high temperature, eg, 50 ° C., for washing. Using sodium / 0.1% sodium dodecyl sulfate; (2) denaturing agents such as formamide during hybridization, eg 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1 at 42 ° C. % Polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 and 750 mM sodium chloride, 75 mM sodium citrate; (3) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaCl, 0.075 M Sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, And 10% dextran sulfate with a high stringency wash consisting of 0.2x SSC (sodium chloride / sodium citrate) at 42 ° C and 50% formamide at 55 ° C followed by 0.1x SSC containing EDTA at 55 ° C Identified by things.
“Moderate stringency conditions” are identified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) Including formation conditions (eg, temperature, ionic strength and% SDS) Moderate stringency conditions include 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7 .6), overnight incubation at 37 ° C. in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / mL denatured sheared salmon sperm DNA, followed by washing the filter at 37-50 ° C. in 1 × SSC. One skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

The term “epitope tag” as used herein refers to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 fused to a “tag polypeptide”. A chimeric polypeptide comprising a peptide. The tag polypeptide has a sufficient number of residues to provide an epitope from which the antibody can be produced, or an epitope that can be identified by several other reagents, but its length is subject to fusion. Short enough not to inhibit the activity of the PRO polypeptide. Also, the tag polypeptide is preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8 to about 50 amino acid residues (preferably about 10 to about 20 residues).
As used herein, the term “immunoadhesin” 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, an immunoadhesin is a fusion of an amino acid sequence (ie, “heterologous”) with a desired binding specificity other than the antigen recognition and binding site of an antibody with an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is a contiguous amino acid sequence that typically includes at least a receptor or ligand binding site. Immunoadhesin immunoglobulin constant domain sequences include IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, such as IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. It can be obtained from any immunoglobulin.

As intended herein, “active” and “activity” refer to natural or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 biology. Means a form of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 that retains target and / or immunological activity, where “biological” The activity refers to natural or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO3. 2, a biological function (inhibitory or stimulatory) produced by a PRO356, PRO509, or PRO866 polypeptide, which is a natural or naturally occurring PRO179, PRO207, PRO320, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, By means of excluding the ability to generate antibodies to an antigenic epitope possessed by PRO362, PRO356, PRO509, or PRO866 polypeptide, “immunological” activity means natural or naturally occurring PRO179, PRO207, PRO320, PRO219. , PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or the antigenic epitope of PRO866 polypeptide It refers to the ability to generate antibodies.
“Biological activity” in the context of antibodies or other agonist molecules (eg, organic or inorganic small molecules, peptides, etc.) that can be identified by the screening assays disclosed herein is those molecules that are “therapeutically effective amounts”. Is used to refer to the ability to exert one or more of the effects listed herein in relation to the definition of In a particular embodiment, “biological activity” is the ability to inhibit neoplastic cell growth or proliferation. A preferred biological activity is inhibition, including slowing or complete arrest of target tumor (eg, cancer) cell growth. Another preferred biological activity is a cytotoxic activity that results in the death of the target tumor (eg, cancer) cell. Yet another preferred biological activity is the induction of apoptosis of target tumor (eg cancer) cells.

The term “immunological activity” refers to an immunological cross-reaction with at least one epitope of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. Means sex.
As used herein, “immunological cross-reactivity” refers to a candidate polypeptide whose PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 has this activity. Polyclonal antiserum raised against the qualitative biological activity of the polypeptide against the known activity PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide It can be competitively inhibited. Such antisera are prepared in a conventional manner, for example, by subcutaneously injecting a known active analog in complete Freund's adjuvant into a goat or rabbit and then boosting intraperitoneally or subcutaneously in incomplete Freund. The The immunological cross-reactivity is preferably “specific”, which corresponds to the corresponding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301 of the identified immunological cross-reactive molecule (eg, antibody). , PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide has a binding affinity that is significantly higher than the binding affinity for any other known native polypeptide of the molecule (preferably at least about 2-fold, more preferably Means at least about 4 times, even more preferably at least about 6 times, and most preferably at least about 8 times higher).

“Tumor” as used herein means all tumorigenic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, adenocarcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, Ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, liver cancer, spawning cancer, thyroid cancer, liver cancer and various types of head and neck cancer Is included.

“Treatment” is an intervention performed in the present invention to prevent or alter the progression of disease pathology. Thus, “treatment” refers to both therapeutic treatment and prophylactic or protective measures. Those in need of treatment include those already with the disease as well as those in which the disease is to be prevented. In tumor (eg, cancer) treatment, the therapeutic agent may directly reduce the pathology of the tumor cells or make the tumor cells more sensitive to other therapeutic mediators such as radiation and / or chemotherapy. May be.
The “pathology” of cancer includes all phenomena that compromise the good survival of the patient. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, inhibition of normal function of neighboring cells, release of cytokines or other secreted products at abnormal levels, suppression of inflammation or immune response, or Including deterioration.

An “effective amount” of a polypeptide or an agonist thereof disclosed herein is an amount capable of inhibiting the growth of target cells to some extent with respect to neoplastic cell growth and tumor growth. The term includes amounts capable of inducing growth inhibition, cell division arrest and / or cytotoxic effects and / or apoptosis of the target cell. An “effective amount” of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof for the purpose of inhibiting neoplastic cell growth Can be determined by routine methods.
A “therapeutically effective amount” has the following effects with respect to tumor treatment: (1) some inhibition of tumor growth, including retardation and complete growth arrest; (2) reduction in tumor cell number; 3) reduction of tumor size; (4) inhibition of tumor cell invasion into peripheral organs (ie, reduction, delay or complete arrest); (5) inhibition of metastasis (ie, reduction, delay or complete arrest). ); (6) Promotion of an anti-tumor immune response, which may lead to tumor regression or rejection, but is not necessarily required; and / or (7) one or more of the symptoms associated with the disease By an amount that can induce one or more of a degree of mitigation. A “therapeutically effective amount” of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof for tumor treatment purposes is empirical Can be determined by routine methods.

A “growth inhibitory amount” of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof is the growth of a cell, particularly a tumor, eg, a cancer cell. An amount that can be inhibited in vitro or in vivo. A “growth inhibitory amount” of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or a PRO866 polypeptide or agonist thereof for the purpose of inhibiting neoplastic cell growth is: Can be determined empirically by routine methods.
A “cytotoxic amount” of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof is defined as a cell, particularly a tumor, eg, a cancer cell, in vitro or An amount that can be destroyed in vivo. A “cytotoxic amount” of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof for the purpose of inhibiting neoplastic cell growth is: Can be determined empirically by routine methods.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioisotopes (eg, I ¹³¹ , I ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ ), chemotherapeutic drugs, and toxins such as enzymatically active toxins from bacteria, fungi, plants or animals, or fragments thereof. Is done.
A “chemotherapeutic agent” is a chemical compound useful in the treatment of tumors, such as cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids such as paclitaxel (Taxol, Bristol- Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rance), toxoter, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vinxanthrone , Carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicin (US Pat. No. 4,675,187), melphalan, and Including other related nitrogen mustards. Also included in this definition are hormone-like drugs that act to regulate or inhibit hormonal effects on tumors such as tamoxifen and onapristone.

  As used herein, “growth inhibitory agent” means a compound or composition that inhibits the growth of cells, particularly cancer cells that overexpress any gene identified herein, in vitro or in vivo. That is, growth inhibitors significantly reduce the proportion of cells that overexpress such genes in the S phase. Examples of growth inhibitory agents include agents that inhibit the cell cycle (at a place other than S phase), such as agents that induce G1 arrest or M-phase arrest. Classic M phase blockers include vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. These agents that cause G1 arrest also overflow S phase arrest, for example DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, ed., Chapter 1, title `` Cell cycle reguration, oncogene, and antineoplastic drugs '', Murakami et al. (WB Saunders: Philadelphia, 1995), especially p13. it can.

  The term “cytokine” is a general term for proteins released from one cell population and acting as intercellular mediators on other cells. Examples of such cytokines are lymphokines, monokines, and traditional 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; relaxin; prorelaxin; glycoprotein such as follicle stimulating hormone (FSH) ), Thyrotropin (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Mueller inhibitor; mouse gonadotropin-related peptide Activin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factor such as NGF-β; Platelet growth factor; Transforming growth factor such as TGF-α and TGF-β; Insulin-like growth factor-I And I Erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-) CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL -7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factors such as TNF-α and TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and is the biologically active equivalent of native sequence cytokines.

The term “prodrug” as used in this application is a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is enzymatically activated or converted to a more active parent form Means form. For example, Wilman, “Prodrugs in Cancer Chemotherapy”, Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting, Belfast (1986), and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug delivery. , Borchardt et al. (Ed.), Pp. 247-267, Human Press (1985). The prodrugs of the present invention are, but not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, optionally substituted Phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to more active, non-cytotoxic drugs. Non-limiting examples of cytotoxic drugs that can be derivatized to the prodrug form used in the present invention include, but are not limited to, the chemotherapeutic agents described above.
The term “agonist” is used in the broadest sense, and the biology of the natural PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides disclosed herein. Includes any molecule that is similar to the pharmacological activity. Suitable agonist molecules are in particular agonist antibodies or antibody fragments, native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide fragments or amino acid sequence variants. , Peptides, small organic molecules and the like. A method for identifying agonists of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, wherein the tumor cells are exposed to a candidate agonist and inhibition of tumor cell growth Can be measured.

“Chronic” administration means administration of the drug in a continuous manner as opposed to an acute manner in order to maintain the initial therapeutic effect (activity) over a long period of time. An “intermittent” administration is a treatment that is not made continuously without interruption, but rather is essentially periodic.
“Mammal” for purposes of treatment means any animal classified as a mammal, humans, farm animals and farm animals, zoos, sports or pet animals such as dogs, horses, cats, cows, etc. including. Preferably the mammal is a human.
Administration of “in combination with” one or more additional therapeutic agents includes simultaneous (temporary) and sequential administration in any order.

  As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to it at the dosages and concentrations employed. is there. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid; polypeptides with low molecular weight (less than about 10 residues); proteins such as Serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides such as glucose, mannose, or dextran; disaccharides and other carbohydrates; Chelating agents; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN (trade name), polyethylene glycol (PEG), and PLURONICS (trade name) Including.

“Natural antibodies” and “natural immunoglobulins” are heterotetrameric glycoproteins of approximately 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. . Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is the heavy chain Is aligned with the variable domain of. Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains.
The term “variable” refers to the fact that certain sites in the variable domain vary widely in sequence within an antibody and are used for the binding and specificity of each particular antibody to that particular antigen. To do. However, variability is not uniformly distributed across the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The natural heavy and light chain variable domains are largely linked by CDRs that take a β-sheet structure, bind the β-sheet structure, and in some cases form a loop bond that forms part of it. Each FR region is included. The CDRs of each chain are bound closer to the FR and contribute to the formation of the antigen-binding site of the antibody together with the CDRs of the other chains (Kabat et al., NIH Publ. No. 91-3242, Vol. I). Pp. 647-669 (1991)). The constant domains are not directly related to antibody binding to antigen, but exhibit various effector functions such as antibody involvement in antibody-dependent cytotoxicity.

“Hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region is a “complementarity determining region” or “CDR” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable region and the heavy chain variable. Regions 31-35 (H1), 50-65 (H2) and 95-102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. 1991)) and / or “hypervariable loops” (eg, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) and heavy chain variable region) and heavy Residues from residues 26-32 (H1), 53-55 (L2) and 96-101 (L3) of the chain variable region; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)) Including. “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
“Antibody fragments” comprise a portion of a native antibody, preferably the antigen binding or variable region of the native antibody. Examples of antibody fragments are Fab, Fab ′, F (ab ′) ₂ and Fv fragments; diabody; linear antibody (Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]. ); Single chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies, called “Fab” fragments, combines two identical antigen-binding fragments, each with a single antigen-binding site, and the remaining “Fc” fragment, whose name reflects its ability to crystallize easily. Generate. Pepsin treatment generates an F (ab ′) ₂ fragment that has two antigen binding sites but can be a cross-linked antigen.

“Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain in tight, non-covalent association. In this arrangement, the three CDRs of each variable region interact to determine the antigen binding site on the surface of the V _H -V _L dimer. To be precise, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs that are antigen specific) has the ability to recognize and bind antigen, but has a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the symbol for Fab ′ where the cysteine residue of the constant domain has a free thiol group. F (ab ′) ₂ antibody fragments originally were produced as pairs of Fab ′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
“Light chains” of antibodies (immunoglobulins) from any vertebrate species are based on the amino acid sequence of their constant domains, one or two distinctly different forms called kappa (κ) and lambda (λ) Can be classified.
Based on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins are divided into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.

The term “monoclonal antibody” as used herein is derived from a substantially homogeneous population of antibodies, ie, a population that is identical except for naturally occurring mutations in which the individual antibodies comprising the population are present in small amounts. Refers to antibody. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Furthermore, unlike conventional (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. . In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and not being construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 [1975], or by recombinant DNA methods (eg, US Pat. No. 4,816,567). You may create it by reference. A “monoclonal antibody” is a technique described in phage antibody libraries, for example, as described in Clackson et al., Nature, 352: 624-628 [1991] and Marks et al., J. Mol. Biol., 222: 581-597 (1991). You may isolate using. See also US Pat. Nos. 5,750,373, 5,571,698, 5,403,484 and 5,223,409 which describe the preparation of antibodies using phagemids and phage vectors.
Here, monoclonal antibodies specifically include “chimeric” antibodies (immunoglobulins), which correspond to antibodies whose heavy or light chain portions are derived from a particular species or belong to a particular antibody class or subclass. Antibodies that are identical or homologous to the sequence, but the remaining part of the chain is identical or homologous to the corresponding sequences of antibodies derived from other species or belonging to a particular antibody class or subclass, as well as the organism in which they are desired As long as they exhibit biological activity (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).

“Humanized” forms of non-human (eg, murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab ′) that contain minimal sequence derived from non-human immunoglobulin. , F (ab ′) ₂ or other antigen binding sequence of the antibody). For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) whose complementarity determining regions (CDRs) are CDRs (donor antibodies) of non-human species such as mice, rats, goats, etc. Substituted with residues having the desired specificity, affinity and capacity derived from In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, a humanized antibody has at least one, typically all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence, typically Will contain substantially all of the two variable domains. An optimal humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). checking ... Humanized antibodies include PRIMATIZED (trade name) antibodies derived from antibodies produced by immunizing macaques with the antigen of interest of the antigen binding region of the antibody.

“Single-chain Fv” or “sFv” antibody fragments comprise antibody fragments comprising the V _H and V _L domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that allows the sFV to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).
The term “diabody” refers to a small antibody fragment having two antigen-binding sites, the fragment being variable in the light chain variable domain (V _L ) within the same polypeptide chain (V _H -V _L ). Domain (V _H ) is bound. Using a linker that is so short that it allows pairing of two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain, creating two antigen binding sites. Diabodies are described in further detail, for example, in EP404097; WO93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

  An “isolated” antibody is one that has been identified and separated or recovered from a component of its natural environment. Contaminants of the natural environment are substances that interfere with the use of antibodies for diagnosis or therapy, and include enzymes, hormones, and other non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) at least 95% by weight or more preferably 99% by weight antibody as determined by the Lowry method, and (2) by using a spinning cup sequenator. To the extent sufficient to obtain 15 N-terminal or internal amino acid sequence residues, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining Purified. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The term “label” when used herein refers to a detectable compound or composition that binds directly or indirectly to the antibody to produce a “labeled” antibody. The label may be detectable by itself (eg, a radioisotope label or a fluorescent label), or in the case of an enzyme label, it may catalyze chemical conversion of the detectable substrate compound or composition.
“Solid phase” means a non-aqueous matrix to which a compound of the invention can adhere. Examples of solid phases encompassed herein are those partially or wholly formed of glass (eg, controlled-pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone. including. In certain embodiments, depending on the context, the solid phase can include the wells of an assay plate; otherwise, a purification column (eg, an affinity chromatography column). The term also includes a discontinuous solid phase of discrete particles, such as those described in US Pat. No. 4,275,149.

A “liposome” is useful for delivery of a drug (PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or an antibody thereto) to a mammal, Various types of small vesicles containing lipids, phospholipids and / or surfactants. The components of the liposome are usually arranged in a bilayer structure similar to the lipid sequence of biological members.
“Small molecule” is defined herein as having a molecular weight of less than about 500 Daltons.

II. Compositions and Methods of the Invention Full length PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide Newly identified and isolated nucleotide sequences encoding polypeptides referred to as PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides are provided. In particular, cDNAs encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides, as disclosed in more detail in the Examples below, are identified. Isolated.
As disclosed in the examples below, cDNA clones encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides have been deposited with the ATCC. [With the exception, the cDNA encoding the PRO509 polypeptide has not been deposited with the ATCC]. The actual nucleotide sequence of the clone is readily determined by those skilled in the art by sequencing the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides and coding sequences described herein, Applicants have included the sequence information currently available. We identified what was considered the most suitable reading frame.

B. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variants The full-length native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO221, PRO24 , PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides, as well as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variants and preparations of PRO866 It is done. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variants are PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO36, PRO236 By introducing appropriate nucleotide changes into PRO356, PRO509, or PRO866 DNA and / or the desired PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide Prepared by synthesizing Kill. One skilled in the art will recognize that appropriate amino acid changes can alter the post-translational process of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides, eg, glycosylation sites It will be understood that the change in the number or modification of the membrane anchoring properties.

  The PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 native full-length sequences described herein or PRO179, PRO207, PRO320, PRO219, PRO221, PRO24, PRO24 , PRO526, PRO362, PRO356, PRO509, or PRO866 mutations may be made using any techniques and guidelines for conservative and non-conservative mutations described, for example, in US Pat. No. 5,364,934. Can do. Mutations result in PRO179, PRO207, PRO320, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221, PRO221 , PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, such that the amino acid sequence of the polypeptide is altered, such as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO356, PRO356, PRO356, PRO356, PRO356, Or encodes a PRO866 polypeptide Substitution of one or more of the codons may be deleted or inserted. In some cases, the mutation is at least one amino acid of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or any other domain of one or more domains of PRO866 polypeptide. Substitution with amino acids. Guidance on which amino acid residues are inserted, substituted or deleted without adversely affecting the desired activity is compared to the sequence of a protein molecule of known homology with the sequence of a polypeptide of the invention, It is found by minimizing amino acid sequence changes made within regions of high homology. An amino acid substitution can be, for example, a substitution of leucine with serine, that is, a conservative amino acid substitution, as a result of substitution of one amino acid with another amino acid having similar structural or chemical properties. Insertions and deletions can optionally be in the range of 1 to 5 amino acids. Acceptable mutations are determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting mutants for activity exerted by the full-length or mature native sequence.

Also provided herein are fragments of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides. Such fragments may be cleaved at the N-terminus or C-terminus, for example, or lack internal residues when compared to a full-length native protein. Certain 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. ing.
PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 fragments can be prepared by any of a number of conventional methods. Desired peptide fragments may be chemically synthesized. Another approach is to treat the protein by enzymatic digestion, for example by treating the protein with an enzyme known to cleave the protein at a site defined by specific amino acid residues, or by digesting DNA with an appropriate restriction enzyme, Producing a PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide fragments and isolating the desired fragments. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding the desired polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that define the desired ends of the DNA fragments 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 shown in FIG. 2 (SEQ ID NO: 15) and FIG. 16 (sequence), respectively. No. 35), FIG. 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. 22 (SEQ ID NO: 55, FIG. 24 (SEQ ID NO: 60), or FIG. 26 (SEQ ID NO: 62). The native PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide polypeptides and at least one biological and Or share immunological activity.

In a particular embodiment, the conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such a substitution results in a change in biological activity, the product will be screened by introducing more significant changes, such as those listed in Table 3 or related to the amino acid classification shown below. Is done.

A significant modification of the function and immunological identity of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide is (a) a polypeptide in the substitution region By selecting substituents that are significantly different in their effect on the maintenance of the backbone structure, eg sheet or helical arrangement, (b) charge or hydrophobicity of the target site, or (c) the majority of the side chains . Naturally occurring residues are grouped based on common side chain properties:
(1) Hydrophobicity: nornoicin, met, ala, val, leu, ile;
(2) Neutral hydrophilicity: cys, ser, thr;
(3) Acidity: asp, glu;
(4) Basicity: asn, gln, his, lys, arg;
(5) Residues affecting chain orientation: gly, pro; and (6) Aromatics: trp, tyr, phe
Non-conservative substitutions will require exchanging one member of these classes for another. Residues so substituted can also be introduced at conservative substitution sites, more preferably at the remaining (non-conserved) sites.

Mutations can be made using techniques well known in the art such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34: 315 (1985)], restricted selective mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other DNA cloning techniques known in the art. In this manner, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 mutant DNA can also be prepared.
Scanning amino acid analysis can also be used to identify one or more amino acids along adjacent sequences. Among them, preferred scanning amino acids are relatively small and neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is a typically preferred scanning amino acid in this group because it eliminates side chains beyond the beta carbon and is unlikely to change the backbone structure of the mutant [Cunninghem and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is often found in both buried and exposed positions [Creighton, The Proteins, (WH Freeman and Co., NY); Chothia, J. Mol. Biol., 150: 1 (1976)]. If alanine substitution does not result in a sufficient amount of mutants, isoteric amino acids can be used.

C. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide modifications Covalent modifications of PRO356, PRO509, and PRO866 polypeptides are included within the scope of the present invention. One type of covalent modification is PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or the target amino acid residue of PRO866 polypeptide, PRO179, PRO207. , PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or an organic derivatization reagent capable of reacting with a selected N- or C-terminal residue of a PRO866 polypeptide Including. Derivatization with bifunctional reagents may include, for example, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides in a water-insoluble support matrix or 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 antibody Useful for crosslinking to surfaces for use in purification methods or vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide ester such as 4-azidosalicylic acid, 3,3′-dithiobis (succinimidyl) Homobifunctional imide esters including disuccinimidyl esters such as propionate), bifunctional maleimides such as bis-N-maleimide-1,8-octane, and methyl-3-[(p-azidophenyl)- Reagents such as dithio] propioimidate.
Other modifications include deamination of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, lysine, arginine, and histidine side chains, respectively. Α-amino group methylation [TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman and Co., San Francisco, pp. 79-86 (1983)], N-terminal amine acetylation, and optional C-terminus Includes amidation of carboxyl groups.

Other types of covalent modifications of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides included within the scope of the present invention are: Including altering the native glycosylation pattern. “Modification of native glycosylation pattern” means, for purposes herein, the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. Deletion of one or more carbohydrate moieties found in (by removal of existing glycosylation sites or deletion of glycosylation by chemical and / or enzymatic means) and / or the native sequences PRO179, PRO207, PRO320, PRO219 Of one or more glycosylation sites not present in the, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides Means pressurized. In addition, the phrase includes qualitative changes in glycosylation of natural proteins, including changes in the nature and proportion of the various carbohydrate moieties present.
Addition of glycosylation sites to the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can be accomplished by altering the amino acid sequence. This change may be, for example, the addition of one or more serine or threonine residues to the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, or Also by substitution with (in the case of O-linked glycosylation sites). In some cases, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 amino acid sequences can be translated into the desired amino acid by changes at the DNA level. By mutating DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides at bases preselected to be produced Be changed.

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 of the present invention are glycosides to the polypeptide. By chemical or enzymatic coupling. These methods are described in International Patent Application No. WO 87/05330 published September 11, 1987 and Aplin and Wriston, CRC Crit. Rev. Biochem., Pp 259-306 (1981).
Removal of carbohydrate moieties present on PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides is a target for chemical or enzymatic or glycosylation This is done by mutational substitution of a codon encoding an amino acid residue. For example, chemical deglycosylation techniques are known in the art, for example, Hakimuddin et al., Arch. Biochem Biophys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981) It is described by. Enzymatic cleavage of carbohydrate moieties on polypeptides can be accomplished using various endo- and exo-glycosidases as described in Thotakura et al., Meth. Enzymol., 138: 350 (1987). .

Other types of covalent modifications of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides are PRO179, PRO207, PRO320, PRO219, PRO221, PRO241, RO241 , PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide to one of a variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene; No. 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
Further, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide of the present invention is a PRO179, PRO207 fused to another heterologous polypeptide or amino acid sequence. Modifications may be made by methods that form chimeric molecules comprising 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 an anti-tag antibody can selectively bind, and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, Fusions with PRO356, PRO509, or PRO866 polypeptides are included. 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 polypeptide. The presence of such epitope-tagged forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide should be detected using an antibody against the tag polypeptide. Can do. Also, the provision of the epitope tag can be accomplished by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, Allows easy purification of PRO356, PRO509, or PRO866 polypeptides. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (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 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and herpes simplex virus sugars A protein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include Flag-peptide [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)]; α- Tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule is a fusion of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or a PRO866 polypeptide and a specific region of an immunoglobulin or immunoglobulin. May include body. For a bivalent form of a chimeric molecule (also referred to as “immunoadhesin”), such a fusion can be the Fc region of an IgG molecule. The Ig fusion preferably solubilizes PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide in place of at least one variable region within the Ig molecule. (Transmembrane domain deletion or inactivation) forms. In particularly preferred embodiments, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions, see US Pat. 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 polypeptides The following description is primarily directed to PRO179, PRO207, PRO320, PRO219, PRO241, PRO241, PRO241 , PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, by culturing the cells transformed or transfected with a vector containing the nucleic acid, PRO207, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 Related to the production. Of course, it is believed that other methods well known in the art can be used to prepare PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. It is done. For example, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide sequences, or portions thereof, are produced by direct peptide synthesis using solid phase technology. [See, for example, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)). In vitro protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Various portions of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides are chemically synthesized separately, using chemical or enzymatic methods. In combination, full-length PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides may be produced.

1. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, isolation of DNA encoding PRO356, PRO509, or PRO866 PRO179, PRO207, PRO320, PRO219, PRO221, PRO226, PRO228, PRO226 , PRO362, PRO356, PRO509, or PRO866 DNA that carries PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 mRNA Is it considered to be expressed at the level? From the prepared cDNA library. Accordingly, human PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 DNA is derived from a cDNA library prepared from human tissue, as described in the Examples. It can be easily obtained. Also, the PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509-, or PRO866--encoding genes can be obtained from a genomic library or It may be obtained by a known synthesis method (for example, automated nucleic acid synthesis).
The library was designed to identify the gene of interest or the protein encoded by that gene (PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or (E.g., antibodies to PRO866 or oligonucleotides of at least about 20-80 bases). Screening of cDNA or genomic libraries with selected probes is performed using standard procedures described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). be able to. Other methods of isolating genes encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 [Sambrook et al., Supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The following examples describe cDNA library screening techniques. The oligonucleotide sequence selected as the probe must be long enough and clear enough to minimize false positives. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Labeling methods are well known in the art and include the use of radiolabels such as ³² P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions including moderate stringency and high stringency are given by Sambrook et al., Supra.
Sequences identified in such library screening methods can be compared and aligned with well-known sequences deposited in public databases such as Genbank or personal sequence databases and made available to the public. Sequence identity (either at the amino acid or nucleic acid level) within a given region or over the entire length of the molecule can be determined using methods known in the art and described herein.
Nucleic acids with protein coding sequences use the deduced amino acid sequences disclosed herein for the first time, and Sambrook et al., Supra, which detects intermediates and precursors of mRNA that were not reverse transcribed into cDNA if necessary. Obtained by screening of selected cDNA or genomic libraries by using conventional primer extension methods as described.

2. Host Cell Selection and Transformation Host cells can be expressed or cloned in the expression or cloning vectors for production of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 as described herein. Transfection or transformation, induction of a promoter, selection of transformants, or cultivation in a conventional nutrient medium appropriately modified to amplify the gene encoding the desired sequence. Culture conditions such as medium, temperature, pH, etc. can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al., Supra. it can.
Methods of eukaryotic cell transfection and prokaryotic transformation, such as CaCl ₂ , CaPO ₄ , liposome-mediated and electroporation are known to those skilled in the art. Depending on the host cell used, transformation is done using standard methods appropriate to that cell. Calcium treatment or electroporation with calcium chloride as described in Sambrook et al., Supra, is used for prokaryotes. Infection with Agrobacterium tumefaciens has been reported in certain plant cells as described in Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published 29 June 1989. Used for transformation. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) is used. General aspects of mammalian cell host system transformations are described in US Pat. No. 4,399,216. Transformation into yeast is typically performed by Van solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. USA, 76: 3829 (1979). Implemented according to the method. However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, intact cells, or bacterial protoplast fusion with polycations such as polybrene, polyornithine can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988).

In the vectors described herein, suitable host cells for cloning or expressing DNA are prokaryotes, yeast, or higher eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria such as Gram negative or Gram positive organisms such as Enterobacteriaceae such as E. coli. Various E. coli strains, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strains W3110 (ATCC 27,325) and K5772 (ATCC 53,635) are available to the public. Other preferred prokaryotic host cells are, for example, Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as spirits, and dysentery Enterobacteriaceae such as fungi; Bacillus subtilis and Neisseria gonorrhoeae such as B. licheniformis (for example, B. licheniformis disclosed in DD 266,710 issued April 12, 1989) 41P); Pseudomonas such as Pseudomonas aeruginosa; and Streptomyces. These examples are illustrative and not limiting. E. coli strain W3110 is a preferred host or parent host because it is a common host strain for recombinant DNA production fermentation. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 may be modified to introduce a genetic mutation in a gene encoding an exogenous protein in the host, examples of such hosts include E. coli W3110 strain 1A2 with the complete genotype tonA; E. coli W3110 strain 9E4 with genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244) with complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r ; complete genotype tonA ptrE phA3E -lac) 169 degP ompT rbs7 ilvG kan coli having ^r W3110 strain 37D6; strain E. coli W3110 strain is a non-kanamycin resistant degP deletion mutants of 37D6 40B4; and issued US Patent August 7, 1990 An E. coli strain having mutant periplasmic protease disclosed in JP 4,946,783. Alternatively, in vitro cloning methods such as PCR or other nucleic acid polymerase reactions are preferred.

  In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast include PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356. Suitable cloning or expression hosts for vectors encoding-, PRO509-, or PRO866-. Saccharomyces cerevisia is a commonly used lower eukaryotic host microorganism. Others are Schizosaccharomyces prombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 issued May 2, 1985); Kluveromyces hosts (US patent) No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-975 (1991)), for example, K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol. 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), Cedro Sophyllum ( K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8: 135 (1990)), K. themotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sheekrishna et al., J. Basic Microbiol, 28: 265-278 [1988]); Candida; Trichodel Malaysia (EP 244,234); Red-skinned mold (Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 [1979]); Schwanniomyces, eg, Schwanniomyces Occidentalis (EP 394,538 published October 31, 1990); and filamentous fungi such as Neurospora, Penicillium, Tolypocladium (WO 91/00357 published January 10, 1991) And Koji fungi, such as pseudonogenic Koji fungi (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and black mold (Kelly and Hynes, EMBO J., 4: 475-479 [1985]). Preferred methylotropic yeasts here include, but are not limited to, Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Includes yeast that can grow on methanol selected from the genus. A list of specific species, illustrative of this yeast classification, is described in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

  Suitable host cells for expression of glycosylated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodospera Sf9, and plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More detailed examples include monkey kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells / -DHFR (CHO), (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)) Mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse breast Contains tumor cells (MMT 060562, ATTC CCL51). The selection of an appropriate host cell is within the common general knowledge of the field.

3. Selection and Use of Replicable Vectors Nucleic acids (eg, cDNA or genomic DNA) encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are cloned ( Inserted into a replicable vector for amplification or expression of DNA. Various vectors are publicly available. The vector can be, for example, in the form of a plasmid, cosmid, virus particle, or phage. The appropriate nucleic acid sequence is inserted into the vector by a variety of techniques. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques well known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to generate suitable vectors containing one or more of these components.
PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are not necessarily produced directly by recombinant techniques, but are also signal sequences or mature proteins or It is also produced as a fusion peptide with a heterologous polypeptide such as another peptide having a specific cleavage site at the N-terminus of the polypeptide. In general, the signal sequence is a component of the vector, or PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- inserted into the vector. Or part of the DNA encoding PRO866-. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or a thermostable enterotoxin II leader. For yeast secretion, signal sequences include, for example, the yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces α factor leader, the latter described in US Pat. No. 5,010,182. ), Or acid phosphatase leader, C. albicans glucoamylase leader (EP 362,179 issued April 4, 1990), or international patent application WO 90 / published on November 15, 1990. It can be the signal described in 13646. In mammalian cell expression, signal sequences from secreted polypeptides of the same or related species, other mammalian signal sequences such as viral secretion leaders may be used for direct secretion of the protein.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for many bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and the various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are Useful for cloning vectors in mammalian cells.
Expression and cloning vectors typically contain a selection gene, also termed a selectable marker. Typical selection genes are (a) conferring resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) compensate for auxotrophic defects, or (c) the gene code D of Bacillus -Encode proteins that supply important nutrients not available from complex media, such as the example of a gene encoding alanine racemase.

Other examples of suitable selectable markers for mammalian cells include PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526, such as DHFR or thymidine kinase. It is possible to identify the ability of a cell to take up nucleic acids encoding-, PRO362-, PRO356-, PRO509-, or PRO866-. Suitable host cells when using wild type DHFR are deficient in DHFR activity prepared and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). CHO cell line. 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); Kingman et al., Gene, 7: 141 (1979); Tschemper et al. Gene, 10: 157 (1980)]. The trp1 gene provides a selectable marker for mutant strains of yeast lacking the ability to grow in the presence of tryptophan, such as, for example, ATCC 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)] .
Expression and cloning vectors are typically PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509-, or PRO866-encoding nucleic acid. A promoter is operably linked to the sequence and directs mRNA synthesis. Promoters that are recognized by a variety of potential host cells are known. Suitable promoters for use in prokaryotic hosts are the β-lactamase and lactose promoter systems [Cahng et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)], alkaline phosphatase, tryptophan (trp ) Promoter system [Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983). )]including. Promoters used in bacterial systems also encode PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509-, or PRO866-. It has a Shine-Dalgarno (SD) sequence operably linked to DNA.

Examples of suitable promoter sequences for use with yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)], eg enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, Glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trioselate isomerase, phosphoglucose isomerase, and glucokinase are included.
Other inducible promoters that have the additional effect that transcription is controlled by growth conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glycerin There are promoter regions of aldehyde-3-phosphate dehydrogenase and the enzymes that govern the utilization of maltose and galactose. Vectors and promoters suitably used for expression in yeast are further described in EP 73,657.

Transcription of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 from a vector in a mammalian host cell is, for example, polyoma virus, infectious epithelioma virus (UK 2,211,504 published July 5, 1989), adenovirus (eg adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40) By promoters obtained from viral genomes, promoters obtained from heterologous mammals, such as actin promoters or immunoglobulin promoters, and promoters obtained from heat shock promoters As long as such a promoter is compatible with the host cell system.
Transcription of DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 by higher eukaryotes inserts an enhancer sequence into the vector. Can be enhanced by. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to enhance its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the late SV40 enhancer (100-270 base pairs) of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancer late of the origin of replication. Enhancers are spliced into the vector at the 5 ′ or 3 ′ position of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 coding sequences, but preferably Located 5 'from the promoter.

Also, expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) are required for transcription termination and mRNA stabilization. Also includes sequences. Such sequences can be obtained from the normally 5 ', sometimes 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of mRNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. Including.
Still other methods, vectors and suitable for adapting the synthesis of PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 in recombinant cell culture Host cells 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). Gene Amplification / Expression Detection Gene amplification or expression is based on the sequences provided here, using appropriately labeled probes, eg, conventional Southern blotting, Northern blotting to quantify mRNA transcription [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, can be measured directly in the sample. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes, can also be used. The antibody can then be labeled and an assay can be performed, where the duplex is bound to the surface, so that the antibody bound to that duplex at the time of formation on the surface of the duplex. The presence can be detected.
Alternatively, gene expression can be measured by immunological methods that directly quantify gene product expression, such as immunohistochemical staining of cells or tissue sections and cell culture or body fluid assays. Antibodies useful for immunohistochemical staining or assay of sample fluids can be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibody is directed against the native sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or the DNA sequence provided herein. An exogenous sequence that encodes a specific antibody epitope fused to PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 DNA Can be prepared.

5. Polypeptide Purification Forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 can be recovered from the culture medium or lysates of host cells. If membrane-bound, it can be detached from the membrane using an appropriate wash solution (eg Triton-X100) or enzymatic cleavage. Cells used for the expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are freeze thaw cycles, sonication, mechanical disruption, or cytolytic agents Can be destroyed by various chemical or physical means.
It is desirable to purify PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 from recombinant cell proteins or polypeptides. Purified by the following procedure, which is an example of a suitable purification procedure: fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or a cation exchange resin such as DEAE; chromatofocusing; PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A sepharose column to remove contaminants such as IgG; and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, A metal chelation column that binds the epitope tag form of PRO356, PRO509, or PRO866. Use various protein purification methods known in the art and described, for example, in Deutcher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). Can do. The purification process chosen depends, for example, on the production method used and in particular the nature of the produced PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866.

E. Antibodies Some of the candidate agents for use in the compositions and methods of the present invention include PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide organisms. Antibodies and antibody fragments that mimic biological activity.
1. Polyclonal antibodies Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies in mammals can be generated by one or more injections of, for example, an immunizing agent and, if desired, an adjuvant. Typically, the mammal is injected with the immunizing agent or adjuvant by multiple subcutaneous or intraperitoneal injections. The immunizing agent may comprise a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or a fusion protein thereof. It is useful to conjugate the immunizing agent to a protein that is known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol will be selected by one skilled in the art without undue experimentation.

2. Monoclonal antibody The antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method as described in Kohler and Milstein, Nature, 256: 495 (1975). In hybridoma methods, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to generate antibodies that specifically bind to the immunizing agent or to generate lymphocytes that can be generated. Trigger. Lymphocytes can also be immunized in vitro.
The immunizing agent typically comprises a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or a fusion protein thereof. Generally, peripheral blood lymphocytes ("PBL") are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. . The lymphocytes are then fused with an immortal cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103. ]. Immortal cell lines are usually transformed mammalian cells, particularly myeloma cells from rodents, cows and humans. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells are preferably cultured in a suitable medium containing one or more substances that inhibit the survival or growth of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxatin, aminopetitin and thymidine ("HAT medium"). This substance prevents the growth of HGPRT-deficient cells.

Preferred immortal cell lines are those that fuse efficiently, support stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. A more preferred immortal cell line is the mouse myeloma line, which is available, for example, from the Salk Institute Cell Distribution Center in San Diego, California and the American Type Culture Collection in Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have also been disclosed [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 culture medium in which the hybridoma cells are cultured is then assayed for the presence of monoclonal antibodies to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is measured by an immunoprecipitation or in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by the Scatchard analysis method by Munson and Pollard, Anal. Biochem., 107: 220 (1980).

After the desired hybridoma cells are identified, the clones can be subcloned by a limiting dilution step and grown by standard methods [Goding, supra]. Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown as ascites in vivo in a mammal.
Monoclonal antibodies secreted by the subclone can be isolated from the culture medium or ascites fluid by conventional immunoglobulin purification methods such as protein A-Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography. Separated or purified.

Monoclonal antibodies can also be made by recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily obtained using a conventional method (for example, using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced. The hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector that is transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulin protein. And monoclonal antibodies can be synthesized in recombinant host cells. DNA can also be obtained by, for example, substituting coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences [US. Patent No. 4,816,567; Morrison et al., Supra], or non-coding immunoglobulin coding sequences. Modification can be made by covalently linking part or all of the coding sequence of an immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide substitutes for the constant domain of the antibody of the invention or substitutes for the variable domain of one antigen binding site of the antibody of the invention to produce a chimeric bivalent antibody. can do. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or can be substituted for the variable domains of one antigen binding site of the antibodies of the invention, producing chimeric bivalent antibodies.
The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is generally cleaved at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Generation of fragments, particularly Fab fragments, by antibody digestion can be accomplished using conventional techniques known in the art.

3. Human and humanized antibodies The antibodies of the present invention further include humanized antibodies or human antibodies. Humanized forms of non-human (eg mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen binding subsequences of antibodies). And contains the minimum sequence derived from non-human immunoglobulin. A humanized antibody is a CDR residue of a non-human species (donor antibody) in which the complementarity determining region (CDR) of the recipient has the desired specificity, affinity and ability, such as mouse, rat or rabbit. Human immunoglobulin (recipient antibody) substituted by In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has at least one, typically all or almost all CDR regions corresponding to those of a non-human immunoglobulin and all or almost all FR regions of a human immunoglobulin consensus sequence, typically Contains substantially all of the two variable domains. Humanized antibodies optimally comprise at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op Struct. Biol., 2: 593-596 (1992)].

  Methods for humanizing non-human antibodies are well known in the art. Generally, one or more amino acid residues derived from non-human are introduced into a humanized antibody. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization essentially involves winter and co-workers [Jones et al., Nature, 321: 522-525 (1986); Riechmann by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)] by replacing the rodent CDR or CDR sequence with the corresponding sequence of a human antibody. To be implemented. Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

  Human antibodies also include phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1992); Marks et al., J. Mol. Biol., 222: 581 (1991)]. It can also be created using various methods known in Also, techniques such as Cole et al. And Boerner et al. Can be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. P. 77 (1985; Boerner et al., J. Immunol. 147 (1): 86-95 (1991); US Pat. No. 5,750,373) Similarly, human antibodies inactivate human immunoglobulin loci in transgenic animals such as endogenous immunoglobulin genes partially or completely inactivated. Production of human antibodies that are very similar to those found in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire during stress testing This approach is described, for example, in US Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and the following scientific literature: Marks et al., Bio / Technology 10, 7 79-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14 , 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

4). Bispecific antibody
Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, the other is any other To an antigen, preferably a cell surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are well known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs with different specificities of the two heavy chains (Milstein and Cuello, Nature, 305: 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar procedures are disclosed in WO 93/08829 published May 13, 1993 and Traunecker et al., EMBO J., 10: 3655-3656 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. Desirably, at least one fusion has a first heavy chain constant region (CH1) containing the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface includes at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). A complementary “cavity” of the same or smaller size as the large side chain is created at the interface of the second antibody molecule by replacing the large amino acid side chain with a smaller one (alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies). Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to produce F (ab ′) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of other Fab′-TNB derivatives to form bispecific antibodies. The produced bispecific antibody can be used as a drug for selective immobilization of an enzyme.
Fab ′ fragments may be recovered directly from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describes the production of fully humanized bispecific antibody F (ab ′) ₂ molecules. Each Fab ′ fragment is secreted separately from E. coli and directional chemical bonds in vitro to form bispecific antibodies. The bispecific antibody thus formed can bind to cells overexpressing ErbB2 receptor and normal human T cells, triggering the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various methods for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab ′ portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided an alternative mechanism for generating bispecific antibody fragments. A fragment consists of a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) by a linker that is short enough to allow pairing between two domains on the same chain. Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of another fragment to form two antigen binding sites. Other bispecific antibody fragment production methods using single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994).

More than bivalent antibodies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
Exemplary bispecific antibodies bind to two different epitopes on the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides provided herein. sell. Alternatively, 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 polypeptide arm is a trigger molecule on leukocytes such as a T cell receptor molecule (eg, CD2, CD3, CD28 or B7), or IgG such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). It binds to the arm that binds to the Fc receptor (FcγR), and the cell defense mechanism is specific for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO50. , Or PRO866 may be concentrated in the polypeptide-expressing cells. Bispecific antibodies localize cytotoxic drugs to cells that express a particular PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. It may be used for These antibodies include PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509-, or PRO866-binding arms and cytotoxic drugs. Alternatively, it has an arm that binds to a chelating agent such as EOTUBE, DPTA, DOTA, or TETA. Other bispecific antibodies of interest bind to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, as well as tissue factor (TF). To join.

5. Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies are used, for example, to target immune system cells to unwanted cells [US Pat. No. 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. Proposed. This antibody could be prepared in vitro using known methods in synthetic protein chemistry, including those related to crosslinkers. For example, immunotoxins can be made using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in US Pat. No. 4,6767,980.

6). Effector Function Design It is desirable to modify the antibodies of the present invention for effector function, for example to enhance the efficacy of the antibody in the treatment of cancer. For example, cysteine residues are introduced into the Fc region to form interchain disulfide bonds in this region. The homodimeric antibody produced in this way may have improved internalization ability and / or increased complement-mediated cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be designed that has a dual Fc region and thus may have enhanced complement lysis and ADCC ability. See Stevenson et al., Anti-cancer Drug Design 3: 219-230 (1989).

7). Immunoconjugates The present invention also provides for chemotherapeutic agents, cytotoxic agents such as toxins (eg, enzymatically active toxins from bacteria, fungi, plants or animals, or fragments thereof), or radioisotopes (ie, radioconjugates). It also relates to an immune complex comprising a conjugated antibody.
Chemotherapeutic agents useful for the generation of such immune complexes have been described above. Enzyme-active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, cholera toxin, botulinum toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPIII, and PAP-S), Momodica charantia ( momordica charantia inhibitor, curcin, crotin, crotin, sapaonaria oficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin ( enomycin) and trichothecene. A variety of radioactive nucleotides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

The conjugates of antibodies and cytotoxic agents are various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoester bifunctionality. Derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium Using derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) Can be created. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to an antibody. See WO 94/11026.
In other embodiments, the antibody may be conjugated to a “receptor” (such as streptavidin) for use in tumor pre-targeting, and the antibody-receptor complex is administered to the patient, followed by a clarifier. Used to remove unbound complexes from the circulation, followed by administration of a “ligand” (eg, avidin) conjugated to a cytotoxic agent (eg, a radionucleotide, etc.).

8). Immunoliposomes The proteins, antibodies and the like disclosed herein may be prepared as immunoliposomes. Liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); Prepared by methods known in the art, such as described in 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in US Pat. No. 5,013,556.
Particularly useful liposomes are generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of a predetermined size to produce liposomes having a desired diameter. Fab ′ fragments of antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982). A chemotherapeutic agent (such as doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989).

F. Identification of Proteins that Can Inhibit Neoplastic Cell Growth or Proliferation The proteins disclosed in this application were assayed in a panel of 60 tumor lines currently used in the National Cancer Society (NCI) experimental, disease-oriented, in vitro drug screening did. The purpose of this screening is to identify molecules with cytotoxic and / or cytostatic activity against different types of tumors. NCI screens over 10,000 new molecules every year (Monks et al., J. Natl. Cancer Inst., 83: 757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol. Update, 3 (10 ): 1-12 [1989]). The tumor cell line used for this experiment is described in Monks et al. Cell lines whose growth was significantly inhibited by the proteins of this application were identified in the examples.

The results revealed that the proteins tested were cytostatic in various cancer cell lines and, in some cases and at concentrations, cytotoxic activity.
Other cell-based assays and animal models for tumors (eg, cancer) also support the discovery of NCI cancer screening and are used to further understand the relationship between the identified proteins and tumor sperm cell growth progression and pathogenic equivalents it can. For example, primary media from tumors in transgenic animals (as described below) can be used in the cell-based assays herein, but stable cell lines are preferred. Techniques for deriving continuous cell lines from transgenic animals are known in the art (see, eg, Small et al., Mol. Cell. Biol., 5: 642-648 [1985]).

G. Animal Model To further understand the role of the genes identified herein in tumor progression and pathogenesis and to test the efficacy of candidate therapeutics including antibodies and other agonists of natural polypeptides including small molecule agonists Various well-known animal models can be used for this purpose. The in vivo nature of these models can predict response, particularly in human patients. Animal models of tumors and cancers (eg, breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents such as mouse models. Such models are based on standard techniques such as subcutaneous injection, tail vein injection, spleen transplantation, intraperitoneal transplantation, subrenal transplantation, or orthopin transplantation, eg, colon cancer cells transplanted into colon tissue. It is created by introducing tumor cells into syngeneic mice. (See PCT publication WO 97/33551 issued on September 18, 1997.)

Probably the most frequently used animal species for oncogene research is immunodeficient mice, especially nude mice. The observation that hypo mice / hypoplastic nude mice act as hosts for human tumor xenografts has led to widespread use for this purpose. An autosomal recessive nu gene is, for example, ASW, A / He, AKR, BALB / c, B10. A large number of different nude mice including LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I / st, NC, NFR, NFS, NFS / N, NZB, NZC, NZW, P, RIII and SJL It was introduced into a common gene line. In addition, a wide range of other animals other than nude mice with genetic immunodeficiency were grown and used as recipients for tumor xenografts. For further details, see The Nude Mouse in Oncology Research, E. Boven and B. Winograd, CRC Press, Inc., 1991.
Cells introduced into these animals include well-known tumor / cancer cell lines, such as the tumor cell lines listed above, and, for example, the B104-1-1 cell line (stable NIH-3T3 cells transfected with the neu proto-oncogene. Ras-transfected NIH-3T3 cells: Caco-2 (ATCC HTB-37), moderately well-differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or tumor And can be derived from cancer. Tumor or cancer cell samples can be obtained from patients undergoing surgery using standard conditions including freezing and storage in liquid nitrogen (Karmali et al., Br. J. Cancer 48, 689- 696 [1983]).

  Tumor cells can be introduced into animals such as nude mice by various techniques. The subcutaneous (sc) space in mice is highly preferred for tumor transplantation. Tumors are obtained as a solid block, as a needle biopsy using a trochar, and as a cell suspension. c. Can be transplanted. For solid block or trochar transplantation, appropriately sized tumor tissue fragments are s. c. Introduced into space. Cell suspensions are freshly prepared from primary tumors or stable tumor cell lines and injected subcutaneously. Tumor cells can also be injected as subcutaneous implants. In this position, the inoculum is s. c. It is deposited between the tissues. Boven and Winograd (1991), supra. Animal models of breast cancer are basically based on, for example, Drebin et al., PNAS by transplanting neuroblastoma cells (from which the neu oncogene is first isolated) or neu transfected NIH-3T3 cells into nude mice. Produced as described in USA 83, 9129-9133 (1986).

Similarly, colorectal cancer animal models are generated by passaging colon cancer cells to animals, such as nude mice, and leading to the development of tumors in these animals. Orthotopic transplantation models for human colon cancer in nude mice are described, for example, in Wang et al., Cancer Research 54, 4726-4728 (1994) and Too et al., Cancer Research 55, 681-684 (1995). This model is based on “METAMOUSE”, commercially available from the so-called AntiCancer, Inc. (SanDiego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from in vitro media can then be passaged to animals. These tumors can be provided as targets for further testing and drug screening. Alternatively, tumors obtained from passages can be isolated, and RNA of pre-passage cells and cells isolated after one or more passages are analyzed for identifiable expression of the gene of interest. Such passaging techniques can be performed on well-known tumor or cancer cell lines.

For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 have been introduced into the fibrosarcoma of BALB / c female mice (DeLeo et al., J. Exp. Med. 146, 720 [1977]), which contains various antigens. Provides a highly controllable model system for the study of anti-tumor activity (Palladino et al., J. Immunol. 138, 4023-4032 [1987]). Conveniently, tumor cells are grown in vitro in cell culture medium. Prior to injection into animals, the cell lines are washed and suspended in buffer at a cell density of about 10 × 10 ⁶ to 10 × 10 ⁷ cells / ml. Animals are then infected subcutaneously with 10 to 100 μl of cell suspension and left for 1 to 3 weeks until tumors appear.

  In addition, the mouse Lewis lung (3LL) carcinoma, one of the most fully studied experimental tumors, can be used as a research tumor model. Efficacy in this tumor model correlated with beneficial effects in the treatment of human patients diagnosed with small cell carcinoma of the lung (SCCL). This tumor can be introduced into normal mice upon injection of tumor fragments from affected mice or cells remaining in the medium (Zupi et al., Br. J. Cancer 41, suppl. 4, 309 [1980]), with evidence , Showing that the tumor starts with an injection of only one cell and that a very high population of infected tumor cells survive. For further information on this tumor model, see Zacharski, Haemostasis 16, 300-320 [1986].

  One way to evaluate the effectiveness of a test compound in an animal model of a transplanted tumor is to measure the size of the tumor before and after treatment. Traditionally, the size of the transplanted tumor is measured with a two or three dimensional slide caliper. Measurements limited to two dimensions do not accurately reflect the size of the tumor and are therefore usually converted to the corresponding volume using mathematical formulas. However, measurement of tumor size is very inaccurate. The therapeutic effect of a candidate drug can be better described as treatment-induced growth delay and specific growth delay. Another important variable in the description of tumor growth is tumor volume doubling time. Computer programs for calculation and description of tumor growth are also available, such as those reported by Rygaard and Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals, Wu and Sheng, Basel, 1989, 301 It is. However, it is noted that the necrosis and inflammatory response following the tumor can actually increase the size of the tumor at least initially. Therefore, these changes need to be carefully monitored using a combination of morphological methods and flow cytometric analysis.

  Recombinant (transgenic) animal models can be processed by introducing the coding portion of the genes identified herein into the genome of the animal of interest using standard techniques for the production of transgenic animals. Animals that can be provided as targets for transgenic manipulation include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates such as baboons, chimpanzees and monkeys. Techniques known in the art for introducing transgenes into these animals include pronuclear microinjection (Hoppe and Wanger, US Pat. No. 4,873,191); retrovirus-mediated gene transfer to the embryonic lineage (eg, Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]); Gene targeting in embryonic hepatocytes (Thompson et al., Cell 56, 313-321 [1989]); (Lo, Mol. Cel. Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 [1989]). For review, see, for example, US Pat. No. 4,736,866.

  For the purposes of the present invention, transgenic animals include those that have the transgene only in part ("mosaic animals"). The transgene is integrated as a single transgene or as a concatamer, eg, head-to-head or head-to-tail tandem. Selective introduction of transgenes into specific cell types is also possible, for example, according to the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992).

  Transgene expression in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification is used to confirm transgene integration. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunohistochemistry. The animals are further tested for signs of tumor or cancer development.

  The effectiveness of antibodies that specifically bind to the polypeptides identified herein and other candidate drugs in the treatment of spontaneous animal tumors can also be tested. A suitable target for such studies is feline oral squamous cell carcinoma (SCC). Cat oral SCC is a highly invasive malignancy, the most common oral malignancy in cats, accounting for more than 60% of oral tumors reported in this species. It hardly metastasizes to distant sites, but the low incidence of this metastasis merely reflects the short survival time of the cat with this tumor. These tumors are usually inoperable, but mainly due to the cat's oral anatomy. There is currently no effective treatment for this tumor. Prior to entering the study, each cat was subjected to a complete clinical and biopsy and scanned by computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors were excluded from the study. The tongue will begin to paralyze due to this tumor, and the animal will not be able to feed itself even after treatment kills the tumor. Treat each cat repeatedly over time. Tumor photographs were taken every day during the treatment period and at the time of subsequent rechecks. After treatment, each cat was again CT scanned. CT scans and radiographs were evaluated every 8 weeks thereafter. Data were evaluated for differences in survival, reactivity and toxicity compared to the control group. A positive response requires tumor shrinkage, preferably improved quality of survival or prolonged survival.

  In addition, other naturally occurring animal tumors such as canine, feline and baboon fibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma can also be tested. Breast cancer in these dogs and cats is a preferred model because its expression and behavior is very similar to that of humans. However, the use of this model is limited by the incidence of this type of tumor in animals.

H. Candidate Drug Screening Assays Candidate drug screening assays are designed to identify compounds that bind competitively or complex with receptors of individually identified polypeptides and signals through such receptors. The Such screening assays include assays that follow high-throughput screening of chemical libraries that make them particularly suitable for the identification of small molecule drug candidates. Small molecules are considered to include antibody synthetic organic or inorganic compounds, which are peptides, preferably soluble peptides, (poly) peptide-immunoglobulin fusions, particularly but not limited to poly- and monoclonal antibodies and antibody fragments. Single-chain antibodies, anti-idiotype antibodies, and chimeric or humanized forms of those antibodies or fragments, and antibodies, including human antibodies and antibody fragments. The assays can be performed in a variety of formats and include well-characterized protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays in the field.

  In binding assays, the interaction is binding and the complex formed is isolated or detected in the reaction mixture. In a specific embodiment, the polypeptide receptor or candidate drug encoded by the gene identified herein is immobilized to a solid phase, eg, a microtiter plate, by covalent or non-covalent bonding. Non-covalent binding is generally achieved by coating a solid surface with a solution of the polypeptide and drying. Alternatively, an immobilized antibody specific for the polypeptide to be immobilized, such as a monoclonal antibody, can be used to anchor it to a solid surface. The assay is performed by adding a non-immobilized component, which may be labeled with a detectable label, to a coated surface containing an immobilized component, eg, an anchoring component. When the reaction is completed, unreacted components are removed, for example, by washing, and the complex adhered to the solid surface is detected. If the first non-immobilized component has a detectable label, detection of the label immobilized on the surface indicates that complex formation has occurred. If the initial non-immobilized component does not have a label, complex formation can be detected, for example, by a labeled antibody that specifically binds to the immobilized complex.

  If a candidate compound interacts but does not bind to a particular receptor, the interaction with that receptor can be assayed by well-known methods to detect protein-protein interactions. Such assays include traditional techniques such as cross-linking, co-immunoprecipitation, and co-purification through a gradient or chromatographic column. Furthermore, protein-protein interactions have been described by Fields and co-workers [Fiels and Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88, 9578-9582. (1991)] is used for monitoring as disclosed in Chevray and Nathans [Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)]. be able to. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one that acts as a DNA binding domain and the other that functions as a transcriptional activation domain. The yeast expression system described in the previous literature (commonly referred to as the “2-hybrid system”) takes advantage of this property and uses two hybrid proteins while the target protein is the DNA binding domain of GAL4. On the other hand, the candidate activation protein is fused to the activation domain. Expression of the GAL1-lacZ reporter gene under the control of a GAL4-activated promoter depends on reconstitution of GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected with a chromogenic substance for β-galactosidase. A complete kit (MATCHMAKER ™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technology is commercially available from Clontech. This system can also be extended to the mapping of protein domains involved in a particular protein interaction as well as the identification of amino acid residues important for this interaction.

I. Pharmaceutical Compositions Polypeptides of the invention, agonist antibodies that specifically bind to the proteins identified herein, as well as other molecules identified in the screening assays disclosed above, are useful for the treatment of tumors, including cancer. It can be administered in the form of a composition.
Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is usually preferred. For example, peptide molecules that retain the ability to bind to a target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be synthesized chemically and / or produced by recombinant DNA techniques (eg, Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]).

  The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may include an agent that improves its function, eg, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. These molecules are suitably present in combination in amounts that are effective for the purpose intended.

  The therapeutic formulations of the polypeptides identified herein, or their agonists, contain the active ingredient with the desired degree of purity in any pharmaceutically acceptable carrier, supplement, in the form of a lipophilic formulation or aqueous solution. Prepared and stored by mixing with a form or stabilizer (Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. [1980]). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, and other organic acids; ascorbic acid and methionine Antioxidant; Preservative (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl paraben such as methyl or propylparaben; catechol; resorcinol; cyclohexanol 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; glycine, glutami Amino acids such as 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; sodium Non-ionic interfaces such as metal complex (eg Zn-protein complex) and / or TWEEN (trade name), PLURONICS (trade name), and polyethylene glycol (PEG) Contains an active agent.

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitor. These molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be used in colloidal drug delivery systems (eg, in microcapsules prepared by coacervation techniques or by interfacial polymerization, eg, hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively , Liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or in microemulsions. These techniques are disclosed in Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. (1980).

The formulation to be used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.
The therapeutic antibody composition is typically placed in a container with a sterile access port, such as an intravenous solution bag or a bag or vial with a stopper pierceable with a hypodermic needle.

  Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of a molded article, such as a film or microcapsule. Examples of sustained release matrices are polyester hydrogels (eg poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate. , Non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D) -3-hydroxy Contains butyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins in a shorter time. When encapsulated antibodies remain in the body for a long time, they denature or aggregate upon exposure to moisture at 37 ° C, resulting in reduced biological activity and possible changes in immunogenicity. . Reasonable methods can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation through thio-disulfide exchange, stabilization may be modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content, appropriate Addition of various additives and the development of specific polymer matrix compositions.

J. et al. Treatment Methods The polypeptides and antibodies, peptides and small molecule agonists of the present invention may be used to treat various tumors such as cancer. Examples of conditions or diseases to be treated include benign or malignant tumors (eg, renal, liver, kidney, bladder, breast, stomach, ovary, colorectal, prostate, pancreas, lung, vulva, Thymus, hepatic carcinoma; sarcoma; glioblastoma; and various head and neck tumors); leukemia and lymphoid malignancies; neurons, glia, astrocytes, hypothalamus and glands, macrophages, epithelium, between Diseases such as quality and blastocoel disease; and inflammation, angiogenesis and immunological diseases. The anti-tumor agents of the present invention (including polypeptides disclosed herein and agonists similar to their activity, including antibodies, peptides and small organic molecules) are known to mammals, preferably humans, in well-known methods, such as boluses. Or intravenously by continuous infusion over time, or intramuscular, intraperitoneal, intracerebral spinal, intraocular, intraarterial, intralesional, subcutaneous, interarticular, intrasynovial, intrathecal, oral, topical, or It is administered by the inhalation route.

  Other therapeutic regimens may be combined with the administration of the anticancer agents of the invention. For example, patients treated with such anti-cancer agents may receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. The preparation method and dosage schedule of such chemotherapeutic agents are either used according to the manufacturer's instructions or determined empirically by skilled practitioners. Preparation methods and dosage schedules for such chemotherapy are also described in Chemotherapy Service M.C. Perry, Williams &amp; Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may be administered prior to or subsequent to the administration of the anti-tumor agent of the present invention, or may be administered simultaneously therewith. The anti-cancer agents of the invention may be combined with doses known for these molecules of anti-estrogenic compounds such as tamoxifen or anti-progesterones such as onapristone (see EP 616812).

  It is also preferred to administer antibodies against tumor-associated antigens, such as antibodies that bind to ErB2, EGFR, ErB3, ErB4, or vascular endothelial factor (VEGF). Sometimes it is also advantageous to administer cytokines to the patient. In a preferred embodiment, the anticancer agent herein is co-administered with a growth inhibitor. For example, the growth inhibitor is first administered, followed by the anticancer agent of the present invention. However, simultaneous administration or administration of the anticancer agent of the present invention first is also conceivable. A suitable dose for a growth inhibitor is the amount currently used, but can be reduced by the combined (synergistic) effect of the growth inhibitor and this antibody.

  The appropriate dose of an anti-tumor agent here for the prevention or treatment of a disease is the type of disease to be treated, the severity and course of the disease as defined above, the drug administered for the purpose of prevention or treatment Whether it is done, prior treatment, the patient's clinical history and response to medication, and the discretion of the attending physician. The drug is suitably administered to the patient at one time or over a series of treatments. Animal studies provide reliable guidance for determining effective doses for human therapy. Effective interspecies scaling of doses is described, for example, in Mordenti J. and Chappell. W. `` The Use of Interspecies Scaling in Toxicokinetics '', Toxicokinetics and New Drug Development, edited by Yacobi et al., Pergamon Press, New York 1989, 42-46. It can be carried out according to the principle as disclosed.

  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 an anti-tumor agent can be administered, eg, one or more separate doses or consecutive This is the first candidate dose to be administered to a patient by infusion. A typical daily dose will be about 1 μg / kg to 100 mg / kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until a desired suppression of disease symptoms appears. However, other dose schedules may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. In particular, guidance on dosage and delivery methods is provided in the literature; see, for example, US Pat. Nos. 4,657,760, 5,206,344 or 5,255,212. Different preparations are effective for different therapeutic compounds and different diseases, for example administration targeted to one organ or tissue needs to be transported differently than other organs or tissues is expected.

K. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided. This product comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a material such as glass or plastic. The container contains a composition effective to diagnose and treat the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). May be). The active agent in the composition is the antitumor agent of the present invention. The label on or on the container indicates that the composition is used for diagnosis or treatment of the selected condition. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The following examples are provided for purposes of illustration only and are not intended to limit the scope of the invention in any way.
All patents and references cited herein are hereby incorporated by reference in their entirety.

(Example)
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The cell source identified by the ATCC registration number throughout the following examples and specification is American Type Culture Collection, Manassas, VA.

Example 1: Extracellular domain homology screening to identify novel polypeptides and cDNAs encoding them
The extracellular domain (ECD) sequences of about 950 known secreted proteins from the Swiss-Prot public database (including secreted signal sequences, if necessary) were used to search the EST database. EST databases include public databases (eg, Dayhoff, GenBank) and proprietary databases (eg, LIFESEQ (trade name), Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altschul et.al., Methods in Enzymology 266: 460-480 (1996)) as a comparison of the ECD protein sequence with the 6-frame translation of the EST sequence. A comparison that does not encode a known protein and has a Blast score of 70 (may be 90) or higher is the program “phrap” (Phil Green, University of Washington, Seattle, WA; http: //bozeman.mbt. washington.edu/phrap.docs/phrap.html) and clustered into consensus DNA sequences.
Using this extracellular domain homology screen, consensus DNA sequences were constructed against other identified EST sequences using phrap. In addition, the resulting consensus DNA sequence is often extended using repeated cycles of BLAST or BLAST2 and phrap (if not all), and the consensus sequence is extended as much as possible using the sources of EST sequences discussed above. It was.
Based on the consensus sequence obtained as described above, oligonucleotides are then synthesized, and a cDNA library containing the sequence of interest is identified by PCR, and a clone of the full-length coding sequence of the PRO polypeptide is isolated. Used for use as a probe. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give PCR products that are about 100-1000 bp long. The probe sequence is typically 40-55 bp long. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5 kbp. To screen several libraries for full-length clones, DNA from the libraries was screened by PCR with PCR primer pairs, such as Ausubel et al., Current Protocols in Molecular Biology. The positive library was then used to isolate clones encoding the gene of interest using one of the probe oligonucleotides and primer pairs.
The cDNA library used for isolation of the cDNA clones was made by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA is primed with oligo dT containing a NotI site, ligated to a SalI hemikinase adapter with blunt ends, cut with NotI, roughly sized by gel electrophoresis, and an appropriate cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain an SfiI site; see Holmes et al., Science, 253: 1278-1280 (1991)), cloned in the specified orientation at unique XhoI and NotI sites.

Example 2: Isolation of cDNA encoding human PRO179 A cDNA clone (DNA16451-1078) encoding native human PRO179 polypeptide was used to express the human fetal kidney preferentially representing the 5 'end of the cDNA clone using yeast screening. Identified from a cDNA library.
The primers used for the identification of DNA16451-1078 are as follows:
OLI114:
5'-CCACGTTGGCTTGAAATTGA-3 '(SEQ ID NO: 3)
OLI115:
5'-CCTTTAGAATTGATCAAGACAATTCATGATTTGATTCTCTATCTCCAGAG-3 '(SEQ ID NO: 4)
OLI116:
5'-TCGTCTAACATAGCAAATC-3 '(SEQ ID NO: 5)
The DNA16451-1078 full-length clone contained a single open reading frame, had an apparent translation start site at nucleotide positions 37-39, and had a stop signal at nucleotide positions 1417-1419 (FIG. 1; SEQ ID NO: 1). The predicted polypeptide precursor is 460 amino acids long. The full-length PRO179 protein is shown in FIG. 2 (SEQ ID NO: 2).
Analysis of the full-length PRO179 sequence shown in FIG. 2 (SEQ ID NO: 2) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO179 sequence demonstrated the following: The signal peptide is approximately at amino acids 1-16. N-glycosylation sites are at approximately amino acids 23-27, approximately 115-119, approximately 296-300, and approximately 357-361. The cAMP- and cGMP-dependent protein kinase phosphorylation sites are at approximately amino acids 100-104, and approximately 204-208. The tyrosine kinase phosphorylation site is approximately at amino acids 342-351. The N-myristoylation site is at approximately amino acids 279-285, approximately amino acids 352-358, approximately amino acids 367-373, and the leucine zipper pattern is approximately at amino acids 120-142, and 127-149.
Clone DNA16451-1078 was deposited with ATCC on September 18, 1997 and was assigned ATCC deposit no. The full-length PRO179 protein shown in FIG. 2 has an estimated molecular weight of approximately 53,637 daltons and a pI of 6.61.
Analysis of the Dayhoff database (version 35.45 SwissProt 35) of the full-length sequence shown in FIG. 2 (SEQ ID NO: 2) shows two known human ligands of the TIE-2 receptor (h-TIE-2L1 and h-TIE-2L2) And the presence of a fibrinogen-like domain showing significant sequence identity. The abbreviation “TIE” is an acronym, meaning “tyrosine kinase-containing Ig and EGF homology domains” and was created to refer to a new family of tyrosine kinase receptors. Therefore, PRO179 was identified as a new member of the TIE ligand family.

Example 3 Isolation of cDNA Encoding Human PRO207 Expression Sequence Tag (EST) DNA database (LIFESEQ (trade name), Incyte Pharmaceuticals, Palo Alto, Calif.) Was searched for ESTs having homology with Apo-2 ligand Was identified. A human fetal kidney cDNA library was screened. MRNA isolated from human fetal kidney tissue (Clontech) was used for the preparation of the cDNA library. This RNA was used to generate a library of oligo dT primed cDNAs in the vector pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plasmid System). In this method, double stranded cDNA was classified into a size exceeding 1000 bp, and SalI / NotI binding cDNA was cloned into an XhoI / NotI digested vector. pRK5D is a cloning vector having a sp6 transcription initiation site, followed by an SfiI restriction enzyme site, and further a XhoI / NotI cDNA cloning site. The library was screened by hybridization with synthetic oligonucleotide probes:
5'-CCAGCCCTCTGCGCTACAACCGCCAGATCGGGGAGTTTATAGTCACCCGG-3 '(SEQ ID NO: 8)
Based on EST.
All cDNA clones were sequenced. The DNA 30879-1152 full-length clone is shown in FIG. 3 (SEQ ID NO: 6). Clone DNA30879-1152 contains a single open reading frame, has an apparent translation start site at nucleotide positions 58-60 (FIG. 3; SEQ ID NO: 6), and an apparent stop signal at nucleotide positions 805-807. had. The predicted polypeptide precursor is 249 amino acids long.
Analysis of the full-length PRO207 sequence shown in FIG. 4 (SEQ ID NO: 7) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO207 sequence (Figure 4; SEQ ID NO: 7) demonstrated the following: The signal peptide is approximately at amino acids 1-40. N-glycosylation site is approximately amino acids 139-143, N-myristoylation site is approximately amino acids 27-33, approximately amino acids 29-35, approximately amino acids 36-42, approximately amino acids 45-51, approximately amino acids 118-124, approximately amino acids. 121-127, approximately amino acids 125-131, and approximately amino acids 128-134, amidation sites are approximately amino acids 10-14 and 97-101, and prokaryotic membrane lipoprotein attachment sites are approximately amino acids 24-35. . Clone DNA30879-1152 was deposited with ATCC on October 10, 1997 and is assigned ATCC deposit number 209358. The full-length PRO207 protein shown in FIG. 4 has an estimated molecular weight of approximately 27,216 daltons and a pI of 9.61.
Based on the BLAST and FastA sequence alignment analysis (using the ALIGN-2 computer program) of the full-length sequence of PRO207 shown in FIG. 4 (SEQ ID NO: 7), PRO207 is amino acid sequence identical to several members of the TNF cytokine family. In particular human lymphotoxin-β (23.4%) and human CD40-ligand (19.8%).

Example 4: Isolation of cDNA encoding human PRO320 Consensus DNA sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA28739. Based on the DNA28739 consensus sequence, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO320. did:
Forward PCR primer
A pair of PCR primers (forward and reverse) were synthesized:
5'-CCTCAGTGGCCACATGCTCA-3 '(SEQ ID NO: 11)
Reverse PCR primer
5'-GGCTGCACGTATGGCTATCCATAG-3 '(SEQ ID NO: 12)
In addition, a synthetic oligonucleotide hybridization probe was made from a DNA28739 sequence having the following nucleic acid sequence:
Hybridization probe
5'-GATAAACTGTCAGTACAGCTGTGAAGACACAGAAGAAGGGCCACAGTGCC-3 '(SEQ ID NO: 13)
In order to screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO320 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal lung tissue (LIB025).
The DNA sequence of the clone isolated as described above gave the full-length sequence of DNA32284-1307 [Figure 5, SEQ ID NO: 9] and the derived protein sequence of PRO320.
The entire nucleic acid sequence of DNA32284-1307 is shown in FIG. 5 (SEQ ID NO: 9). Clone DNA32284-1307 contains a single open reading frame, contains an apparent translation start site at nucleotide positions 135-137, and an apparent stop codon at nucleotide positions 1149-1151. The predicted polypeptide precursor is 338 amino acids long. Analysis of the full-length PRO320 sequence shown in FIG. 6 (SEQ ID NO: 10) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are It is an outline. Analysis of the full-length PRO320 sequence shown in FIG. 6 demonstrated the following: The signal peptide is approximately at amino acids 1-21. The amidation site is approximately amino acids 330-334, the aspartate and asparagine hydroxylation sites are approximately amino acids 109-121, approximately amino acids 191-203, and approximately amino acid sites 236-248, and the cysteine pattern signature of the EGF-like domain is approximately amino acid 80. -91, a calcium binding EGF-like domain is approximately at amino acids 103-125, approximately amino acids 230-252, and approximately amino acids 185-207. Clone DNA32284-1307 has been deposited with ATCC on March 11, 1998 and is assigned ATCC deposit no. The full-length PRO320 protein shown in FIG. 6 has an estimated molecular weight of approximately 37,143 daltons and a pI of 8.92.

Example 5: Isolation of cDNA encoding human PRO219 Consensus DNA sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA28729. Based on the DNA28729 consensus sequence, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO219. did:
A set of PCR primers (forward and reverse) were synthesized:
Forward PCR primer
5'-GTGACCCTGGTTGTGAATACTCC-3 '(SEQ ID NO: 16)
Reverse PCR primer
5'-ACAGCCATGGTCTATAGCTTGG-3 '(SEQ ID NO: 17)

Furthermore, a synthetic oligonucleotide hybridization probe was prepared from the DNA28729 sequence having the following nucleic acid sequence.
Hybridization probe
5'-GCCTGTCAGTGTCCTGAGGGACACGTGCTCCGCAGCGATGGGAAG-3 '(SEQ ID NO: 18)
In order to screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO219 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal kidney tissue.
The DNA sequence of the clone isolated as described above gave the full length sequence of DNA32290-1164 [FIG. 7, SEQ ID NO: 14] and the derived protein sequence of PRO219.
The entire nucleic acid sequence of DNA32290-1164 is shown in FIG. 7 (SEQ ID NO: 14). Clone DNA32290-1164 contains a single open reading frame and contains an apparent translation start site at nucleotide positions 204-206 and an apparent stop codon at nucleotide positions 2949-2951. The predicted polypeptide precursor is 1005 amino acids long. Analysis of the full-length PRO219 sequence shown in FIG. 8 (SEQ ID NO: 15) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO219 sequence shown in FIG. 8 demonstrated the following: The signal peptide is approximately at amino acids 1-23. The N-glycosylation site is approximately amino acids 221-225. The cAMP and cGMP dependent protein kinase phosphorylation sites are approximately amino acids 115-119, approximately amino acids 606-610, and approximately amino acids 892-896. The N-myristoylation sites are approximately amino acids 133-139, approximately 258-264, approximately 299-305, approximately 340-346, approximately 453-459, approximately 494-500, approximately 639-645, approximately 690-694, approximately 752- 758, approximately 792-798. The amidation sites are approximately amino acids 314-318, approximately 560-564, and approximately 601-605. The hydroxylation sites of aspartic acid and asparagine are approximately amino acids 253-265, approximately 294-306, approximately 335-347, approximately 376-388, approximately 417-429, approximately 458-470, approximately 540-552, and approximately 581-593. It is. Clone DNA32290-1164 was deposited with ATCC on October 17, 1997 and is assigned ATCC deposit no. The full-length PRO219 protein shown in FIG. 8 has an estimated molecular weight of approximately 102,233 daltons and a pI of 6.02.
Analysis of full-length PRO219 shown in FIG. 8 (SEQ ID NO: 15) suggests that certain portions have high homology with mouse and human tomatolin-2 precursor polypeptides.

Example 6: Isolation of cDNA encoding human PRO221 Consensus DNA sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA28756. Based on the DNA28756 consensus sequence, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO221. did:
A set of PCR primers (forward and reverse) were synthesized:
Forward PCR primer
5'-CCATGTGTCTCCTCCTACAAAG-3 '(SEQ ID NO: 21)
Reverse PCR primer
5'-GGGAATAGATGTGATCTGATTGG-3 '(SEQ ID NO: 22)
In addition, a synthetic oligonucleotide hybridization probe was made from the DNA28756 sequence having the following nucleic acid sequence:
Hybridization probe
5'-CACCTGTAGCAATGCAAATCTCAAGGAAATACCTAGAGATCTTCCTCCTG-3 '(SEQ ID NO: 23)
In order to screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO221 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal lung tissue.
The DNA sequence of the clone isolated as described above gave the full-length sequence of DNA33089-1132 [Figure 9, SEQ ID NO: 19] and the derived protein sequence of PRO221.
The entire nucleic acid sequence of DNA33089-1132 is shown in FIG. 9 (SEQ ID NO: 19). Clone DNA33089-1132 contains a single open reading frame and contains an apparent translation start site at nucleotide positions 179-181 and an apparent stop codon at nucleotide positions 956-958. The predicted polypeptide precursor is 259 amino acids long. Analysis of the full-length PRO221 sequence shown in FIG. 10 (SEQ ID NO: 20) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are It is an outline. Analysis of the full-length PRO221 sequence shown in FIG. 10 demonstrated the following: The signal peptide is approximately at amino acids 1-33. The transmembrane domain is approximately amino acids 204-219; the N-glycosylation site is approximately amino acids 47-51, approximately amino acids 94-98; cAMP and cGMP-dependent protein kinase phosphorylation sites are approximately amino acids 199-203; N-myristoylation sites. Is approximately at amino acids 37-43, approximately amino acids 45-51, and approximately amino acids 110-116. Clone DNA33089-1132 was deposited with ATCC on September 16, 1997 and is assigned ATCC deposit number 209262. The full-length PRO221 protein shown in FIG. 10 has an estimated molecular weight of approximately 29,275 daltons and a pI of 6.92.
Analysis of the full-length PRO221 shown in FIG. 10 (SEQ ID NO: 20) shows that full-length PRO221 has significant homology with the leucine-rich repeat protein spar family that includes the SLIT protein.

Example 7: Isolation of cDNA encoding human PRO224 Consensus DNA sequences were constructed against other EST sequences using pharp as described in Example 1 above. This consensus sequence is herein designated DNA30845. Based on the DNA30845 consensus sequence, synthesized oligonucleotides for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO224. did:
A set of PCR primers (forward and reverse) were synthesized:
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 was made from DNA30845 sequence with the following nucleic acid sequence:
Hybridization probe
5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAATGCCCACC-3 '(SEQ ID NO: 28)
In order to screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO224 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal liver tissue.
Determination of the DNA sequence of the clone isolated as described above yielded the full-length sequence of DNA33221-1133 [FIG. 11, SEQ ID NO: 24] and the derived protein sequence of PRO224.
The entire nucleic acid sequence of DNA33221-1133 is shown in FIG. 11 (SEQ ID NO: 24). Clone DNA33221-1133 contains a single open reading frame and contains an apparent translation start site at nucleotide positions 33-35 and an apparent stop codon at nucleotide positions 879-881. The predicted polypeptide precursor is 282 amino acids long. Analysis of the full-length PRO224 sequence shown in FIG. 12 (SEQ ID NO: 25) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above It is an outline. Analysis of the full-length PRO224 sequence shown in FIG. 12 demonstrated the following: The signal peptide is approximately at amino acids 1-30. The transmembrane domain is approximately at amino acids 231-248. The N-glycosylation site is at approximately amino acids 126-130, approximately 195-199, and approximately 213-217. N-myristoylation sites are approximately amino acids 3-9, approximately 10-16, approximately 26-32, approximately 30-36, approximately 112-118, approximately 166-172, approximately 212-218, approximately 224-230, approximately 230- 236, and approximately 263-269. Prokaryotic membrane lipoprotein lipid attachment sites are approximately at amino acids 44-55. The leucine zipper pattern is approximately at amino acids 17-39. Clone DNA33221-1133 was deposited with ATCC on September 16, 1997 and is assigned ATCC deposit number 209263. The full-length PRO224 protein shown in FIG. 12 has an estimated molecular weight of approximately 28,991 daltons and a pI of 4.62.
Analysis of full-length PRO224 shown in FIG. 12 (SEQ ID NO: 25) shows that full-length PRO224 has homology with low density lipoprotein receptor, apolipoprotein E receptor, and chicken oocyte receptor P95. Suggest. Based on BLAST and FastA sequence alignment analysis of the full-length sequences, PRO224 has amino acid identity with portions of these proteins in the range of 28% to 45% and overall with these proteins in the range of 33% to 39%. Have identity.

Example 8: Isolation of cDNA clones encoding human PRO328 DNA consensus sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence was herein designated DNA35615. Based on this DNA35615 consensus sequence, oligonucleotides can be used to 1) identify a cDNA library containing the sequence of interest by PCR, and 2) use as a probe to isolate a clone of the full-length coding sequence of PRO328. Synthesized.
A pair of PCR primers (forward and reverse) were synthesized:
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 generated from the consensus DNA35615 sequence.
Hybridization probe
5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3 '(SEQ ID NO: 33)
To screen several libraries for a source of full-length clones, DNA from the libraries was screened by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate clones encoding the PRO328 gene using one of the probe oligonucleotides and PCR primers. RNA for construction of the cDNA library was isolated from human fetal kidney tissue.
DNA sequencing of the clones isolated as described above resulted in the full-length DNA sequence of DNA40587-1231 [Figure 13, (SEQ ID NO: 29)] and the derived protein sequence of PRO328.
The entire nucleotide sequence of DNA40587-1231 is shown in FIG. 13 (SEQ ID NO: 29). Clone DNA40587-1231 contains a single open reading frame, has an apparent translation start site at nucleotide positions 15-17, and has an apparent stop codon at nucleotide positions 1404-1406. The predicted polypeptide precursor is 463 amino acids long. Analysis of the full-length PRO328 sequence shown in FIG. 14 (SEQ ID NO: 30) demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO328 sequence shown in FIG. 14 demonstrated the following: The signal peptide is approximately at amino acids 1-22. The N-glycosylation site is at approximately amino acids 114-118, approximately 403-407, approximately 409-413. The glycosaminoglycan attachment site is approximately amino acids 439-443. N-myristoylation sites are at approximately amino acids 123-129, approximately 143-149, approximately 152-158, approximately 169-175, approximately 180-186, approximately 231-237, and approximately 250-256. The amidation sites are approximately amino acids 82-88 and 172-176. The peroxidase proximal heme ligand signature is approximately at amino acids 287-298. The extracellular protein SCP / Tpx-1 / Ag5 / PR-1 / Sc7 signature 1 domain is approximately amino acids 127-138, and the extracellular protein SCP / Tpx-1 / Ag5 / PR-1 / Sc7 signature 2 domain is approximately 160-172. It is in. Clone DNA40587-1231 was deposited with ATCC on November 7, 1997 and was assigned ATCC deposit no. The full-length PRO328 protein shown in FIG. 14 has an estimated molecular weight of approximately 49,471 daltons and a pI of 5.36.
Analysis of full-length PRO328 shown in FIG. 14 (SEQ ID NO: 30) suggests that a portion of the full-length PRO328 sequence has significant homology with human glioblastoma and cysteine-rich secreted proteins, thereby Are novel glioblastomas or cysteine-rich secreted proteins.

Example 9: Isolation of cDNA encoding human PRO301 DNA consensus sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence is designated herein as DNA35936. Based on the DNA35936 consensus sequence, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO301. did.
The oligonucleotides used in the above means are as follows.
Forward PCR primer 1:
5'-TCGCGGAGCTGTGTTCTGTTTCCC-3 '(SEQ ID NO: 36)
Forward PCR primer 2:
5'-ACACCTGGTTCAAAGATGGG-3 '(SEQ ID NO: 37)
Forward 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 as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO301 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal kidney tissue.
Determination of the DNA sequence of the clone isolated as described above yielded the full-length sequence of DNA40628-1216 [FIG. 15, SEQ ID NO: 34] and the derived protein sequence of PRO301.
The entire nucleic acid sequence of DNA40628-1216 is shown in FIG. 15 (SEQ ID NO: 34). Clone DNA40628-1216 contains a single open reading frame, an apparent translation start site at nucleotide positions 52-54, and an apparent stop codon at nucleotide positions 949-951. The predicted polypeptide precursor is 299 amino acids long. Analysis of the full-length PRO301 sequence shown in FIG. 16 (SEQ ID NO: 35) demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO301 sequence shown in FIG. 16 demonstrated the following: The signal peptide is approximately at amino acids 1-27. The transmembrane domain is approximately at amino acids 235-256. The N-glycosylation site is approximately at amino acids 185-189 and the cAMP- and cGMP-dependent protein kinase phosphorylation sites are approximately at amino acids 270-274. The N-myristoylation sites are at approximately amino acids 105-111, approximately 116-122, approximately 158-164, approximately 219-225, approximately 237-243, and approximately 256-262. Clone DNA40628-1216 was deposited with ATCC on November 7, 1997 and is assigned ATCC deposit number 209432. The full-length PRO301 protein shown in FIG. 16 has an estimated molecular weight of approximately 32,583 daltons and a pI of 8.29.
Based on the BLAST and FastA sequence alignment analysis of the full-length PRO301 sequence shown in FIG. 16 (SEQ ID NO: 35), PRO301 is identical in amino acid sequence to A33 antigen precursor (30%), coxsackie and adenovirus (29%). Showing gender.

Example 10: Isolation of cDNA encoding human PRO526 DNA consensus sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence is designated herein as DNA39626.init. In addition, using the source of EST sequences as discussed above, the initial consensus sequence was extended using BLAST and repeated cycles of the program “phrap” to extend the initial consensus sequence as much as possible. The constructed consensus sequence was named <consen01> here. <consen01> Based on the consensus sequence, 1) to identify a cDNA library containing the sequence of interest by PCR, and 2) to use as a probe to isolate a clone of the full-length coding sequence of PRO526 Was synthesized.
A pair of PCR primers (forward and reverse) were synthesized.
Forward PCR primer:
5'-TGGCTGCCCTGCAGTACCTCTACC-3 '(SEQ ID NO: 44)
Reverse PCR primer:
5'-CCCTGCAGGTCATTGGCAGCTAGG-3 '(SEQ ID NO: 45)
In addition, a <consen01> consensus sequence was constructed in which the synthetic oligonucleotide hybridization probe had the nucleotide sequence shown below:
Hybridization probe:
5'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCACAC-3 '(SEQ ID NO: 46)
In order to screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO526 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal liver tissue (LIB228).
Determination of the DNA sequence of the clone isolated as described above yielded the full-length sequence of DNA44184-1319 [FIG. 17, SEQ ID NO: 42] and the derived protein sequence of PRO526.
The entire nucleic acid sequence of DNA44184-1319 is shown in FIG. 17 (SEQ ID NO: 42). Clone DNA44184-1319 contains a single open reading frame, contains an apparent translation start site at nucleotide positions 514-516, and an apparent stop codon at nucleotide positions 1933-1935. The predicted polypeptide precursor is 473 amino acids long. Analysis of the full-length PRO526 sequence shown in FIG. 18 (SEQ ID NO: 43) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO526 sequence shown in FIG. 18 demonstrated the following: The signal peptide is approximately at amino acids 1-26. The leucine zipper pattern is approximately amino acids 135-157. The glycosaminoglycan attachment site is approximately amino acids 436-440. The N-glycosylation sites are approximately amino acids 82-86, approximately 179-183, approximately 237-241, approximately 372-376, and approximately 423-427. The von Willebrand factor (VWF) type C domain is found at approximately amino acids 411-427. Clone DNA44184-1319 was deposited with ATCC on March 26, 1998 and was assigned ATCC deposit number 209704. The full-length PRO526 protein shown in FIG. 18 has an estimated molecular weight of approximately 50,708 daltons and a pI of 9.28.
Analysis of the full-length PRO526 sequence shown in FIG. 18 (SEQ ID NO: 43) shows that a portion thereof has significant homology with leucine repeat proteins including ALS, SLIT, carboxypeptidase, and platelet glycoprotein V. This suggests that PRO526 is a novel protein involved in protein-protein interactions.

Example 11: Isolation of cDNA encoding human PRO362 DNA consensus sequences were constructed against other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA42257. Based on the DNA42257 consensus sequence, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO362. did.
A set of PCR primers (forward and reverse orientation) were synthesized:
Forward 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 was constructed from the DNA42257 consensus sequence having the nucleotide sequence shown below.
Hybridization probe:
5'-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCTGAT-3 '(SEQ ID NO: 53)
In order to screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO362 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal brain tissue (LIB153).
Determination of the DNA sequence of the clone isolated as indicated above yielded the full-length sequence of DNA45416-1251 [FIG. 19, SEQ ID NO: 47] and the derived protein sequence of PRO362.
The entire nucleic acid sequence of DNA45416-1251 is shown in FIG. 19 (SEQ ID NO: 47). Clone DNA45416-1251 contains a single open reading frame, contains an apparent translation start site at nucleotide positions 119-121, and an apparent stop codon at nucleotide positions 1082-1108. The predicted polypeptide precursor is 321 amino acids long. Analysis of the full-length PRO362 sequence shown in FIG. 20 (SEQ ID NO: 48) demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO362 sequence shown in FIG. 20 demonstrated the following: The signal peptide is approximately at amino acids 1-19. The transmembrane domain is approximately at amino acids 281-300. The attachment site for glycosaminoglycans is approximately at amino acids 149-153. The cAMP- and cGMP-dependent protein kinase phosphorylation sites are approximately at amino acids 308-312. The N-myristoylation sites are at approximately amino acids 2-8, approximately 148-154, approximately 158-164, approximately 207-213, and approximately 215-221. Clone DNA45416-1251 was deposited with ATCC on February 5, 1998 and was assigned ATCC deposit no. The full-length PRO362 protein shown in FIG. 20 has an estimated molecular weight of approximately 35,544 daltons and a pI of 8.51.
Analysis of the full-length PRO362 sequence shown in FIG. 20 (SEQ ID NO: 48) suggests that the full-length PRO362 sequence has significant similarity to the A33 antigen protein and the HCAR protein. More specifically, analysis of the Dayhoff database (version 33.45 SwissProt 35) shows that the PRO362 amino acid sequence and the following Dayhoff sequences: AB002341_1, HSU55258_1, HSC7NRCAM_1, RNU81037_1, A33_HUMAN, P_W14158, NMNCAMRI_1, HSTITINN2_1, S71824_1, and HSU63041_1 Significant sequence similarity was demonstrated.

Example 12: Isolation of cDNA Encoding Human PRO356 The sequence tag (EST) DNA database expressed (LIFESEQ (trade name), Incyte Pharmaceuticals, Palo Alto, CA) was searched and EST (# 29939340) is PRO179 [ Identified in Example 2 and identified as DNA 16451-1078 (FIG. 1; SEQ ID NO: 1)]. A human fetal lung separation library prepared with mRNA purchased from Clontech Inc. (Palo Alto, CA), catalog # 65528-1 was used according to the manufacturer's instructions for cloning PRO356.
The cDNA library used to isolate the cDNA clone encoding human PRO356 was generated by standard methods using, for example, reagents commercially available from Invitrogen, San Diego, CA. The cDNA is primed with oligo dT containing a NotI site, ligated to a SalI hemikinase adapter with blunt ends, cut with NotI, approximated by gel electrophoresis, and the appropriate cloning vector (pRKB or pRKD; pRK5B Is a precursor of pRK5D that does not contain an SfiI site; see Holmes et al., Science, 253: 1278-1280 (1991)), cloned in the specified orientation at unique XhoI and NotI sites.
Based on the ESTs shown above, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO356. did. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give PCR products that are about 100-1000 bp long. The probe sequence is typically 40-55 bp long. To screen several libraries for full-length clones, DNA from the libraries was screened by PCR amplification with PCR primer pairs, such as Ausubel et al., Current Protocols in Molecular Biology. A positive library was used to isolate clones encoding the gene of interest using one of the probe oligonucleotides and 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 cDNA clone was identified and fully sequenced. The complete nucleotide sequence of DNA47470-1130-P1 is shown in FIG. 21 (SEQ ID NO: 54). Clone DNA47470-1130-P1 contains a single open reading frame, an apparent translation start site at nucleotide positions 215-217, and an apparent stop codon at nucleotide positions 1253-1255 (FIG. 21, SEQ ID NO: 54). ). The predicted polypeptide precursor is 346 amino acids long, has a calculated molecular weight of approximately 40,018 daltons, and an estimated pI of 8.19. The full-length PRO356 protein is shown in FIG. 22 (SEQ ID NO: 55).
Analysis of the full-length PRO356 sequence shown in FIG. 22 (SEQ ID NO: 55) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO356 sequence shown in Figure 22 demonstrated the following: The signal peptide is approximately at amino acids 1-26. N-glycosylation sites are approximately at amino acids 58-62, approximately 253-257, and approximately 267-271. The glucosaminoglycan adhesion site is approximately 167-171 and the cAMP- and cGMP-dependent protein kinase phosphorylation sites are approximately at amino acids 176-180. N-myristoylation sites are at approximately amino acids 168-174, approximately 196-202, approximately 241-247, approximately 252-258, approximately 256-262, approximately 327-333. The cell adhesion site is approximately in the amino acid sequence 199-202.
Clone DNA47470-1130-P1 was deposited with ATCC on October 28, 1997 and was assigned ATCC deposit number 209422. The deposited clone actually has a more accurate sequence than that shown here.
Analysis of the Dayhoff database (version 35.45 SwissProt 35) using the ALIGN-2 sequence alignment analysis of the full-length sequence shown in FIG. 22 (SEQ ID NO: 55) Amino acid sequence identity between both 2L2 (34%) is shown. The abbreviation “TIE” stands for “tyrosine kinase containing Ig and EGF homology domains” and was created to refer to a new family of receptor tyrosine kinases.

Example 13: Isolation of cDNA Encoding Human PRO509 To isolate the DNA 50148-1068 cDNA, a bacteriophage library of human retinal cDNA (commercially available from Clontech) exhibits some homology to the TNFR family. Screening was performed by hybridization with a synthetic oligonucleotide probe based on the EST sequence (GenBank locus AA021617). The oligonucleotide probe used in this screening was 60 bp long. Five positive clones (including a 1.8-1.9 Kb cDNA insert) were obtained from the cDNA library, and these five positive clones were targeted by PCR using the hybridization probes shown above as PCR primers. It was confirmed that Single phage plaques containing each positive clone were isolated by limiting dilution and the DNA was purified using the Wizard Lambda Prep DNA purification kit (commercially available from Promega).
CDNA from three of the five bacteriophage clones was excised from the vector arm by EcoRI digestion and gel purification and further subcloned into pRK5 and sequenced on both strands. The three clones had the same open reading frame (with the exception that an intron was found in one clone).
The complete nucleotide sequence of DNA50148-1068 is shown in Figure 23 (SEQ ID NO: 59). The cDNA contains one open reading frame, which has a start site assigned to the ATG codon at nucleotide positions 82-84. This open reading frame ends with a stop codon TGA at nucleotide positions 931-933.
The predicted full-length amino acid sequence of PRO509 is 283 amino acids long. The full-length PRO509 protein is shown in FIG. 24 (SEQ ID NO: 60) and has an estimated molecular weight of approximately 30,420 daltons and approximately pI7.34.
Analysis of the full-length PRO509 sequence shown in FIG. 24 (SEQ ID NO: 60) demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO509 sequence shown in Figure 24 (Figure 24 (SEQ ID NO: 60)) demonstrated the following: The signal peptide is approximately at amino acids 1-36. The transmembrane protein is approximately at amino acids 205-221. N-glycosylation sites are at approximately amino acids 110-114, and approximately 173-177. , N-myristoylation sites are at approximately amino acids 81-87, approximately 89-95, approximately 104-110, approximately 120-126, approximately 153-159, approximately 193-199, approximately 195-201, and approximately 220-226. . The cell adhesion site is approximately in the amino acid sequence 231-234.
Alignment of PRO509's 58 amino acid long cytoplasmic region with members of the known human TNF receptor family (using the ALIGN ^™ computer program) reveals several sequence similarities, particularly CD40 (12 identities) and LT -Showed beta receptor (11 identity).

Example 14: Isolation of cDNA encoding PRO866 DNA consensus sequences were constructed relative to other EST sequences using phrap as described in Example 1 above. This consensus sequence is herein designated DNA42257. Based on the DNA44708 consensus sequence, oligonucleotides were synthesized for 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO866. did.
PCR primers (forward and reverse) were synthesized:
Forward 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 was constructed from DNA44708 having the following nucleotide sequence:
Hybridization probe:
5'-CAGCCCCTTCTCCTCCTTTCTCCCACGTCCTATCTGCCTCTC-3 '(SEQ ID NO: 69)
To screen several libraries as a source of full-length clones, DNA from the libraries was selected by PCR amplification with the PCR primer pairs identified above. The positive library was then used to isolate a clone encoding the PRO866 gene using one of the probe oligonucleotide and PCR primer pair. RNA for construction of the cDNA library was isolated from human fetal kidney tissue (LIB228).
Determination of the DNA sequence of the clone isolated as indicated above yielded the full-length sequence of DNA53971-1359 [Figure 25, SEQ ID NO: 61] and the derived protein sequence of PRO866.
The entire nucleic acid sequence of DNA53971-1359 is shown in FIG. 25 (SEQ ID NO: 61). Clone DNA53971-1359 contains a single open reading frame, contains an apparent translation start site at nucleotide positions 275-277, and an apparent stop codon at nucleotide positions 1268-1270. The predicted polypeptide precursor is 331 amino acids long. Analysis of the full-length PRO866 sequence shown in FIG. 26 (SEQ ID NO: 62) has demonstrated the existence of various important polypeptide domains, but the positions given for those important polypeptide domains are as described above. It is an outline. Analysis of the full-length PRO866 sequence shown in FIG. 26 demonstrated the following: The signal peptide is approximately at amino acids 1-26. The glycosaminoglycan adhesion site is approximately at amino acids 131-135. The cAMP- and cGMP-dependent protein kinase phosphorylation sites are approximately at amino acids 144-148. N-myristoylation sites are at approximately amino acids 26-32, approximately 74-80, approximately 132-138, approximately 134-140, approximately 190-196, approximately 287-293, and approximately 290-296. Clone DNA53971-1359 was deposited with ATCC on April 7 , 1998 and was assigned ATCC deposit no. The full-length PRO866 protein shown in FIG. 26 has an estimated molecular weight of approximately 35,844 daltons and a pI of 5.45.
Analysis of the amino acid sequence of the full-length PRO866 polypeptide shown in FIG. 26 (SEQ ID NO: 62) suggests that it has significant sequence similarity with the mindin / spondin family, which Show that PRO866 can be a novel Mindin homologue. More specifically, the Dayhoff database (version 35.45 SwissProt 35) is a significant homology between the PRO866 amino acid sequence and the following Dayhoff sequences: AB006085_1, AB006084_1, AB006086_1, AF017267_1, CWU42213_1, AC004160_1, CPMICRP_1, S49108, A48569 and I46687. Demonstrated sex.

Example 15: In situ hybridization In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences in cell or tissue preparations. It is useful, for example, for identification of gene expression sites, analysis of tissue distribution of transcripts, identification and localization of viral infection, specific mRNA synthesis and chromosomal mapping.
Situ hybridization, Lu and Gillett, Cell Vision 1: according to the optimal deformation of the protocol 169-176 (1994), using PCR-generated ^{33 P-} labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissue was sectioned, deparaffinized, deproteinized with proteinase K (20 g / ml) for 15 minutes at 37 ° C., and as described in Lu and Gillett, supra. In situ hybrid formation. [ ³³ P] UTP-labeled antisense riboprobe is generated from the PCR product and hybridized at 55 ° C. overnight. The slides are immersed in Kodak NTB2 ^™ nuclear track emulsion and exposed for 4 weeks.

³³ P- riboprobe synthesis
6.0μl of (125mCi) of ^{33 P-UTP (Amersham BF 1002} , SA <2000 Ci / mmol) were speed vacuum drying. The following ingredients were added to a tube containing dry ³³ P-UTP:
2.0 μl of 5x transcription buffer
1.0 μl of DTT (100 mM)
NTP mixture 2.0μl (2.5mM: 10μ; 10mM of GTP, CTP; ATP + 10μl of H ₂ O)
1.0 μl UTP (50 μM)
1.0 μl of RNAsin
1.0 μl DNA template (1 μg)
1.0 μl H ₂ O
1.0 μl of RNA polymerase (T3 = AS, T7 = S, normal for PCR products)
Tubes were incubated at 37 ° C. for 1 hour, 1.0 μl RQ1 DNase was added, and then incubated at 37 ° C. for 15 minutes. 90 μl TE (10 mM Tris pH 7.6 / 1 mM EDTA pH 8.0) was added and the mixture was pipetted onto DE81 paper. The remaining solution was loaded onto a Microcon-50 ^™ ultrafiltration unit and spun using program 10 (6 minutes). The filtration unit was converted to a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE was added. 1 μl of final product was pipetted onto DE81 paper and counted with 6 ml of Biofluor II ^™ .
The probe was run on a TBE / urea gel. 1-3 μl probe or 5 μl RNA MrkIII was added to 3 μl loading buffer. After heating on a heating block to 95 ° C. for 3 minutes, the gel was immediately placed on ice. Gel wells were flushed, sample loaded and run at 180-250 volts for 45 minutes. The gel was wrapped in plastic wrap (SARAN ^TM brand) and exposed overnight from 1 hour to XAR film with an intensifying screen at -70 ° C. freezer.

³³ P-hybridization Pretreatment of frozen sections Slides were removed from the refrigerator, placed on an aluminum tray and thawed at room temperature for 5 minutes. The tray was placed in a 55 ° C. incubator for 5 minutes to reduce condensation. The slides were fixed for 5 min in 4% paraformaldehyde in the fume hood and washed at room temperature for 5 minutes with 0.5xSSC (25ml 20xSSC + 975ml SQ H 2 O). After deproteinization for 10 minutes at 37 ° C. in 0.5 μg / ml proteinase K (12.5 μl of a 10 mg / ml stock in 250 ml preheated RNase-free RNase buffer), the sections were washed with 0.5 × SSC for 10 minutes at room temperature. Sections were dehydrated in 70%, 95%, 100% ethanol for 2 minutes each.
B. Pretreatment of paraffin-embedded sections Slides were deparaffinized, placed in SQ H ₂ O and rinsed twice with 2 × SSC for 5 minutes each at room temperature. Sections are 20 μg / ml proteinase K (500 μl 10 mg / ml in 250 ml RNase-free RNase buffer; 37 ° C., 15 minutes) -human embryo or 8x proteinase K (100 μl in 250 ml RNase buffer, 37 ° C., 30 minutes) — Deproteinized with formalin tissue. Subsequent rinsing and dehydration with 0.5 × SSC was performed as described above.
C. Prehybridization Slides were arranged in plastic boxes lined with Box buffer (4 × SSC, 50% formamide) -saturated filter paper. The tissue was coated with 50 μl of hybridization buffer (3.75 g dextran sulfate + 6 ml SQ H ₂ O), vortexed, uncapped and heated in the microwave for 2 minutes. After cooling on ice, 18.75 ml formamide, 3.75 ml 20 × SSC and 9 ml SQ H ₂ O were added and the tissue was vortexed well and incubated at 42 ° C. for 1-4 hours.

D. Hybridization 1.0 × 10 ⁶ cpm probe and 1.0 μl tRNA (50 mg / ml stock) per slide were heated at 95 ° C. for 3 minutes. Slides were chilled on ice and 48 μl of hybridization buffer was added per slide. After vortexing, 50 μl of ³³ P mixture was added to 50 μl of prehybrid on the slide. Slides were incubated overnight at 55 ° C.
E. Washing:
Washing was performed with 2 × SSC, EDTA for 2 × 10 minutes at room temperature (400 ml 20 × SSC + 16 ml 0.25 M EDTA, V _f = 4 L), followed by RNase A treatment for 30 minutes at 37 ° C. (500 μl of 10 mg / ml in 250 ml RNase buffer = 20 μg / ml). Slides were washed 2 x 10 minutes with EDTA at room temperature. Stringent wash conditions are as follows: 2 hours at 55 ° C., 0.1 × SSC, EDTA (20 ml 20 × SSC + 16 ml EDTA, V _f = 4 L).

F. Oligonucleotides In situ analysis was performed on two of the DNA sequences disclosed herein. The oligonucleotides used for these analyzes are as follows:
(1) DNA30879-1152 (PRO207)
p1:
5'-GGATTCTAATACGACTCACTATAGGGCTCCTGCGCCTTTCCTGAACC-3 '(SEQ ID NO: 70)
p2:
5'-CTATGAAATTAACCCTCACTAAAGGGAGACCCATCCTTGCCCACAGAG-3 '(SEQ ID NO: 71)
(2) DNA33089-1132 (PRO221)
p1:
5'-GGATTCTAATACGACTCACTATAGGGCTGTGCTTTCATTCTGCCAGTA-3 '(SEQ ID NO: 72)
p2:
5'-CTATGAAATTAACCCTCACTAAAGGGAGGGTACAATTAAGGGGTGGAT-3 '(SEQ ID NO: 73)
(3) DNA33221-1133 (PRO224)
p1:
5'-GGATTCTAATACGACTCACTATAGGGCGCAGCGATGGCAGCGATGAGG-3 '(SEQ ID NO: 74)
p2:
5'-CTATGAAATTAACCCTCACTAAAGGGACAGACGGGGCAGCAGGGAGTG-3 '(SEQ ID NO: 75)
(4) DNA40628-1216 (PRO301)
p1:
5'-GGATTCTAATACGACTCACTATAGGGCGAGTCCTTCGGCGGCTGTT-3 '(SEQ ID NO: 76)
p2:
5'-CTATGAAATTAACCCTCACTAAAGGGACGGGTGCTTTTGGGATTCGTA-3 '(SEQ ID NO: 77)
(5) DNA45416-1251 (PRO362)
p1:
5'-GGATTCTAATACGACTCACTATAGGGCCTCCAAGCCCACAGTGACAA-3 '(SEQ ID NO: 78)
p2:
5'-CTATGAAATTAACCCTCACTAAAGGGACCTCCACATTTCCTGCCAGTA-3 '(SEQ ID NO: 79)

G. Results In situ analysis was performed on the five DNA sequences disclosed herein. The results from these analyzes are as follows:
(1) DNA30879-1152 (PRO207) (Apo2L Homolog)
Low levels of expression were observed in chondroblastoma and one other soft tissue sarcoma. In all other organizations, it was negative.
Human fetal tissues tested (E12-E16) include: placenta, umbilical cord, liver, kidney, adrenal gland, thyroid, lung, heart, large blood vessel, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, Spinal cord, body wall, pelvis and lower limbs.
Adult human tissues tested included: kidney (normal and end stage), adrenal gland, myocardium, aorta, spleen, lymph node, gallbladder, pancreas, lung, skin, eye (including retina), placenta, bladder, and liver ( Normal, chronic, acute).
Non-human primates tested include:
Chimpanzee tissues: salivary glands, stomach, thyroid, parathyroid, skin, thymus, ovaries, lymph nodes Rhesus monkey tissues: cerebral cortex, hippocampus, cerebellum, penis.
(2) DNA33089-1132 (PRO221) (1 TM receotor)
Specific expression was observed across fetal brain and gray matter and spinal cord neurons. The probe appears to cross react with the rat. Low levels of expression in cerebellar neurons of adult rhesus monkey brain. All other tissues are negative.
Human fetal tissues examined (E12-E16 weeks) include: placenta, umbilical cord, liver, kidney, adrenal gland, thyroid, lung, heart, large blood vessel, canteen, stomach, small intestine, spleen, thymus, pancreas, brain , Eyes, spinal cord, body wall, pelvis and lower limbs.
Adult tissues examined: liver, kidney, adrenal gland, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rhesus monkey), hippocampus (rhesus monkey), cerebellum (rhesus monkey), penis, eye, bladder, stomach Gastric cancer, colon, colon cancer and chondrosarcoma. Acetominophen-induced liver injury and cirrhosis.

(3) DNA33221-1133 (PRO224) (LDLR homolog-1 TM)
Expression was limited to the vascular endothelium of fetal spleen, adult spleen, fetal liver, adult thyroid and adult lymph node (chimpanzee). Additional sites of expression were found in the developing spinal ganglia. All other tissues were negative.
Human fetal tissues tested (E12-E16) include: placenta, umbilical cord, liver, kidney, adrenal gland, thyroid, lung, heart, large blood vessel, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, Spinal cord, body wall, pelvis and lower limbs.
Adult human tissues tested include: kidney (normal and end stage), adrenal gland, myocardium, aorta, spleen, lymph node, gallbladder, pancreas, lung, skin, eye (including retina), bladder, liver (normal, chronic) Acute disorder).
Non-human primates tested include:
Chimpanzee tissues: salivary glands, stomach, thyroid, parathyroid, skin, thymus, ovaries, lymph nodes Rhesus monkey tissues: cerebral cortex, hippocampus, cerebellum, penis.
(4) DNA40628-1216 (PRO301) (CD22 homolog (JAM hlog, A33 Ag hlog)
Expression in inflammatory human tissues (psoriasis, IBD, inflammatory kidney, inflammatory lung, hepatitis, normal tonsils, adults and chimpanzee multiblock):
Expression was assessed primarily in inflammatory human tissues and in a few normal human and non-human primate tissues. Expression is found in colonic mucosal epithelium, tracheal airway epithelium, oral mucosa (tongue), tonsil crypt mucosa, placenta mucosa, prostate mucosa, glandular gastric mucosa, epithelial cells, thymic Hassal body, hepatocytes, bile duct epithelium, and Found in each epithelial structure evaluated including placental epithelium. The only evidence of expression outside the epithelial structure was weak, low, inconsistent expression at the germinal center of the follicle in tonsils with reactive thickening.
In non-human primate organizations:
Chimpanzee tissue: weak diffusible expression in epidermis of tonsillar epithelium; weak expression in thymic epithelium of Hassal body; mild diffusible expression observed in glandular mucosal epithelium in stomach .
In human tissues: Diffusible, low to moderate expression was present in hepatocytes and bile duct epithelium in the liver (multiblock including chronic cholangitis, lobular hyperplasia, acetaminophen toxicity). Expression was most prevalent in perilobular / periportal hepatocytes. It was the most prevalent in the bile duct epithelium in liver fragments with chronic sclerosing cholangitis.
Weak expression was observed in epidermis psoriasis samples.
In lungs with chronic interstitial pneumonia or chronic bronchitis, low diffusible expression was seen in the mucosal epithelium of the airways; weak diffusible expression was also seen in the alveolar epithelium. There was no expression in the epithelium of bronchial / bronchiolar submucosal glands.
Moderate diffuse expression was observed in both the placental epithelium and the mucosal epithelium of the gallbladder.
Low diffusible expression was seen in the prostate epithelium.
High diffusive expression was observed in the tonsil mucosa and crypt epithelium; the signal was highest in the mucosal cells that lined the tonsil crypt. There was weak, inconsistent diffusible expression in the germinal center of the cortical follicle (B lymphocyte region); however, in other tissues evaluated with lymphatic structure or lymphocytic inflammation, expression in B lymphocytes There was no.
In colons with inflammatory bowel disease and polyp / adenomatous changes, low expression in mucosal epithelium was seen, with expression highest at the villi tip. In one specimen with a polyp, there was no evidence of increased expression in the dysplastic epithelium when compared to the adjacent mucosa. There was no obvious expression in reactive mucosal lymphoid tissue present in many fragments. Tissue without expression includes: heart, peripheral nerve, and muscle (heart, smooth).

(5) DNA45416-1251 (PRO362) (Ig domain homolog)
Expression in inflammatory human tissues (psoriasis, IBD, inflammatory kidney, inflammatory lung, hepatitis, normal tonsils, adults and chimpanzee multiblock):
The expression of this novel protein has been evaluated in various human and non-human primate tissues and found to be highly restricted. Expression was only present in lung alveolar macrophages and liver sinusoidal Kupffer cells. Expression in these cells was significantly increased when these distinguishable cell populations were activated. These two subpopulations of tissue macrophages are localized in different organs and they have similar biological functions. Both these types of phagocytic cells act as filters to remove substances from the bloodstream or respiratory tract, including pathogens, senescent cells and proteins, and can both secrete a wide range of important proinflammatory cytokines.
In inflammatory lung (7 patient samples), expression is prominent in the reactive alveolar macrophage cell population, determined by large, pale, often vacuolar cells present in the alveoli as single or aggregates, normal And non-reactive macrophages (normal size single scattered cells) were weak to negative. Expression in alveolar macrophages increased during inflammation when these cells increased (activated) in number and size. Despite the presence of histocytes in the areas of interstitial inflammation and peribronchial lymphocyte hyperplasia in these tissues, expression was restricted to alveolar macrophages. Many inflammatory lungs also had some inflammation suppression; expression was absent from neurotrophic granulocytes.
In the liver, there was strong expression of reactive / activated Kupffer cells in the liver with acute central lobular necrosis (acetaminophen toxicity) or fairly significant periportal inflammation. However, there was no expression in Kupffer cells in normal liver or liver with only mild to moderate lobular hyperplasia / hypertrophy. That is, there was an increase in expression in activated / reactive cells, as in the lung.
There was no expression of this molecule in histocytes / macrophages present in inflammatory bowel disease, hyperplasia / reactive tonsils or normal lymph nodes. The lack of expression in these tissues, all including histiocytic inflammation or resident macrophage populations, strongly supports expression restricted to a unique macrophage subset population defined as alveolar macrophages and liver Kupffer cells. Spleen or bone marrow could not be evaluated.
Human tissues evaluated that did not show detectable expression included: inflammatory bowel disease (7 moderate to severe patient samples), tonsils with reactive hyperplasia, peripheral lymph nodes, psoriatic skin (mild to moderate) 2 patient samples with moderate disease), heart, peripheral nerve.
The chimpanzee tissues evaluated that did not show detectable expression included: tongue, stomach, thymus.

Example 16: Use of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 as hybridization probes The following methods are used for PRO179, PRO207, PRO320, PRO219, Describes the use of nucleic acid sequences encoding PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 as hybridization probes.
PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 code sequences (FIGS. 1, 3, 5, 7, 9, 11, 13, 15, 17, respectively) 19, 21, 23, and 25, each of which includes SEQ ID NOs: 1, 6, 9, 14, 19, 24, 29, 34, 42, 47, 54, 59, and 61), or a fragment thereof, , Homologous DNA in human tissue cDNA library or human tissue genomic library (PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866) Natural used as a probe for screening the occurrence such as variants).
Hybridization and washing of the filter containing any library DNA was performed under the following high stringency conditions. Hybridization to filters of radiolabeled probes derived from genes encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide is 50% formaldehyde, 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 at 42 ° C. for 20 hours. The filter was washed at 42 ° C. in an aqueous solution of 0.1 × SSC and 0.1% SDS.
The DNA having the desired sequence identity with the DNA encoding the full-length native sequence can then be identified using standard methods known in the art.

Example 17: Expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866PRO in E. coli This example shows the desired PRO179, PRO207 by recombinant expression in Illustrates the preparation of non-glycosylated forms of PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866.
DNA sequences encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 were first amplified using selected PCR primers. The primer must have a restriction enzyme site corresponding to the restriction enzyme site of the selected expression vector. Various expression vectors are used. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)), which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into a vector. The vector is preferably an antibiotic resistance gene, trp promoter, poly-His leader (including the first 6 STII codons, poly-His sequence, and enterokinase cleavage site), specific PRO179, PRO207, PRO320, PRO219, PRO221. , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 coding region, lambda transcription terminator, and argU gene.
The ligation mixture is then used to transform selected E. coli using the method described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant clones are selected. Plasmid DNA is isolated and confirmed by restriction analysis and DNA sequence analysis.
Selected clones can be grown overnight in liquid media such as LB broth supplemented with antibiotics. The overnight medium is then used for seeding the large-scale medium. The cells are then grown to optimal optical density, during which the expression promoter is activated.
Furthermore, after several hours of culture, collection by centrifugation is possible. The cell pellet obtained by centrifugation is solubilized using various reagents known in the art and then solubilized PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356. , PRO509, or PRO866 protein was purified using a metal chelation column under conditions in which the protein binds tightly.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are expressed in E. coli in the form of a poly-His (poly-histidine) tag using the following method. Also good. DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 was first amplified using selected PCR primers. Primers provide restriction enzyme sites that correspond to the restriction enzyme sites of the selected expression vector, and efficient and reliable translation initiation, rapid purification with metal chelate columns, and proteolytic removal with enterokinase Includes other useful sequences. The PCR-amplified poly-His tag sequence was then ligated to an expression vector, which was used to transform an E. coli host based on strain 52 (W3110 fuhA (tonA) longalE rpoHts (htpRts) clpP (lacIq)). Transformants were initially treated with 3-5 O.D. with shaking at 30 ° C. in LB containing 50 mg / ml carbenicillin. D. Grow to ₆₀₀ . The medium was then CRAP medium (3.57 g (NH ₄ ) ₂ SO ₄ , 0.71 g sodium citrate 2H 2 O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, and Diluted 100-100 times in 110 mM MPOS, pH 7.3, mixed with 0.55% (w / v) glucose and 7 mM MgSO ₄ ) and grown for about 20-30 hours with shaking at 30 ° C. . A sample was taken out and expression was confirmed by SDS-PAGE, and the bulk medium was centrifuged to obtain a cell pellet. Cell pellets were frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentation (6-10 g pellet) was resuspended in 10 volumes (w / v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfate and sodium tetrathionate were added to final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred at 4 ° C. overnight. This process resulted in a denatured protein in which all cysteine residues were blocked with sulfite. The solution was concentrated in a Beckman Ultracentrifuge at 40,000 rpm for 30 minutes. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6M guanidine, 20 mM Tris, pH 7.4) and filtered through a 0.22 micron filter to clarify. The clarified extract was loaded onto a 5 ml Qiagen Ni ^{+ 2-} NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with an addition buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4 ° C. The protein concentration was estimated by its absorption at 280 nm using the extinction coefficient calculated based on its amino acid sequence.
Proteins are diluted by gradual dilution of the sample into freshly prepared regeneration 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. Regenerated. The refolding volume was selected so that the final protein concentration was 50-100 microgram / ml. The refolding solution was slowly stirred at 4 ° C. for 12-36 hours. The refolding reaction was stopped by adding TFA at a collection concentration of 0.4% (pH of about 3). Prior to further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added at a final concentration of 2-10%. The renatured protein was chromatographed on a Poros R1 / H reverse phase column using a 0.1% TFA transfer buffer and elution with a 10-80% acetonitrile gradient. Aliquots of fractions with _A280 absorption were analyzed on SDS polyacrylamide gel and fractions containing homologous regenerative proteins were pooled. In general, most correctly regenerated protein species are eluted at the lowest concentration of acetonitrile because these species are the most compact and their hydrophobic inner surface is shielded from interaction with the reverse phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to removing the incorrectly regenerated protein from the desired form, the reverse phase process also removes endotoxin from the sample.
Pool the fractions containing the desired regenerated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides and generate a weak nitrogen stream to the solution. Used to remove acetonitrile. The protein was prepared to 20 mM Hepes, pH 6.8 containing 0.14 M sodium chloride and 4% mannitol by gel filtration and sterile filtration on G25 Superfine (Pharmacia) resin equilibrated with dialysis or preparation buffer.
PRO207, PRO224, and PRO301 polypeptides were successfully expressed in E. coli in poly-His tag form by the method described above.

Example 18: Expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide in mammalian cells This example is by recombinant expression in mammalian cells Illustrates the preparation of glycosylated forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides.
PRK5 (see EP 307,247 issued on March 15, 1989) was used as an expression vector. In some cases, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 DNA, selected restriction enzymes capable of inserting these DNAs into pRK5, Sambrook, etc. , And inserted into pRK5 using a ligation method as described above. The obtained vectors were pRK5-PRO179, pKR5-PRO207, pKR5-PRO320, pKR5-PRO219, pKR5-PRO221, pKR5-PRO224, pKR5-PRO328, pKR5-PRO301, pKR5-PRO526, pKR5-PRO362, pKR5-PRO362, , PKR5-PRO509, or pKR5-PRO866 polypeptide.
In one embodiment, the selected host cell can be a 293 cell. Human 293 cells (ATCC CCL 1573) were grown and confluent in tissue culture plates in media such as DMEM supplemented with fetal bovine serum and optionally nourishing ingredients and / or antibiotics. About 10 μg of pRK5-PRO179, pRK5-PRO207, pRK5-PRO320, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO301, pRK5-PRO526, pRK5-PRO50, PRO362-PRO56, PRO36-PRO56 Alternatively, pRK5-PRO866 DNA was mixed with about 1 μg of VA RNA gene-encoding DNA [Thimmappaya et al., Cell, 31: 543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl ₂ . To this mixture, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added dropwise and a precipitate formed at 25 ° C. for 10 minutes. The precipitate was suspended, added to 293 cells and allowed to settle at 37 ° C. for about 4 hours. The culture medium was aspirated and 2 ml of 20% glycerol in PBS was added for 30 seconds. The 293 cells were then washed with serum free medium, fresh medium was added and the cells were incubated for about 5 days.
Approximately 24 hours after transfection, the culture medium was removed and replaced with culture medium (only) or culture medium containing 200 μCi / ml ³⁵ S-cysteine and 200 μCi / ml ³⁵ S-methionine. After 12 hours of incubation, the conditioned medium was collected, concentrated with a spin filter and added to a 15% SDS gel. Treated gels were dried and exposed to film for a time selected to reveal the presence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. The medium containing the transformed cells was further incubated (in serum-free medium) and the medium was tested in the selected bioassay.
In an alternative technique, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are Somparyrac et al., Proc. Natl. Acad. Sci., 12: 7575. 1981), and is transiently introduced into 293 cells using the dextran sulfate method described in 1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO179, pKR5-PRO207, pKR5-PRO320, pKR5-PRO219, pKR5-221, pKR5-PRO224, pKR5-PRO328, pKR5-PRO301, pKR5- Add PRO526, pKR5-PRO362, pKR5-PRO356, pKR5-PRO509, or pKR5-PRO866. The cells were first concentrated from the spinner flask by centrifugation 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 reintroduced into a spinner flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and cell debris. The expressed sample containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 is then concentrated and by a selected method such as dialysis and / or column chromatography. Purified.
In other embodiments, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO328, PRO526, PRO362, PRO356, PRO509, or PRO866 can be expressed in CHO cells. pRK5-PRO179, pRK5-PRO207, pRK5-PRO320, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO301, pRK5-PRO526, pRK5-PRO362, pRK5-K-PRO356R -PRO866 can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE- dextran. As described above, the cell culture medium can be incubated and the culture medium can be replaced with culture medium (only) or a medium containing a radioactive label such as ³⁵ S-methionine. After identifying the presence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, the culture medium may be replaced with serum-free medium. Preferably, the medium is incubated for about 6 days and then the conditioned medium is collected. The medium containing the expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 peptide can then be concentrated and purified for the selected method.
In addition, the epitope tags PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 may be expressed in host CHO cells. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 were subcloned from the pRK5 vector. The subclone insert can then be subjected to PCR and fused to a frame with a selected epitope tag such as a poly-His tag in a baculovirus expression vector. The poly-His tag PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 inserts then select markers such as DHFR for selection of stable clones. It can be subcloned into the SV40 derived vector. Finally, CHO cells were transfected with the SV40 derived vector (as described above). In order to confirm expression, labeling may be performed as described above. The culture medium containing the expressed poly-His tag PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 is then concentrated and Ni ²⁺ -chelate affinity chromatography It can be purified by selected methods such as.
In addition, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are transiently expressed in CHO and / or COS cells by other stable expression methods. It may be expressed in CHO cells.

Stable expression in CHO cells was performed using the following method. The protein is an IgG construct in which the coding sequence (eg, extracellular domain) in the solubilized and / or poly-His tag form of the corresponding protein is fused to a constant region sequence comprising the IgG1, hinge, CH2 and CH2 domains. (Immunoadhesin) or expressed as a poly-His tag form.
Following PCR amplification, the corresponding DNA was subcloned into a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.1, John Wiley and Sons (1997). The CHO expression vector has restriction sites compatible with 5 ′ and 3 ′ of the DNA of interest and is constructed so that the cDNA can be conveniently shuttled. The vector was expressed in CHO cells as described in Lucas et al., Nucl. Acids Res. 24: 9, 1774-1779 (1996) and used for expression of the target cDNA and dihydrofolate reductase (DHFR). The SV40 early promoter / enhancer is used for control. DHFR expression allows selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA was introduced into approximately 10 million CHO cells of the commercially available transfection reagents Superfect® (Quiagen), Dosper® and Fugene® (Boehringer Mannheim). Cells were grown as described in Lucas et al. About 3x10 ^-7 cells are frozen in an ampule for further growth and production as described below.
An ampoule containing plasmid DNA was placed in a water bath, thawed, and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 ml of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were suspended in 10 ml selective medium (0.2 μm filtered PS20, 5% 0.2 μm diafiltered fetal bovine serum added). Cells were then divided into 100 ml spinners containing 90 ml selection medium 1-2 days later, the cells were transferred to a 250 ml spinner filled with 150 ml selection medium and incubated at 37 ° C. After a further 2-3 days, 250 ml, 500 ml and 2000 ml spinners were seeded at 3 × 10 ⁵ cells / mL. The cell medium was replaced with fresh medium by centrifugation and resuspended in production medium. Any suitable CHO medium may be used, but in practice the production medium described in US Pat. No. 5,122,469 issued June 16, 1992 was used. A 3 L production spinner was seeded at 1.2 × 10 ⁶ cells / ml. On day 0, cell number and pH were measured. On the first day, the spinner was sampled and sprayed with filtered air. On the second day, sample the spinner, change the temperature to 33 ° C, and add 500 g / l glucose and 30 ml of 0.6 ml of 10% antifoam (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) did. Throughout production, the pH was adjusted and maintained near 7.2. Cell medium was collected by centrifugation after 10 days or until viability was below 70% and filtered through a 0.22 μm filter. The filtrate was stored at 4 ° C. or immediately loaded onto a purification column.

For poly-His tag constructs, the protein was purified using a Ni ²⁺ -NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned media was pumped at 4 ° C. at a flow rate of 4-5 ml / min onto a 6 ml Ni ²⁺ -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 further washed with equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted with 25 ml G25 Superfine (Pharmacia) in storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol and stored at −80 ° C.
Immunoadhesin (Fc-containing) constructs were purified from conditioned media as follows. The conditioned medium was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into a tube containing 275 μl of 1M Tris buffer, pH 9. The highly purified protein was subsequently desalted in the storage buffer described above for the poly-His tag protein. Uniformity was tested on SDS polyacrylamide gels and N-terminal amino acid sequencing was determined by Edman degradation.
The PRO179, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO356, PRO509, and PRO866 polypeptides disclosed herein were stably expressed in CHO cells by the methods described above. Furthermore, PRO224, PRO328, PRO301, and PRO356 were expressed by transient expression methods in CHO cells.

Example 19: Expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 in yeast The following methods are used to produce PRO179, PRO207, Describes recombinant expression of PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866.
First, yeast expression vectors for intracellular production or secretion of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 from the ADH2 / GAPDH promoter create. DNAs and promoters encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are inserted into the appropriate restriction enzyme sites of the selected plasmid and PRO179, PRO207, Directs intracellular expression of PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. DNA encoding the ADH2 / GAPDH promoter in a plasmid in which DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 is selected for secretion, Native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or a PRO866 signal peptide or other mammalian signal peptide or, for example, a yeast alpha factor secretion signal / leader sequence, And (if necessary) PRO179, PRO207, PRO320, PRO219, PR 221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or may be cloned with linker sequences for expression of PRO866.
Yeast strains such as yeast strain AB110 can then be transformed with the above expression plasmid and cultured in the selected fermentation medium. Transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gel with Coomassie blue staining.
Recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides were substantially separated from the yeast cell fermentation liquor and selected. It is isolated and purified by concentrating the culture solution with a cartridge filter. The concentrate containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 is further purified by using a 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 methods are performed in baculovirus-infected insect cells. 1 shows recombinant expression of PRO. A sequence encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 was fused upstream of an epitope tag contained in a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (such as the Fc region of IgG). Various plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Navogen). Briefly, a sequence encoding a predetermined part of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 coding sequence, eg, the extracellular domain of a transmembrane protein or A sequence encoding a mature protein when the protein is extracellular is amplified by PCR with primers complementary to the 5 ′ and 3 ′ regions. The 5 ′ primer may include flanking (selected) restriction enzyme sites. The product is then digested with selected restriction enzymes and subcloned into an expression vector.

Recombinant baculovirus is obtained by co-transfecting the above plasmid and BaculoGold (trade name) viral DNA (Pharmingen) into lipopodin (commercially available from GIBCO-BRL) in Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711). Created by populating. After incubation at 28 ° C. for 4-5 days, the released virus was collected and used for further amplification. Viral infection and protein expression were performed as described in O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).
Next, the expressed poly-His tags PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 are expressed as follows by, for example, Ni ²⁺ -chelate affinity chromatography. To be purified. Extracts were prepared from virus-infected recombinant Sf9 cells as described in Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells were washed and sonicated buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) Resuspended in and sonicated twice on ice for 20 seconds. The sonicated product was clarified by centrifugation, and the supernatant was diluted 50-fold with loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni ²⁺ -NTA agarose column (commercially available from Qiagen) was prepared in a total volume of 5 ml, washed with 25 ml water and equilibrated with 25 ml loading buffer. The filtered cell extract was loaded onto the column at 0.5 ml per minute. The column was washed with loading buffer to A ₂₈₀ baseline where fraction collection began. The column was then washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0) that elutes non-specifically bound protein. After reaching A ₂₈₀ baseline again, the column was developed with a 0 to 500 ml imidazole gradient in the secondary wash buffer. 1 ml fractions were collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ²⁺ -NTA conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His ₁₀ -tags PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 were pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tag (or Fc tag) PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 can be accomplished using, for example, protein A or protein G column chromatography. It can be performed using known chromatographic techniques including.

Following PCR amplification, each coding sequence is subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusion and pb.PH.His.c for poly-His tag protein) Baculogold (trade name) baculovirus DNA (Pharmingen) was co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (Gibco-BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen) and have a modified polylinker region containing a His or Fc tag sequence. Cells were grown in Hink's TNM-FH medium (Hyclone) supplemented with 10% FBS. Cells were incubated for 5 days at 28 ° C. The supernatant was collected and used for initial virus amplification by subsequent infection of Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS with an approximate infection efficiency (MOI) of 10. Cells were incubated for 3 days at 28 ° C. The supernatant is collected and expression of the construct in baculovirus expression vectors is transferred to 25 ml Ni ²⁺ -NTA beads (QIAGEN) for histidine-tagged protein or protein-A sepharose CL-4B for IgG-tagged protein The 1 ml supernatant was batch bound to a bead (Pharmacia) followed by SDS-PAGE analysis and determined by comparison to a known concentration of protein standard by Coomassie blue staining.
The initial virus amplification supernatant was used to infect a stirred culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28 ° C. The supernatant was collected and filtered. Batch binding and SDS-PAGE analysis were repeated as necessary until expression of the agitated culture was confirmed.

Conditioned medium (0.5-3 L) from the transfected cells was collected by centrifugation to remove the cells and filtered through a 0.22 micron filter. For poly-His tag constructs, protein constructs were purified using Ni ²⁺ -NTA columns (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. The conditioned medium, 20 mM Hepes at a flow rate of 4-5 ml / min at 4 ° C., was pumped to the Ni ²⁺ -NTA column equilibrated with 6ml with buffer containing imidazole NaCl and 5mM of PH7.4,0.3M. After packing, 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 desalted in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8 on a 25 ml G25 Superfine (Pharmacia) column and stored at −80 ° C. .
Protein immunoadhesin (Fc-containing) constructs were purified from conditioned media as follows. Conditioned media was pumped onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was fully equilibrated with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions in a tube containing 275 mL of 1M Tris buffer, pH 9. The highly purified protein was desalted in poly-His tag protein storage buffer as described above. Protein uniformity was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
PRO301, PRO362, PRO356, PRO509, and PRO866 were expressed in baculovirus infected Sf9 insect cells.

  Alternatively, the modified baculovirus method may be used for High5 cell uptake. In this method, DNA encoding the desired sequence may be amplified in an appropriate system such as Pfu (Stratagene) or fused upstream (5′-) of the epitope tag contained in the baculovirus expression vector. Good. Such epitope tags include a poly-His tag and an immunoglobulin tag (such as an Fc region of IgG). A variety of plasmids can be used, including plasmids derived from commercially available plasmids such as pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors are designed for constitutive expression of the recombinant protein from the baculovirus ie1 promoter in stably transformed insect cells (1). This plasmid differs only in the direction of multiple cloning sites and contains all the promoter sequences known to be important for ie1-mediated gene expression in uninfected insect cells as well as the hr5 enhancer component. pIE1-1 and pIE1-2 contain a translation initiation site and can be used to produce fusion proteins. Briefly, a desired portion of the PRO polypeptide (such as a sequence encoding the extracellular domain of a transmembrane protein) is amplified by PCR with primers complementary to the 5 'and 3' regions. The 5 'primer may introduce flanking (selected) restriction enzyme sites. The product is then digested with selected restriction enzymes and subcloned into an expression vector. For example, a derivative of pIE1-1 can comprise the Fc region of human IgG (pb.PH.IgG) or the 8 histidine (pb.PH.His) tag downstream (3′-). Preferably, the sequence of the construction vector is confirmed for confirmation.

High-5 cells were grown to 50% confluence under conditions of 27 ° C., no CO _2, no pen / strept. For each 150 mm plate, 1 ml of Ex-cell medium containing 30 μg of PRO polypeptide (media: Ex-cell 401 + 1/100 L-Glu JRH Biosciences # 14401-78P (Note: this medium is lightly sensitive) In a separate tube, 100 μl of cellfectin (CellFECTIN (Gibco BRL # 10362-010) (mixed by vortex)) was mixed with 1 ml of Ex-cell medium. The two solutions were mixed and incubated for 15 minutes at room temperature. 8 ml of Ex-cell medium was added to 2 ml of DNA / cellfectin mixture and layered on high-5 cells washed once with Ex-cell medium. The plates were then incubated in the dark at room temperature. The DNA / cellfectin mixture was then aspirated and the cells were struck once with Ex-cells to remove excess cellfectin. 30 ml of fresh Ex-cell medium was added and the cells were incubated at 28 ° C. for 3 days. The supernatant is collected and expression of the PRO polypeptide in a baculovirus-infected vector is performed using 1 ml of supernatant, 25 mL of Ni ²⁺ -NTA beads (QIAGEN) for histidine tag protein or Protein A Sepharose CL for IgG tag protein. Measured by SDS-PAGE analysis by batch binding to -4B beads (Pharmacia) followed by Coomassie blue staining with known concentrations of protein standards.
Conditioned medium (0.5-3 L) from transfected cells was collected by removing the cells by centrifugation and filtering through a 0.22 micron filter. For the poly-His tag construct, the protein construct was purified using a Ni ²⁺ -NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned medium was pumped at 48 ° C. at a flow rate of 4-5 ml / min onto a 6 ml Ni ²⁺ -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 further washed with equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted with 25 ml G25 Superfine (Pharmacia) in storage buffer, pH 6.8, containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, at −80 ° C. Stored in
Protein immunoadhesin (Fc-containing) constructs were purified from conditioned media as follows. The conditioned medium was loaded onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed extensively with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fraction into a tube containing 275 ml 1M Tris buffer, pH 9. The highly purified protein was subsequently desalted in the storage buffer described above for the poly-His tag protein. The homogeneity of the PRO polypeptide can be assessed by SDS polyacrylamide gel and N-terminal amino acid sequencing by Edman degradation and other analytical techniques as desired or required.
PRO179, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, and PRO356 were expressed by the baculovirus method using high-5 cells described above.

Example 21: Preparation of antibodies that bind to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 This example includes PRO179, PRO207, PRO320, PRO21, RO22 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or the preparation of monoclonal antibodies that can specifically bind to PRO866.
Techniques for the production of monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used include PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 containing purified PRO179, PRO207, PRO320, PRO219, PRO241, PRO241, PRO241 , PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 fusion protein, and recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO509, PRO509 or PRO509 on the cell surface Cells that contain. The selection of the immunogen can be made without undue experimentation by one skilled in the art.

Mice such as Balb / c were emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally at 1-100 micrograms PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, Immunize with PRO509 or PRO866 immunogen. Alternatively, the immunogen may be emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind footpad. The immunized mice are then boosted 10 to 12 days later with an additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice are boosted with additional immunization injections. 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 -Serum samples from retro-orbital bleeding may be taken periodically from mice for testing in an ELISA assay for detection of PRO866 antibody.
After a suitable antibody titer has been detected, the animals that are “positive” for antibodies are those of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. Intravenous injections can be made. Three to four days later, the mice were sacrificed and spleen cells were removed. The spleen cells were then (using 35% polyethylene glycol), P3X63AgU. Fused to 1st selected mouse myeloma cell line. Hybridoma cells were generated by fusion and then plated on 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the growth of unfused cells, myeloma hybrids, and spleen cell hybrids.
Hybridoma cells are screened in an ELISA for reactivity against PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. Determination of “positive” hybridoma cells that secrete the desired monoclonal antibody to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 is within the scope of common general knowledge. Is within.
Positive hybridoma cells were injected intraperitoneally into syngeneic Balb / c mice, anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO301 Ascites fluid is generated containing PRO526, anti-PRO362, anti-PRO356, anti-PRO509, or anti-PRO866 monoclonal antibodies. Alternatively, hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of monoclonal antibodies produced in ascites can be performed using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the affinity of antibody for protein A or protein G can also be used.

Example 22: Purification of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides using specific antibodies Native or recombinant PRO179, PRO207, PRO320 , PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can be purified by various standard protein purification methods in the field. For example, pro-PRO179, pro-PRO207, pro-PRO320, pro-PRO219, pro-PRO221, pro-PRO224, pro-PRO328, pro-PRO301, pro-PRO526, pro-PRO362, pro-PRO356, pro-PRO509, Or pro-PRO866 polypeptide, mature PRO179, mature PRO207, mature PRO320, mature PRO219, mature PRO219, mature PRO224, mature PRO328, mature PRO301, mature PRO526, mature PRO362, mature PRO356, mature PRO509, or mature PRO866 polypeptide, or pre-PRO179, pre-PRO207, pre-PRO320, pre-PRO219, pre-PR 221, pre-PRO224, pre-PRO328, pre-PRO301, pre-PRO526, pre-PRO362, pre-PRO356, pre-PRO509, or pre-PRO866 polypeptide are the target PRO179, PRO207, PRO320, PRO219, PRO221. , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or purified by immunoaffinity chromatography using an antibody specific for the PRO866 polypeptide. Generally, the immunoaffinity columns are 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 polypeptide antibodies are made by covalent attachment to an activated chromatography resin.

Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or purification with immobilized protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Similarly, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography with immobilized protein A. The partially purified immunoglobulin is covalently coupled to a chromatographic resin such as CnBr-activated Sepharose ^™ (Pharmacia LKB Biotechnology). The antibody is bound to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
Such an immunoaffinity column is a fraction from cells containing solubilized forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. It is used in the purification of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides by preparation. This preparation is derived by solubilization of whole cells or cell component fractions obtained via addition of detergents or differential centrifugation by methods known in the art. Alternatively, soluble PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide containing a signal sequence is secreted in an effective amount into the medium in which the cells are growing. The

  Solubilized PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide-containing preparations are passed through an immunoaffinity column and the columns are PRO179, PRO207, PRO320. , PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide (eg, a high ionic strength buffer in the presence of a detergent). The columns are then antibody / PRO179, antibody / PRO207, antibody / PRO320, antibody / PRO219, antibody / PRO221, antibody / PRO224, antibody / PRO328, antibody / PRO301, antibody / PRO526, antibody / PRO362, antibody / PRO356, antibody / PRO509, or antibody / PRO866 polypeptide binding conditions (eg, low pH, about 2-3, chaotropes such as high concentrations of urea or thiocyanate ions), and eluted with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide is recovered.

Example 23: Drug screening
The present invention is particularly useful for screening compounds by using PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 or binding fragments thereof in various drug screening techniques. Useful. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides or fragments used in such stinging authority, even in the free state in solution, become solid supports. It may be fixed, located on the cell surface, or localized in the cell. One method of drug screening is stably transfected with recombinant nucleic acids expressing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides or fragments. Eukaryotic or prokaryotic host cells. Agents are screened against such transfected cells in a competitive binding assay. Such cells can be used in standard binding assays in either viable or immobilized form. For example, complex formation between a PRO179, PRO207, PRO320, PRO219, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or fragment and the agent being tested may be measured. Alternatively, test the decrease in complex formation between the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide and its target cells caused by the reagent being tested You can also do it.
That is, the present invention relates to a drug or any other reagent that may affect a PRO179, PRO207, PRO320, PRO219, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide related diseases or disorders. A screening method is provided. These methods involve contacting the reagent with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or fragment, and (i) the reagent and PRO179, PRO207. , PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or the presence of a complex between PRO866 polypeptides or fragments, or (ii) PRO179, PRO207, PRO320, PRO219, PRO221 , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PR Assaying for the presence of a complex between the O866 polypeptide or fragment and the cell. In these competitive binding assays, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides or fragments are typically labeled. After appropriate incubation, the free PRO polypeptide or fragment is separated from the bound form and the amount of free or unconjugated label is determined by the specific reagents PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, Binds to PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 It is a measure of the ability to inhibit the body.

Other techniques for drug screening provide high-throughput screening for compounds with appropriate binding affinity for polypeptides and are described in detail in WO 84/03564 issued September 13, 1984. Has been. Briefly, a number of different small peptide test compounds are synthesized on a solid support such as plastic pins or some other surface. When applied to a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, the peptide test compounds are PRO179, PRO207, PRO320, PRO219, PRO219, PRO219, PRO241, PRO242, PRO241 Washed in reaction with PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. Bound PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides are detected by methods well known in the art. Purified PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can also be coated directly onto plates for use in the above drug screening techniques. it can. Furthermore, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
The present invention also relates to a neutralizing antibody capable of binding to PRO179, PRO207, PRO320, PRO219, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. Also contemplated are competitive drug screening assays that specifically compete with a test compound for a PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or fragment thereof. In this method, the antibody is a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, of any peptide having one or more antigenic determinants. Can be used to detect presence.

Example 24: Rational Drug Design The purpose of rational drug design is to target biologically active polypeptides (eg, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides) or small molecules with which they interact, such as agonists, antagonists, or structural analogs of inhibitors. These examples include the more active and stable forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides or in vivo PRO179, PRO207, PRO320, PRO219. , PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can be used to create agents that promote or inhibit function (see Hodgson, Bio / Technology, 9: 19-21 (1991). )).
In one method, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or PRO179, PRO207, PRO320, PRO219, PRO219, PRO219, PRO241, PRO24 , PRO526, PRO362, PRO356, PRO509, or PRO866-inhibitor complex is determined by X-ray crystallography, by computer modeling, most typically by a combination of the two methods. To elucidate the structure of the molecule and determine the active site, both the shape and charge of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide Must be confirmed. A few but useful information regarding the structure of PRO179, PRO207, PRO320, PRO219, PRO221, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide is modeled based on the structure of homologous proteins. Sometimes obtained. In both cases, relevant structural information is used in the design of similar PRO polypeptide-like molecules or the identification of inhibitors to go. Useful examples of rational drug design are molecules with improved activity or stability as shown in Braxton and Wells, Biochemistry, 31: 7796-7801 (1992), or Athauda et al., J. Biochem. , 113: 742-746 (1993), including molecules that act as inhibitors, agonists or antagonists of natural peptides.

It is also possible to isolate a target-specific antibody selected by the functional assay as described above and elucidate its structure. This method in principle produces a pharmacore on which subsequent drug design can be based. By generating anti-idiotype antibodies (anti-ids) against functional pharmacologically active antibodies, protein crystallography can be bypassed. As a mirror image of the mirror image, the anti-ids binding site can be predicted to be an analog of the original receptor. Anti-ids can then be used to identify and isolate peptides from a bank of chemically or biologically produced peptides. The isolated peptide will function as a pharmacore.
In accordance with the present invention, a sufficient amount of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide to perform analytical experiments such as X-ray crystallography. It is available. Furthermore, the knowledge of the PRO polypeptide amino acid sequence provided herein provides the knowledge used in computer modeling techniques to replace or add to X-ray crystallography.

Example 25: In vitro anti-proliferation assay The anti-proliferative activity of various PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides, essentially by Skehan et al., National Cancer Institute (NCI) experimental, disease-oriented in vitro anticancer drug discovery assay using the sulforhodamine B (SRB) dose binding assay described in J. Natl. Cancer Inst. 82: 1107-1112 (1990) Measured in The 60 tumor cell lines ("NCI panel") used in this assay, and their maintenance and in vitro culture conditions, are described in Monks et al., J. Natl. Cancer Inst. 83: 757-766 (1991). Yes. The purpose of this screening is to first assess the cytotoxicity and / or mitotic activity of the test compound against different types of tumors (Monks et al., Supra; Boyd, Cancer: Princ. Pract. Oncol. Update 3 (10): 1-12 [1989]).
Cells from approximately 60 human tumor cell lines were harvested with trypsin / EDTA (Gibco), washed once, resuspended in IMEM, and their survival was measured. The cell suspension was pipetted (100 μL volume) into another 96-well plate. The cell density of the 6 day incubation was smaller than the 2 day incubation and overexpression was prevented. After seeding, preincubation was performed at 37 ° C. for 24 hours for stabilization. Two-fold dilutions of the desired concentration were added to microtiter plate wells in 100 μl aliquots at zero (1: 2 dilution). Test compounds were evaluated at 5 log dilutions (1000 to 100,000 times). Incubations were performed for 2 and 6 days with 5% CO ₂ gas and 100% humidification.
After incubation, the medium was removed and the cells were fixed with 0.1 ml of 10% trichloroacetic acid at 40 ° C. The plate is washed 5 times with demineralized water, dried, stained with 0.1 ml of 0.4% sulforhodamine B (Sigma) dissolved in 1% acetic acid for 30 minutes and washed 4 times with 1% acetic acid to remove unbound dye. The dyeing was extracted with 0.1 ml of 10 mM Tris base [Tris (hydroxymethyl) aminomethane], pH 10.5 for 5 minutes. Absorption (OD) of sulforhodamine B at 492 nm was measured with a computer-connected 96-well plate reader.
A test sample is considered positive if it exhibits a growth inhibitory effect of at least 40% at one or more concentrations. The results are shown in Table 4 below, where the abbreviations for the tumor cell types are as follows:
NSCL = non-small cell lung cancer; CNS = central nervous system

Deposits of Materials The following materials were deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209 USA (ATCC):
Material ATCC Deposit Number Deposit Date
DNA16451-1078 209281 September 18, 1997
DNA30879-1152 209358 October 10, 1997
DNA32284-1307 209670 Mar 11, 1998
DNA32290-1164 209384 October 17, 1997
DNA33089-1132 209262 September 16, 1997
DNA33221-1133 209263 September 16, 1997
DNA40587-1231 209438 Nov 7, 1997
DNA40628-1216 209432 1997/11/7
DNA44184-1319 209704 March 26, 1998
DNA45416-1251 209620 Feb 5, 1998
DNA47470-11130-P1 209422 Oct 28, 1997
DNA53971-1359 209750 April 7, 1998

These deposits were made in accordance with the provisions of the Budapest Treaty and its regulations (Budapest Convention) on the international recognition of the deposit of microorganisms in the patent procedure. This guarantees that the live culture of the deposit will be maintained for 30 years from the date of deposit. Deposits may be obtained from the ATCC in accordance with the terms of the Budapest Treaty and in accordance with an agreement between Genentech and ATCC, whichever comes first, at the time of issuance of the relevant US patent or any Ensures that the progeny of the deposited culture are made available permanently and unrestricted at the time of publication of a US or foreign patent application, and is subject to 35 USC 122 and the Patent Office Commissioner Regulation (specifically 37 CFR of reference number 886OG638). Guarantees that the offspring will be made available to those who have been determined to have the rights in accordance with (including Section 1.14).
The assignee of this application will promptly replace the material with the same other at the time of notification if the culture of the deposited material dies or is lost or destroyed when cultured under appropriate conditions. Agree to The availability of deposited materials is not to be considered a license to practice the present invention in violation of rights granted under any governmental authority in accordance with patent law.
The above written description is considered to be sufficient to enable one skilled in the art to practice the invention. The deposited embodiments are intended as an illustration of certain aspects of the invention, and since any functionally equivalent product is within the scope of the invention, the deposited product will limit the scope of the invention. It is not limited. The deposition of materials herein does not acknowledge that the written description contained herein is insufficient to allow implementation of any aspect of the present invention, including its best mode, and that It is not to be construed as limiting the scope of the claims for the particular illustrations represented. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and are intended to be within the scope of the following claims.

FIG. 2 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO179, which is a clone designated herein as DNA16451-1078. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 2 is a view showing an amino acid sequence (SEQ ID NO: 2) of a native sequence PRO179 polypeptide derived from the coding sequence of SEQ ID NO: 1 shown in FIG. 1. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 6) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO207, the nucleotide sequence (SEQ ID NO: 6) being a clone designated herein as DNA30879-1152. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 4 is a view showing an amino acid sequence (SEQ ID NO: 7) of a native sequence PRO207 polypeptide derived from the coding sequence of SEQ ID NO: 6 shown in FIG. 3. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 9) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO320, the nucleotide sequence (SEQ ID NO: 9) being a clone designated herein as DNA32284-13407. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 6 is a view showing an amino acid sequence (SEQ ID NO: 10) of a native sequence PRO320 polypeptide derived from the coding sequence of SEQ ID NO: 9 shown in FIG. 5. FIG. 2 shows the nucleotide sequence of a cDNA (SEQ ID NO: 14) comprising a nucleotide sequence encoding the native sequence PRO219, the nucleotide sequence (SEQ ID NO: 14) being a clone designated herein as DNA32290-1164. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 8 shows the amino acid sequence (SEQ ID NO: 15) of native sequence PRO219 polypeptide derived from the coding sequence of SEQ ID NO: 14 shown in FIG. 7. FIG. 2 shows a nucleotide sequence (SEQ ID NO: 19) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO221, the nucleotide sequence (SEQ ID NO: 19) being a clone designated herein as DNA330892-1132. Further, bold and underlined positions are the respective start and stop codon positions. It is a figure which shows the amino acid sequence (sequence number: 20) of native sequence PRO221 polypeptide induced | guided | derived from the coding sequence of sequence number: 19 shown in FIG. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 24) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO224, the nucleotide sequence (SEQ ID NO: 24) being a clone designated herein as DNA33221-1133. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 12 is a view showing an amino acid sequence (SEQ ID NO: 25) of a native sequence PRO224 polypeptide derived from the coding sequence of SEQ ID NO: 24 shown in FIG. 11. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 29) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO328, the nucleotide sequence (SEQ ID NO: 29) being a clone designated herein as DNA40587-1231. Further, bold and underlined positions are the respective start and stop codon positions. It is a figure which shows the amino acid sequence (sequence number: 30) of native sequence PRO328 polypeptide induced | guided | derived from the coding sequence of sequence number: 29 shown in FIG. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 34) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO301, the nucleotide sequence (SEQ ID NO: 34) being a clone designated herein as DNA40628-1216. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 16 is a view showing an amino acid sequence (SEQ ID NO: 35) of a native sequence PRO301 polypeptide derived from the coding sequence of SEQ ID NO: 34 shown in FIG. 15. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 42) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO526, the nucleotide sequence (SEQ ID NO: 42) being a clone designated herein as DNA44184-1319. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 18 shows the amino acid sequence (SEQ ID NO: 43) of native sequence PRO526 polypeptide derived from the coding sequence SEQ ID NO: 42 shown in FIG. 17. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 47) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO362, the nucleotide sequence (SEQ ID NO: 479) being a clone designated herein as DNA45416-1251. Bold and underlined positions are the respective start and stop codon positions. FIG. 20 shows the amino acid sequence (SEQ ID NO: 48) of native sequence PRO362 polypeptide derived from the coding sequence SEQ ID NO: 47 shown in FIG. 19. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 54) of a cDNA comprising the nucleotide sequence encoding the native sequence PRO356, the nucleotide sequence (SEQ ID NO: 54) being a clone designated herein as DNA47470-1130-P1 . Further, bold and underlined positions are the respective start and stop codon positions. FIG. 22 is a view showing an amino acid sequence (SEQ ID NO: 55) of a native sequence PRO356 polypeptide derived from the coding sequence of SEQ ID NO: 54 shown in FIG. 21. FIG. 5 shows the nucleotide sequence (SEQ ID NO: 59) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO509, which is a clone designated herein as DNA50148-1068. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 24 shows the amino acid sequence (SEQ ID NO: 60) of native sequence PRO509 polypeptide derived from the coding sequence of SEQ ID NO: 59 shown in FIG. 23. FIG. 2 shows a nucleotide sequence (SEQ ID NO: 61) of a cDNA comprising a nucleotide sequence encoding the native sequence PRO866, the nucleotide sequence (SEQ ID NO: 61) being a clone designated herein as DNA53971-1359. Further, bold and underlined positions are the respective start and stop codon positions. FIG. 26 shows the amino acid sequence (SEQ ID NO: 62) of native sequence PRO866 polypeptide derived from the coding sequence of SEQ ID NO: 61 shown in FIG. 25.

Claims (24)

  The PRO224 poly having the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25) or the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25) having one, several amino acids deleted, substituted, or inserted. A composition useful for tumor cell growth inhibition comprising an effective amount of a peptide in admixture with a pharmaceutically acceptable carrier.   2. The composition of claim 1, comprising a growth inhibitory amount of the PRO224 polypeptide.   A composition according to claim 1 or 2 for the treatment of tumors in mammals.   The composition according to claim 3, wherein the tumor is cancer.   The composition according to claim 4, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, kidney cancer, colorectal cancer, prostate cancer, lung cancer, central nervous cancer, melanoma, and leukemia.   In vitro, the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25) or one or several amino acids in the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25) is deleted, substituted or inserted in the tumor cell in vitro. A method of inhibiting tumor cell growth comprising exposing to an effective amount of a PRO224 polypeptide having a defined amino acid sequence. A manufactured product comprising (a) a container and (b) a composition contained in the container, wherein the composition is the composition according to any one of claims 1 to 5.   8. The article of manufacture of claim 7, wherein a label referring to the use of the composition in inhibiting growth of neoplastic cells is further attached to the container, or a package insert is included in the container.   12 encodes a polypeptide comprising the amino acid sequence shown in FIG. 12 (SEQ ID NO: 25), or encodes a polypeptide that hybridizes with the nucleic acid under highly stringent conditions and has tumor cell growth inhibitory activity An isolated nucleic acid.   An isolated nucleic acid comprising the nucleic acid sequence shown in FIG. 11 (SEQ ID NO: 24) or a polypeptide that hybridizes with the nucleic acid under highly stringent conditions and has a tumor cell growth inhibitory activity Nucleic acid.    A vector comprising the nucleic acid according to claim 9 or 10.   12. The vector of claim 11 operably linked to a control sequence recognized by a host cell transformed with the vector.   A host cell comprising the vector of claim 12.   The host cell of claim 13, wherein the cell is a CHO cell.   14. The host cell of claim 13, wherein the cell is E. coli.   The host cell of claim 13, wherein the cell is a yeast cell.   14. The host cell of claim 13, wherein the cell is a baculovirus infected insect cell.   It consists of the amino acid sequence shown in Fig. 12 (SEQ ID NO: 25), or from the amino acid sequence in which one or several amino acids are deleted, substituted or inserted in the amino acid sequence shown in Fig. 12 (SEQ ID NO: 25) An isolated polypeptide comprising and having tumor cell growth inhibitory activity.   19. A method for producing the polypeptide of claim 18, comprising culturing the host cell of claim 13 under conditions suitable for expression of the polypeptide, and recovering the polypeptide from a cell culture medium. How to   A chimeric molecule comprising the polypeptide according to claim 18 fused to a heterologous amino acid sequence.   21. The chimeric molecule of claim 20, wherein the heterologous amino acid sequence is an epitope tag sequence.   The chimeric molecule of claim 21, wherein the heterologous amino acid sequence is an immunoglobulin Fc region.   An antibody that specifically binds to the polypeptide of claim 18.   24. The antibody of claim 23, wherein the antibody is a monoclonal antibody, a humanized antibody, or a single chain antibody.

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