KR100499600B1 - Methods and Compositions for Inhibiting Neoplastic Cell Growth - Google Patents

Abstract

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

Description

Methods and Compositions for Inhibiting Neoplastic Cell Growth

The present invention relates to methods and compositions for inhibiting neoplastic growth. In particular, the present invention relates to antitumor compositions and methods of treating tumors. The invention also relates to screening methods for identifying growth inhibition, i.e., identifying anti-tumor compounds.

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

Cancer increases in the number of abnormal or neoplastic cells derived from normal tissue, which proliferate to form tumor masses, infiltration of adjacent tissue by the neoplastic tumor cells and malignant cells that eventually spread to local lymph nodes and extremities through the blood or lymphatic system. It is characterized by generation (transition). In the cancerous phase, cells grow under conditions in which normal cells do not grow. Cancers come in various forms with different degrees of invasiveness and invasiveness.

Despite recent advances in cancer therapies, there is a great demand for new therapeutic agents that can inhibit neoplastic growth. Accordingly, it is an object of the present invention to identify compounds that can inhibit the growth of neoplastic cells, such as cancer cells.

Summary of the Invention

A. Embodiment

The present invention relates to methods and compositions for inhibiting neoplastic growth. More specifically, the present invention relates to the treatment of tumors in mammalian patients, preferably humans, including cancer, such as breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, kidney cancer and central nervous system cancer, leukemia, melanoma, and the like. To methods and compositions for the same.

In one aspect, the invention provides an effective amount of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist as defined herein with a pharmaceutically acceptable carrier. Together, it relates to a composition useful for the inhibition of neoplastic growth. In a preferred embodiment, the composition of the present invention comprises a growth inhibitory amount of PPRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist thereof. In another preferred embodiment, the composition comprises a cytotoxic amount of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist thereof. Optionally, the compositions of the present invention may further comprise one or more growth inhibitors and / or cytotoxic agents and / or chemotherapeutic agents.

In a further aspect, the invention provides a mammal comprising a therapeutically effective amount of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist thereof as defined herein. A composition useful for treating tumors in an animal. The tumor is preferably cancer.

In another aspect, the present invention provides a method for exposing an effective amount of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist thereof to tumor cells. It relates to a method of inhibiting tumor cell growth, including. 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- PRO356, anti-PRO509 or anti-PRO866 agonist antibody. In another embodiment, the agonist is a small molecule that mimics the biological activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. The method can be performed in vitro or in vivo.

In another embodiment, the present invention

(a) a container,

(b) neoplastic cell growth, eg, in a container, comprising PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof as an active substance, for example A composition for inhibiting tumor cell growth, and

(c) the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, or PRO179, PRO207, PRO320, PRO219, PRO221, for inhibiting neoplastic growth To refer to the use of an agonist, which may be an antibody capable of binding to a PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, a label attached to the container, or a package insert contained within the container To provide a product comprising a. In one specific embodiment, the agonist is anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, It is an anti-PRO356, anti-PRO509 or anti-PRO866 agonist antibody. In another embodiment, the agonist is a small molecule that mimics a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Similar products comprising PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides described herein, or agonists thereof in a therapeutically effective amount for the treatment of tumors, are also within the scope of the present invention. Is in. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 Polypeptide or Agonist Defined Here, and Additional Growth Inhibitors, Cytotoxic or Chemotherapeutic Agents Products to be included are also within the scope of the present invention.

B. Additional Embodiments

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

In one aspect, an isolated nucleic acid molecule comprises (a) a full length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, or a transmembrane protein with or without a signal peptide as disclosed herein. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides having an extracellular domain or any other specifically defined fragment of the full length amino acid sequence as disclosed herein At least about 80%, preferably at least about 81%, more preferably at least about 82%, and more preferably at about 83% of the DNA molecule encoding (b) or the complement of the DNA molecule (a) At least%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferred Preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94% At least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%. do.

In another aspect, an isolated nucleic acid molecule is (a) a coding sequence of full length PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide cDNA, as disclosed herein, Coding sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide lacking a signal peptide as disclosed herein, with or without signal peptide as disclosed herein Transgenic coding sequence of the extracellular domain of the transmembrane PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, or any other of the full length amino acid sequence as disclosed herein At least about 80%, preferably at least about 81%, more preferably at least about 80% sequence identity with the DNA molecule comprising the coding sequence of the specifically defined fragment or (b) the complement of the DNA molecule (a) At least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, More preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, even more preferably about At least 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more Preferably at least about 99%.

In a further aspect, the invention provides a complement of a DNA molecule or (b) a DNA molecule encoding the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein. At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably At least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, More preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably about At least 96%, more preferably at least about 97%, more preferably at least about 98%, Preferably it relates to an isolated nucleic acid molecule comprising a nucleotide sequence at least about 99%.

In another aspect, the invention provides nucleotides encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides that have deleted or inactivated transmembrane domains. Provided are isolated nucleic acid molecules comprising a sequence, or the complement of such a coding nucleotide sequence, and disclosed herein are transmembrane domain (s) of such polypeptide. Thus, soluble extracellular domains of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides described herein are contemplated.

Another embodiment is for example an antisense oligonucleotide probe, or anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, which may optionally encode a polypeptide comprising a binding site for an anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibody Of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 Polypeptide Coding Sequence or Complements thereof That Can Be Used as Hybridization Probes for Coding Fragments of PRO356, PRO509, or PRO866 Polypeptides It's about fragments. Such nucleic acid fragments generally have at least about 20 nucleotides in length, preferably at least about 30, more preferably at least about 40, more preferably at least about 50, more preferably at least about 60, more Preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 100, more preferably at least about 110, more preferably about 120 At least about 130, more preferably at least about 140, more preferably at least about 140, more preferably at least about 150, more preferably at least about 160, more preferably at least about 170, more preferred Preferably at least about 180, more preferably at least about 190, more preferably at least about 200, more preferably at least about 250, more preferably at least about 300, and more preferably about 350 More preferably about 400 , More preferably about 450 or more, more preferably about 500 or more, more preferably about 600 or more, more preferably about 700 or more, more preferably about 800 or more, more preferably At least about 900, more preferably at least about 1000, wherein the term "about" in this context means 10% of the length of the nucleotide sequence referred to herein. New fragments of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide coding nucleotide sequences can be prepared using any of a number of well-known sequence alignment programs. , PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 Polypeptide Coding Nucleotide Sequences Are Aligned with Other Known Nucleotide Sequences and Any PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, It should be appreciated that the determination may be made in a routine manner by determining whether a PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide coding nucleotide sequence fragment (s) is new. All such PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide coding nucleotide sequences are all contemplated. In addition, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide fragments encoded by such nucleotide molecular fragments, preferably anti-PRO179, anti-PRO207, anti- PRO179 comprising a binding site for PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibodies Also contemplated are PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide fragments.

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

In certain aspects, the invention relates to an extracellular domain of a transmembrane protein with or without a full length amino acid sequence as described herein, an amino acid sequence lacking a signal peptide as described herein, a signal peptide as described herein, or Sequence identity with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides with any other specifically defined fragment of the full length amino acid sequence as described herein At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably about At least 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more Isolated PRO179, PRO207, PRO320, PRO219, PRO221 comprising amino acid sequences that are preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%. , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide.

In a further aspect, the invention provides at least about 80%, preferably at least about 81%, more preferably sequence identity with an amino acid sequence encoded by any of the human protein cDNAs deposited at ATCC as described herein Preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87% At least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably Is at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98% More preferably about 99% An isolated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide comprising the above amino acid sequence.

In a further aspect, the invention provides an extracellular domain of a transmembrane protein with or without a full length amino acid sequence as described herein, an amino acid sequence lacking a signal peptide as described herein, a signal peptide as described herein, Or the amino acid sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide having any other specifically defined fragment of the full length amino acid sequence as described herein By comparison, at least about 80% positive, preferably at least about 81% positive, more preferably at least about 82% positive, more preferably at least about 83% positive, more preferably at least about 84% positive, more preferably Is at least about 85% positive, more preferably at least about 86% positive, more preferably at least about 87% positive, more preferably Crab is at least about 88% positive, more preferably at least about 89% positive, more preferably at least about 90% positive, more preferably at least about 91% positive, more preferably at least about 92% positive, more preferred Preferably at least about 93% positive, more preferably at least about 94% positive, more preferably at least about 95% positive, more preferably at least about 96% positive, more preferably at least about 97% positive, more preferred Isolated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 comprising amino acid sequences that record at least about 98% positive, more preferably at least about 99% positive Or to a PRO866 polypeptide.

In a specific aspect, the invention provides an isolated PRO179, PRO207, PRO320, PRO219, PRO221 which is encoded by a nucleotide sequence encoding said amino acid sequence as described herein above and does not have an N-terminal signal peptide and / or initiating methionine. , PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Also described herein are methods of preparing the isolated PRO polypeptides, which comprise PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526 which comprise pluripotent cells comprising a vector comprising a suitable coding nucleic acid molecule. , PRO362, PRO356, PRO509, or PRO866 polypeptides are cultured under conditions suitable for expression, and from these cell cultures PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides Recovery.

In another aspect, the invention provides isolated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides that have deleted or are inactivated transmembrane domains. . Also described herein are methods of preparing the isolated PRO polypeptides, which comprise PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526 which comprise pluripotent cells comprising a vector comprising a suitable coding nucleic acid molecule. , PRO362, PRO356, PRO509, or PRO866 polypeptides are cultured under conditions suitable for expression, and from these cell cultures PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides Recovery.

In another embodiment, the invention relates to agonists of native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides as defined above. In a particular embodiment, the agonist is 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 antibody or small molecule.

In a further embodiment, the present invention provides a method of contacting a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide with a candidate molecule to provide the PRO179, PRO207, PRO320, PRO219, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, including monitoring biological activity mediated by a PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide A method of identifying agonists for, PRO356, PRO509, or PRO866 polypeptides. Preferably, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide.

In still further embodiments, the present invention provides for a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or PRO179, PRO207, PRO320, PRO219, PRO221 as defined above. , Agonist of PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or anti-PRO179, anti-PRO179, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, A composition comprising an anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 agonist antibody with a carrier. In some cases, the carrier is a pharmaceutically acceptable carrier.

Other embodiments of the invention include PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonists thereof, or anti-PRO179, anti-PRO207, anti-PRO320 For the treatment of symptoms reactive to 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, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides in the manufacture of a medicament, or agonists thereof as described above, or anti-PRO179, anti-PRO207, Regarding the use of 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 will be.

In another embodiment of the invention, the invention provides a vector comprising DNA encoding any of the polypeptides described herein. Also provided are host cells comprising any of these vectors. By way of example, the host cell may be a CHO cell, E. coli. E. coli , yeast or baculovirus-infected insect cells. Methods of making any of the polypeptides described herein are also provided, which include culturing the host cell under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

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

In other embodiments, the invention also provides antibodies that specifically bind to any of the polypeptides described above or below. Optionally this antibody is a monoclonal antibody, humanized antibody, antibody fragment or single chain antibody.

In another embodiment, the present invention provides oligonucleotide probes or antisense probes useful for isolating the nucleotide sequences of the genome and cDNA, wherein the probes may be derived from any of the above or following nucleotide sequences.

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

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

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

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

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

FIG. 6 is the amino acid sequence of the native sequence PRO320 polypeptide derived from the coding sequence of SEQ ID NO: 9 shown in FIG. 5 (SEQ ID NO: 10).

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

FIG. 8 is the amino acid sequence of the native sequence PRO219 polypeptide derived from the coding sequence of SEQ ID NO: 14 shown in FIG. 7 (SEQ ID NO: 15).

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

FIG. 10 is an amino acid sequence of a native sequence PRO221 polypeptide derived from the coding sequence of SEQ ID NO: 19 shown in FIG. 9 (SEQ ID NO: 20).

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

FIG. 12 is an amino acid sequence of a native sequence PRO224 polypeptide derived from the coding sequence of SEQ ID NO: 24 shown in FIG. 11 (SEQ ID NO: 25).

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

FIG. 14 is an amino acid sequence of a native sequence PRO328 polypeptide derived from the coding sequence of SEQ ID NO: 29 shown in FIG. 13 (SEQ ID NO: 30).

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

FIG. 16 is an amino acid sequence of a native sequence PRO301 polypeptide derived from the coding sequence of SEQ ID NO: 34 shown in FIG. 15 (SEQ ID NO: 35).

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

FIG. 18 is an amino acid sequence of a native sequence PRO526 polypeptide derived from the coding sequence of SEQ ID NO: 42 shown in FIG. 17 (SEQ ID NO: 43).

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

FIG. 20 is an amino acid sequence of a native sequence PRO362 polypeptide derived from the coding sequence of SEQ ID NO: 47 shown in FIG. 19 (SEQ ID NO: 48).

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

FIG. 22 is the amino acid sequence of the native sequence PRO356 polypeptide derived from the coding sequence of SEQ ID NO: 54 shown in FIG. 21 (SEQ ID NO: 55).

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

FIG. 24 is an amino acid sequence of a native sequence PRO509 polypeptide derived from the coding sequence of SEQ ID NO: 59 shown in FIG. 23 (SEQ ID NO: 60).

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

FIG. 26 is an amino acid sequence of a native sequence PRO866 polypeptide derived from the coding sequence of SEQ ID NO: 61 shown in FIG. 25 (SEQ ID NO: 62).

The terms "PRO179", "PRO207", "PRO320", "PRO219", "PRO221", "PRO224", "PRO328", "PRO301", "PRO526", "PRO362", "PRO356", "PRO509" or " The PRO866 "polypeptide refers to the native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328 as used herein. , PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variants (as further defined herein). 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 types or other sources, or produced by recombinant and / or synthetic methods. Can be.

"Natural sequence PRO179", "natural sequence PRO207", "natural sequence PRO320", "natural sequence PRO219", "natural sequence PRO221", "natural sequence PRO224", "natural sequence PRO328", "natural sequence PRO301", "natural sequence Sequence PRO526 "," Native Sequence PRO362 "," Native Sequence PRO356 "," Native Sequence PRO509 "or" Native Sequence PRO866 "refers to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, Polypeptides having the same amino acid sequence as the PRO362, PRO356, PRO509 or PRO866 polypeptide. Such native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be isolated from nature or can be prepared by recombinant and / or synthetic methods. The term "natural sequence" PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide specifically refers to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301 , Naturally occurring truncated or secreted forms (eg, extracellular domain sequences), naturally occurring variant forms (eg, other spliced forms) of the PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides And naturally occurring allelic variants. In one embodiment of the invention, the native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7) 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). , Mature or full-length native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, shown in FIG. 20 (SEQ ID NO: 48), FIG. 22 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 60), or FIG. PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Also, 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), and Fig. 14 (SEQ ID NO: 30). , PRO179, PRO207, shown in 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. The PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides appear to begin with methionine residues designated amino acid position 1 in the figures, but FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 20), Figure 12 (SEQ ID NO: 25), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), FIG. 20 (SEQ ID NO: 48), FIG. 22 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 60), or other methionine residues located upstream or downstream from amino acid position 1 in FIG. , PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or It is conceivable and possible that it can be used as the starting amino acid residue of the PRO866 polypeptide.

An "extracellular domain" or "ECD" of a polypeptide disclosed herein means a polypeptide form that is essentially free of transmembrane and cytoplasmic domains. Typically, the polypeptide ECD will comprise less than 1% of the transmembrane and / or cytoplasmic domains, preferably less than 0.5% of said domains. It should be understood that all transmembrane domain (s) identified in the polypeptides of the invention have been identified according to criteria commonly used to identify hydrophobic domain types in the art. The exact boundaries of the transmembrane domains can vary, but most are within the range of about 5 amino acids on either end of the transmembrane domain as first identified herein and as shown in the accompanying figures. Likewise, in one embodiment of the invention, the extracellular domain of the polypeptide of the invention comprises amino acids 1 to amino acid X of the mature amino acid sequence, wherein X is within 5 of either side of the extracellular domain / membrane domain boundary. Is any amino acid.

Approximate positions of the "signal peptides" of the various PRO polypeptides described herein are shown in the accompanying drawings. However, it should be borne in mind that although the C-terminal boundary of the signal peptide may vary, most are about 5 amino acids in either interface of the signal peptide C-terminal first identified herein, wherein C-terminal boundaries can be identified according to criteria commonly used to identify the type of amino acid sequence element in the art (see, eg, Nielsen et al., Prot. Eng. 10: 1-6 (1997). ) And [von Heinje et al., Nucl.Acids. Res. 14: 4683-4690 (1986)]. In addition, it should be recognized that in some cases the cleavage of the signal sequence of the secreting polypeptide is not entirely identical resulting in one or more secreted polypeptides. Those mature polypeptides whose signal peptides are cleaved within the range of about 5 or less amino acids at either interface of the C-terminus of the signal peptides identified herein, and polynucleotides encoding them, are included in the present invention.

“PRO179 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 17-460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2), (b) the amino acid sequence of X-460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2) ( Wherein X is any amino acid residue from 12 to 21 of FIG. 2 (SEQ ID NO: 2) or (c) about 80% of the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 2 (SEQ ID NO: 2) By active PRO179 polypeptide as defined below (excluding PRO179 polypeptide of natural sequence) having the above amino acid sequence identity.

“PRO207 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 41 to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7), (b) the amino acid sequence of X to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7) ( Wherein X is any amino acid residue of 36 to 45 of FIG. 4 (SEQ ID NO: 7) or (c) about 80% of the amino acid sequence of another fragment of the amino acid sequence of specifically derived FIG. 4 (SEQ ID NO: 7) By active PRO207 polypeptide as defined below (excluding PRO207 polypeptide of natural sequence) having the above amino acid sequence identity.

“PRO320 variant polypeptide” refers to (a) the residue 1 or about 22 to 338 amino acid sequence of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10), (b) the amino acid sequence of X to 338 of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10) , X is any amino acid residue from 17 to 26 of FIG. 6 (SEQ ID NO: 10) or (c) at least about 80% of the amino acid sequence of another fragment of the amino acid sequence of specifically derived FIG. 6 (SEQ ID NO: 10) By active PRO320 polypeptide as defined below, excluding amino acid sequence identity, a PRO320 polypeptide of natural sequence is meant.

“PRO219 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 24 to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15), (b) the amino acid sequence of X to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15) ( Wherein X is any amino acid residue of 19 to 28 of FIG. 8 (SEQ ID NO: 15) or (c) about 80% of the amino acid sequence of another fragment of the amino acid sequence of specifically derived FIG. 8 (SEQ ID NO: 15) It means an active PRO219 polypeptide (except the PRO219 polypeptide of natural sequence) defined below having the above amino acid sequence identity.

“PRO221 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 34 to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20), (b) the amino acid sequence of X to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20) ( Wherein X is any amino acid residue from 29 to 38 of FIG. 10 (SEQ ID NO: 20), (c) the amino acid sequence of 1 or about 34 to X of FIG. 10 (SEQ ID NO: 20), where X is FIG. Any amino acid of 199 to 208 of 20), or (d) having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 10 (SEQ ID NO: 20), By active PRO221 polypeptide as defined in (excluding PRO221 polypeptide of natural sequence).

"PRO224 variant polypeptide" refers to (a) the amino acid sequence of residue 1 or about 31 to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25), (b) the amino acid sequence of X to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25) ( Wherein X is any amino acid residue of 26 to 35 of FIG. 12 (SEQ ID NO: 25), (c) the amino acid sequence of 1 or about 31 to X of FIG. 12 (SEQ ID NO: 25), where X is FIG. 25) 226 to 235) or (d) having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 12 (SEQ ID NO: 25), By active PRO224 polypeptide is defined (excluding the PRO224 polypeptide of natural sequence).

“PRO328 variant polypeptide” refers to (a) residue 1 or about 23 to 463 amino acid sequence of the PRO328 polypeptide of FIG. 14 (SEQ ID NO: 30), (b) X to 463 amino acid sequence of the PRO328 polypeptide of FIG. 14 (SEQ ID NO: 30), wherein , X is any amino acid residue from 18 to 27 of FIG. 14 (SEQ ID NO: 30) or (c) at least about 80% of the amino acid sequence of another fragment of the amino acid sequence of specifically derived FIG. 14 (SEQ ID NO: 30) By active PRO328 polypeptide as defined below (except PRO328 polypeptide of natural sequence), having amino acid sequence identity.

“PRO301 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 28 to 299 of the PRO301 polypeptide of FIG. 16 (SEQ ID NO: 35), (b) the amino acid sequence of X to 299 of the PRO301 polypeptide of FIG. 16 (SEQ ID NO: 35) ( Wherein X is any amino acid residue of 23 to 32 of FIG. 16 (SEQ ID NO: 35), (c) the amino acid sequence of 1 or about 28 to X of FIG. 16 (SEQ ID NO: 35), where X is FIG. 35) of 230) to 239) or (d) having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 16 (SEQ ID NO: 35), By active PRO301 polypeptide is defined (excluding PRO301 polypeptide of natural sequence).

“PRO526 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 27 to 473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43), (b) the amino acid sequence of X to 473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43) ( Wherein X is any amino acid residue from 22 to 31 of FIG. 18 (SEQ ID NO: 43) or (c) about 80% of the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 18 (SEQ ID NO: 43) By active PRO526 polypeptide as defined below (excluding PRO526 polypeptide of natural sequence) having the above amino acid sequence identity.

“PRO362 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 20 to 321 of the PRO362 polypeptide of FIG. 20 (SEQ ID NO: 48), (b) the amino acid sequence of X to 321 of the PRO362 polypeptide of FIG. 20 (SEQ ID NO: 48) ( Wherein X is any amino acid residue from 15 to 24 of FIG. 20 (SEQ ID NO: 48), (c) the amino acid sequence of 1 or about 20 to X of FIG. 20 (SEQ ID NO: 48), where X is FIG. 20 (SEQ ID NO: 48) of (276) to 285) or (d) having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 20 (SEQ ID NO: 48), By active PRO362 polypeptide is defined (excluding the PRO362 polypeptide of natural sequence).

“PRO356 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 27 to 346 of the PRO356 polypeptide of FIG. 22 (SEQ ID NO: 55), (b) the amino acid sequence of X to 346 of the PRO356 polypeptide of FIG. 22 (SEQ ID NO: 55) ( Wherein X is any amino acid residue from 22 to 31 of FIG. 22 (SEQ ID NO: 55) or (c) about 80% of the amino acid sequence of another fragment of the amino acid sequence of specifically derived FIG. 22 (SEQ ID NO: 55) By active PRO356 polypeptide as defined below (excluding PRO356 polypeptide of natural sequence) having the above amino acid sequence identity.

“PRO509 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 37 to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60), (b) the amino acid sequence of X to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60) ( Wherein X is any amino acid residue from 32 to 41 of FIG. 24 (SEQ ID NO: 60), (c) the amino acid sequence of 1 or about 37 to X of FIG. 24 (SEQ ID NO: 60), where X is FIG. 60 to 209 of 60) or (d) having at least about 80% amino acid sequence identity with the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 24 (SEQ ID NO: 60), By active PRO509 polypeptide is defined, excluding the PRO509 polypeptide of natural sequence.

“PRO866 variant polypeptide” refers to (a) the amino acid sequence of residue 1 or about 27 to 331 of the PRO866 polypeptide of FIG. 26 (SEQ ID NO: 62), (b) the amino acid sequence of X to 331 of the PRO866 polypeptide of FIG. 26 (SEQ ID NO: 62) ( Wherein X is any amino acid residue from 22 to 31 of FIG. 26 (SEQ ID NO: 62) or (c) about 80% of the amino acid sequence of another fragment of the specifically derived amino acid sequence of FIG. 26 (SEQ ID NO: 62) By active PRO866 polypeptide as defined below (except for PRO866 polypeptide of natural sequence) having the above amino acid sequence identity.

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

Typically, the PRO179 variant polypeptide comprises (a) residues 1 or about 17-460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2), (b) X-460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2), where X is FIG. (C) any amino acid residue from 12 to 21 of (SEQ ID NO: 2) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 2 (SEQ ID NO: 2) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably about At least 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, a PRO207 variant polypeptide comprises (a) residues 1 or about 41 to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7), (b) X to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7), where X is FIG. (C) any amino acid residue from 36 to 45 of (SEQ ID NO: 7) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 4 (SEQ ID NO: 7) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably about At least 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO320 variant polypeptide comprises (a) residues 1 or about 22-338 of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10), (b) X-338 of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10), wherein X is FIG. (C) any amino acid residue from 17 to 26 of (SEQ ID NO: 10) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 6 (SEQ ID NO: 10) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably At least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO219 variant polypeptide comprises (a) residues 1 or about 24 to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15), (b) X to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15), where X is FIG. (C) any amino acid residue of 19 to 28 of (SEQ ID NO: 15) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 8 (SEQ ID NO: 15) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably About 95% or more, more preferably is a is from about 98% or more, more preferably about 96% or more, more preferably at least about 97%, more preferably to at least about 99%.

Typically, the PRO221 variant polypeptide comprises (a) residues 1 or about 34 to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20), (b) X to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20), where X is (C) any amino acid residue from 29 to 38), (c) 1 or about 34 to X of FIG. 10 (SEQ ID NO: 20), where X is any of 199 to 208 of FIG. 10 (SEQ ID NO: 20) Amino acid sequence) or (d) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of FIG. 10 (SEQ ID NO: 20) of at least about 80%, more preferably at least about 81%, more preferably about At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% Phase, more preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably At least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO224 variant polypeptide comprises (a) residues 1 or about 31 to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25), (b) X to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25), where X is (Optional) 26 to 35 of any amino acid residue), (c) 1 or about 31 to X of FIG. 12 (SEQ ID NO: 25), where X is any of 226 to 235 of FIG. 12 (SEQ ID NO: 25) Amino acid sequence) or (d) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of FIG. 12 (SEQ ID NO: 25) of at least about 80%, more preferably at least about 81%, more preferably about At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably about At least 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO328 variant polypeptide comprises (a) residue 1 of the PRO328 polypeptide of Figure 14 (SEQ ID NO: 30) or about 23-463, (b) X-463 of the PRO328 polypeptide of Figure 14 (SEQ ID NO: 30), where X is Figure 14 (C) any amino acid residue from 18 to 27 of (SEQ ID NO: 30) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 14 (SEQ ID NO: 30) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably Is at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO301 variant polypeptide comprises (a) residues 1 or about 28 to 299 of the PRO301 polypeptide of FIG. 16 (SEQ ID NO: 35), (b) X to 299 of the PRO301 polypeptide of FIG. 16 (SEQ ID NO: 35), where X is FIG. (Any amino acid residue from 23 to 32 of SEQ ID NO: 35), (c) 1 or about 28 to X of Figure 16 (SEQ ID NO: 35), where X is any of 230-239 of Figure 16 (SEQ ID NO: 35) Amino acid sequence) or (d) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of FIG. 16 (SEQ ID NO: 35) of at least about 80%, more preferably at least about 81%, more preferably about At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably about At least 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO526 variant polypeptide comprises (a) residues 1 or about 27-473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43), (b) X-473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43), wherein X is FIG. 18. (C) any amino acid residue from 22 to 31 of (SEQ ID NO: 43) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 18 (SEQ ID NO: 43) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably Is at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO362 variant polypeptide is selected from (a) residues 1 or about 20-321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48), (b) X-321 of the PRO362 polypeptide of Figure 20 (SEQ ID NO: 48), where X is Figure 20 (Optional) 15 to 24 of any amino acid residue), (c) 1 or about 20 to X of FIG. 20 (SEQ ID NO: 48), where X is any of 276 to 285 of FIG. 20 (SEQ ID NO: 48) Amino acid sequence) or (d) specifically derived amino acid sequence identity with another fragment of the amino acid sequence of FIG. 20 (SEQ ID NO: 48) of at least about 80%, more preferably at least about 81%, more preferably about At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% , More preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably about At least 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO356 variant polypeptide comprises (a) residues 1 or about 27 to 346 of the PRO356 polypeptide of Figure 22 (SEQ ID NO: 55), (b) X to 346 of the PRO356 polypeptide of Figure 22 (SEQ ID NO: 55), where X is Figure 22 (C) any amino acid residue from 22 to 31 of (SEQ ID NO: 55) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 22 (SEQ ID NO: 55) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably Is at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO509 variant polypeptide comprises (a) the amino acid sequence of residue 1 or about 37 to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60), (b) the amino acid sequence of X to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60) ( Wherein X is any amino acid residue of 32 to 41 of FIG. 24 (SEQ ID NO: 60), (c) 1 or about 37 to X of FIG. 24 (SEQ ID NO: 60), where X is of FIG. 24 (SEQ ID NO: 60) Any amino acid from 200 to 209) or (d) at least about 80%, more preferably at least about 81% amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 24 (SEQ ID NO: 60) , More preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably about 9 At least 0%, more preferably at least about 91%, more preferably at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferred Preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

Typically, the PRO866 variant polypeptide comprises (a) residues 1 or about 27 to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: 62), (b) X to 331 of the PRO866 polypeptide of Figure 26 (SEQ ID NO: 62), where X is Figure 26 (C) any amino acid residue from 22 to 31 of SEQ ID NO: 62) or (c) at least about 80%, more preferably, amino acid sequence identity with another fragment of the specifically derived amino acid sequence of FIG. 26 (SEQ ID NO: 62) Preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86% At least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably Is at least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably Is at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variant polypeptides are native to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356 It does not cover the PRO509 and PRO866 polypeptides. Typically, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variant polypeptides are about 10 or more amino acids in length, often about 20 or more amino acids, more often At least about 30 amino acids, more often at least about 40 amino acids, more often at least about 50 amino acids, more often at least about 60 amino acids, more often at least about 70 amino acids, more often at least about 80 amino acids , More often at least about 90 amino acids, more often at least about 100 amino acids, more often at least about 150 amino acids, more often at least about 200 amino acids, more often at least about 250 amino acids, more often about At least 300 amino acids or more.

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

Tables 2a-2d also use the methods described below to determine amino acid sequence identity (%) (Tables 2a and 2b) and nucleic acid sequence identity (%) (Tables 2c and 2d) using the ALIGN-2 sequence comparison computer program. Is an example of a hypothesis to be used, where "PRO" represents the amino acid sequence of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide of the family of interest, and the "comparison" Protein "indicates the amino acid sequence of the polypeptide compared to the" PRO "polypeptide of interest, and" PRO-DNA "indicates PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509-, or PRO866-encoding nucleic acid sequences, "comparative DNA" refers to the nucleotide sequence of a nucleic acid molecule compared to the "PRO-DNA" nucleic acid molecule of interest, and "X" "," Y "and" Z "each represent a different amino acid residue Naemyeo other, "N", "L" and "V" represents a nucleotide of a different household, respectively.

“Amino acid sequence identity (%)” for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide sequence as identified herein means PRO179, PRO207, PRO320, PRO219, No conservative substitutions are considered as part of sequence identity after aligning the PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 sequences and introducing a gap if necessary to obtain maximum sequence identity Is defined as the ratio of amino acid residues in a candidate sequence that is identical to amino acid residues in a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide sequence. Alignment to determine percent amino acid sequence identity can be accomplished using various methods known in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Can be achieved. One skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to maximally align over the entire length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values can be obtained using the sequence comparison computer program ALIGN-2 as described below, with the complete source code for the ALIGN-2 program shown in Table 1. . The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code shown in Table 1 is kept as a user document of the U.S. Copyright Office (20559 Washington, DC), which is registered under U.S. Copyright Registration TXU510087. It is. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be edited from the source code shown in Table 1. The ALIGN-2 program can be edited and used in a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For the purposes herein, amino acid sequence identity (%) of a given amino acid sequence A to B, or B for a given amino acid sequence B (constant amino acid sequence identity to a given amino acid sequence B, B, or B (%) Can be expressed differently by a given amino acid sequence A with or including

X / Y × 100

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

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

When NCBI-BLAST-2 is used to compare amino acid sequences, the amino acid sequence identity (%) of a given amino acid sequence A to B, or B to a given amino acid sequence B (for a given amino acid sequence B, B, or B Can be expressed differently with a given amino acid sequence A having or including a certain percent amino acid sequence identity to).

X / Y × 100

Where X is the number of amino acid residues recorded as identical matches by the sequence alignment program NCBI-BLAST-2 in the A and B alignments of the sequence alignment program NCBI-BLAST-2, and Y is the total number of amino acid residues in B . If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be appreciated that the amino acid sequence identity (%) of A to B is not equal to the amino acid sequence identity (%) of B to A.

Amino acid sequence identity values (%) can also be obtained using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266 : 460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to default values. Those not set as default values, ie adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word limit (T) = 11, and score matrix = BLOSUM62. To achieve the objectives herein, the percent amino acid sequence identity value is determined by (a) the target amino acid sequence compared to the amino acid sequence of the target PRO polypeptide having a sequence derived from the native PRO polypeptide (ie, the sequence compared to the target PRO polypeptide). The number of identical amino acid residues matched with WU-BLAST-2 is determined by dividing by (b) the total number of amino acid residues of the desired PRO polypeptide. For example, the term "polypeptide comprising amino acid sequence A having at least 80% amino acid sequence identity with amino acid sequence B", amino acid sequence A is the target amino acid sequence to be compared, and amino acid sequence B is the amino acid sequence of the target PRO polypeptide. .

“PRO179 variant polynucleotide” or “PRO179 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 17 to 460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2), (b) of FIG. 2 (SEQ ID NO: 2) A nucleic acid sequence encoding the amino acids X to 460 of the PRO179 polypeptide, wherein X is any amino acid residue of 12 to 21 of FIG. 2 (SEQ ID NO: 2) or (c) specifically derived FIG. 2 (SEQ ID NO: 2) A nucleic acid molecule encoding an active PRO179 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of. Typically, the PRO179 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 17-460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2), and (b) X-460 of the PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue of 12 to 21 of FIG. 2 (SEQ ID NO: 2) or (c) specifically derived amino acid sequence of FIG. 2 (SEQ ID NO: 2) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably at least about 92% At least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably Will be at least about 98%, more preferably at least about 99%. PRO179 polynucleotide variants do not encompass the entirety of the native PRO179 nucleotide sequence.

“PRO207 variant polynucleotide” or “PRO207 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 41 to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7), (b) of FIG. 4 (SEQ ID NO: 7) A nucleic acid sequence encoding an amino acid from X to 249 of the PRO207 polypeptide, wherein X is any amino acid residue from 36 to 45 of FIG. 4 (SEQ ID NO: 7) or (c) specifically derived from FIG. 4 (SEQ ID NO: 7) A nucleic acid molecule encoding an active PRO207 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of. Typically, the PRO207 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 41 to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7), and (b) X to 249 of the PRO207 polypeptide of FIG. 4 (SEQ ID NO: 7). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 36 to 45 of FIG. 4 (SEQ ID NO: 7) or (c) specifically derived amino acid sequence of FIG. 4 (SEQ ID NO: 7) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably at least about 92% At least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably Will be at least about 98%, more preferably at least about 99%. PRO207 polynucleotide variants do not encompass the entirety of the native PRO207 nucleotide sequence.

“PRO320 variant polynucleotide” or “PRO320 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 22-338 of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10), (b) of FIG. 6 (SEQ ID NO: 10) A nucleic acid sequence encoding the amino acids X-338 of the PRO320 polypeptide, wherein X is any amino acid residue of 17-26 of FIG. 6 (SEQ ID NO: 10) or (c) specifically derived FIG. 6 (SEQ ID NO: 10) By a nucleic acid molecule encoding an active PRO320 polypeptide as defined below having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of an amino acid sequence of. Typically, the PRO320 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 22-338 of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10), and (b) X-338 of the PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 17 to 26 of FIG. 6 (SEQ ID NO: 10) or (c) specifically derived amino acid sequence of FIG. 6 (SEQ ID NO: 10) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably about At least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more Preferably at least about 98%, more preferably at least about 99%. PRO320 polynucleotide variants do not encompass the entirety of the native PRO320 nucleotide sequence.

“PRO219 variant polynucleotide” or “PRO219 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 24 to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15), (b) of FIG. 8 (SEQ ID NO: 15) A nucleic acid sequence encoding X to 1005 amino acids of the PRO219 polypeptide, wherein X is any amino acid residue from 19 to 28 of FIG. 8 (SEQ ID NO: 15) or (c) specifically derived FIG. 8 (SEQ ID NO: 15) By a nucleic acid molecule encoding an active PRO219 polypeptide as defined below having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of an amino acid sequence of. Typically, the PRO219 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 24 to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15), and (b) X to 1005 of the PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 19 to 28 of FIG. 8 (SEQ ID NO: 15) or (c) specifically derived amino acid sequence of FIG. 8 (SEQ ID NO: 15) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably At least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more Preferably at least about 98%, more preferably at least about 99%. PRO219 polynucleotide variants do not cover the entirety of the native PRO219 nucleotide sequence.

“PRO221 variant polynucleotide” or “PRO221 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 34 to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20), (b) of FIG. 10 (SEQ ID NO: 20) A nucleic acid sequence encoding the amino acids X to 259 of the PRO221 polypeptide, wherein X is any amino acid residue from 29 to 38 of FIG. 10 (SEQ ID NO: 20), (c) 1 or about 34 of FIG. 10 (SEQ ID NO: 20) To X, wherein X is any amino acid of 199 to 208 of FIG. 10 (SEQ ID NO: 20) or (d) a nucleic acid sequence encoding another fragment of the amino acid sequence of specifically derived FIG. 10 (SEQ ID NO: 20). A nucleic acid molecule encoding an active PRO221 polypeptide as defined below having at least about 80% nucleic acid sequence identity with a. Typically, the PRO221 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 34 to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20), and (b) X to 259 of the PRO221 polypeptide of FIG. 10 (SEQ ID NO: 20). A nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 29 to 38 of FIG. 10 (SEQ ID NO: 20), (c) 1 or about 34 to X of FIG. 10 (SEQ ID NO: 20), wherein X is Nucleic acid sequence identity with any nucleic acid sequence encoding any fragment of the amino acid sequence of FIG. 10 (SEQ ID NO: 20) of SEQ ID NO: 199 to 208) or (d) specifically derived At least 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more Preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably about 94 At least about 95%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%. PRO221 polynucleotide variants do not encompass the entirety of the native PRO221 nucleotide sequence.

“PRO224 variant polynucleotide” or “PRO224 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 31 to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25), (b) of FIG. 12 (SEQ ID NO: 25) A nucleic acid sequence encoding the amino acids X to 282 of the PRO224 polypeptide, wherein X is any amino acid residue from 26 to 35 of FIG. 12 (SEQ ID NO: 25), (c) 1 or about 31 of FIG. 12 (SEQ ID NO: 25) To X, wherein X is any amino acid of 226 to 235 of FIG. 12 (SEQ ID NO: 25) or (d) a nucleic acid sequence encoding another fragment of the specifically derived amino acid sequence of FIG. 12 (SEQ ID NO: 25) A nucleic acid molecule encoding an active PRO224 polypeptide as defined below having at least about 80% nucleic acid sequence identity with a. Typically, the PRO224 variant polynucleotide comprises (a) the nucleic acid sequence encoding residue 1 or about 31 to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25), and (b) X to 282 of the PRO224 polypeptide of FIG. 12 (SEQ ID NO: 25). A nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue of 26 to 35 of FIG. 12 (SEQ ID NO: 25), (c) 1 or about 31 to X of FIG. 12 (SEQ ID NO: 25), wherein X is Nucleic acid sequence identity with any nucleic acid sequence encoding any fragment of the amino acid sequence of FIG. 12 (SEQ ID NO: 25) of Figure 12 (SEQ ID NO: 25) or (d) specifically derived At least 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more Preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably about 94 At least about 95%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%. PRO224 polynucleotide variants do not encompass the entirety of the native PRO224 nucleotide sequence.

“PRO328 variant polynucleotide” or “PRO328 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 23 to 463 of the PRO328 polypeptide of FIG. 14 (SEQ ID NO: 30), (b) of FIG. 14 (SEQ ID NO: 30). A nucleic acid sequence encoding X to 463 amino acids of the PRO328 polypeptide, wherein X is any amino acid residue from 18 to 27 of FIG. 14 (SEQ ID NO: 30) or (c) specifically derived FIG. 14 (SEQ ID NO: 30) A nucleic acid molecule encoding an active PRO328 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of the amino acid sequence of. Typically, the PRO328 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 23 to 463 of the PRO328 polypeptide of FIG. 14 (SEQ ID NO: 30), and (b) X to 463 of the PRO328 polypeptide of FIG. 14 (SEQ ID NO: 30). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 18 to 27 of Figure 14 (SEQ ID NO: 30) or (c) specifically derived amino acid sequence of Figure 14 (SEQ ID NO: 30) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably At least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, More preferably at least about 98%, more preferably at least about 99%. PRO328 polynucleotide variants do not encompass the entirety of the native PRO328 nucleotide sequence.

“PRO301 variant polynucleotide” or PRO301 variant nucleic acid sequence ”refers to (a) a nucleic acid sequence encoding residue 1 or about 28 to 299 of the PRO301 polypeptide of FIG. 16 (SEQ ID NO: 35), (b) PRO301 of FIG. 16 (SEQ ID NO: 35) A nucleic acid sequence encoding residues X to 299 of the polypeptide, wherein X is any amino acid residue from 23 to 32 of FIG. 16 (SEQ ID NO: 35), (c) 1 or about 28 of FIG. 16 (SEQ ID NO: 35) To X, wherein X is any amino acid of amino acids 230 to amino acids 239 of FIG. 16 (SEQ ID NO: 35) or (d) encodes any fragment of the specifically derived amino acid sequence of FIG. 16 (SEQ ID NO: 35) A nucleic acid molecule encoding an active PRO301 polypeptide as defined below having at least about 80% nucleic acid sequence identity with a nucleic acid sequence, typically, a PRO301 variant polynucleotide is a residue of the PRO301 polypeptide of Figure 16 (SEQ ID NO: 53). 1 or about 28 to 299 A nucleic acid encoding the nucleic acid sequence encoding (b) an amino acid of residues X to 299 of the PRO301 polypeptide of FIG. 16 (SEQ ID NO: 35), wherein X is any amino acid residue of 23 to 32 of FIG. 16 (SEQ ID NO: 35) A sequence, (c) 1 or about 28 to X of FIG. 16 (SEQ ID NO: 35), wherein X is any amino acid of amino acids 230 to amino acid 239 of FIG. 16 (SEQ ID NO: 35), or (d) specifically derived Nucleic acid sequence identity with a nucleic acid sequence encoding any fragment of the amino acid sequence of FIG. 16 (SEQ ID NO: 35) is at least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably Is at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88% , More preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% , More preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably At least about 97%, more preferably at least about 98%, more preferably at least about 99%. PRO301 polynucleotide variants do not encompass the entirety of the native PRO301 nucleotide sequence.

“PRO526 variant polynucleotide” or “PRO526 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 27-473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43), (b) of FIG. 18 (SEQ ID NO: 43) Nucleic acid sequences encoding amino acids X-473 of the PRO526 polypeptide, wherein X is any amino acid residue from 22-31 of FIG. 18 (SEQ ID NO: 43) or (c) specifically derived FIG. 18 (SEQ ID NO: 43) A nucleic acid molecule encoding an active PRO526 polypeptide as defined below having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of an amino acid sequence of. Typically, the PRO526 variant polynucleotide is selected from (a) a nucleic acid sequence encoding residue 1 or about 27-473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43), and (b) X-473 of the PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 22 to 31 of FIG. 18 (SEQ ID NO: 43) or (c) specifically derived amino acid sequence of FIG. 18 (SEQ ID NO: 43) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably At least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, More preferably at least about 98%, more preferably at least about 99%. PRO526 polynucleotide variants do not encompass the entirety of the native PRO526 nucleotide sequence.

“PRO362 variant polynucleotide” or “PRO362 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 20-321 of the PRO362 polypeptide of FIG. 20 (SEQ ID NO: 48), (b) of FIG. 20 (SEQ ID NO: 48) A nucleic acid sequence encoding X to 321 amino acids of the PRO362 polypeptide, wherein X is any amino acid residue from 15 to 24 of FIG. 20 (SEQ ID NO: 48), (c) 1 or about 20 of FIG. 20 (SEQ ID NO: 48) To X, wherein X is any amino acid of amino acids 276 to amino acid 285 of FIG. 20 (SEQ ID NO: 48) or (d) any fragment of the specifically derived amino acid sequence of FIG. 20 (SEQ ID NO: 48) By a nucleic acid molecule encoding an active PRO362 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with the nucleic acid sequence. Typically, the PRO362 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 20-321 of the PRO362 polypeptide of FIG. 20 (SEQ ID NO: 48), and (b) X-321 of the PRO362 polypeptide of FIG. 20 (SEQ ID NO: 48). A nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue of 15 to 24 of FIG. 20 (SEQ ID NO: 48), (c) 1 or about 20 to X of FIG. 20 (SEQ ID NO: 48), wherein X is Nucleic acid sequence identity with the nucleic acid sequence encoding any fragment of the amino acid sequence of FIG. 20 (SEQ ID NO: 48) from amino acids 276 to amino acid 285) or (d) specifically derived At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85% Or more, more preferably about 86% or more, more preferably about 87% or more, more wind Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93 At least%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferred More than 99%. PRO362 polynucleotide variants do not cover the entirety of the native PRO362 nucleotide sequence.

“PRO356 variant polynucleotide” or “PRO356 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 27 to 346 of the PRO356 polypeptide of FIG. 22 (SEQ ID NO: 55), (b) of FIG. 22 (SEQ ID NO: 55) A nucleic acid sequence encoding X-346 amino acids of PRO356 polypeptide, wherein X is any amino acid residue from 22-31 of FIG. 22 (SEQ ID NO: 55) or (c) specifically derived FIG. 22 (SEQ ID NO: 55) A nucleic acid molecule encoding an active PRO356 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of an amino acid sequence of. Typically, the PRO356 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 27 to 346 of the PRO356 polypeptide of FIG. 22 (SEQ ID NO: 55), (b) X to 346 of the PRO356 polypeptide of FIG. 22 (SEQ ID NO: 55). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 22 to 31 of Figure 22 (SEQ ID NO: 55) or (c) specifically derived amino acid sequence of Figure 22 (SEQ ID NO: 55) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably At least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, More preferably at least about 98%, more preferably at least about 99%. PRO356 polynucleotide variants do not encompass the entirety of the native PRO356 nucleotide sequence.

“PRO509 variant polynucleotide” or “PRO509 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 37 to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60), (b) of FIG. 24 (SEQ ID NO: 60) A nucleic acid sequence encoding an amino acid from X to 283 of the PRO509 polypeptide, wherein X is any amino acid residue from 32 to 41 of FIG. 24 (SEQ ID NO: 60), (c) 1 or about 37 of FIG. 24 (SEQ ID NO: 60) To X, wherein X is any amino acid from amino acid 200 to amino acid 209 of FIG. 24 (SEQ ID NO: 60) or (d) encodes any fragment of the amino acid sequence of specifically derived FIG. 24 (SEQ ID NO: 60). By a nucleic acid molecule encoding an active PRO509 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with the nucleic acid sequence. Typically, a PRO509 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 37 to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60), and (b) X to 283 of the PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60). A nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 32 to 41 of FIG. 24 (SEQ ID NO: 60), (c) 1 or about 37 to X of FIG. 24 (SEQ ID NO: 60), wherein X is Nucleic acid sequence identity with a nucleic acid sequence encoding any fragment of amino acid sequence of amino acid 200 to amino acid 209 of FIG. 24 (SEQ ID NO: 60) or (d) specifically derived At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85% Or more, more preferably about 86% or more, more preferably about 87% or more, more wind Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93 At least%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferred More than 99%. PRO509 polynucleotide variants do not encompass the entirety of the native PRO509 nucleotide sequence.

“PRO866 variant polynucleotide” or “PRO866 variant nucleic acid sequence” refers to (a) a nucleic acid sequence encoding residue 1 or about 27 to 331 of the PRO866 polypeptide of FIG. 26 (SEQ ID NO: 62), (b) of FIG. 26 (SEQ ID NO: 62) A nucleic acid sequence encoding the amino acids X to 331 of the PRO866 polypeptide, wherein X is any amino acid residue from 22 to 31 of FIG. 26 (SEQ ID NO: 62) or (c) specifically derived FIG. 26 (SEQ ID NO: 62) A nucleic acid molecule encoding an active PRO866 polypeptide as defined below, having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding another fragment of an amino acid sequence of. Typically, the PRO866 variant polynucleotide comprises (a) a nucleic acid sequence encoding residue 1 or about 27 to 331 of the PRO866 polypeptide of FIG. 26 (SEQ ID NO: 62), and (b) X to 331 of the PRO866 polypeptide of FIG. 26 (SEQ ID NO: 62). Any fragment of a nucleic acid sequence encoding an amino acid, wherein X is any amino acid residue from 22 to 31 of Figure 26 (SEQ ID NO: 62) or (c) specifically derived amino acid sequence of Figure 26 (SEQ ID NO: 62) At least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84% of the nucleic acid sequence identity to the nucleic acid sequence encoding Or more, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably Is at least about 90%, more preferably at least about 91%, more preferably At least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, More preferably at least about 98%, more preferably at least about 99%. PRO866 polynucleotide variants do not encompass the entirety of 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, more often at least about 60 nucleotides, more often Is at least about 90 nucleotides, more often at least about 120 nucleotides, more often at least about 150 nucleotides, more often at least about 180 nucleotides, more often at least about 210 nucleotides, more often at least about 240 Nucleotides, more often at least about 270 nucleotides, more often at least about 300 nucleotides, more often at least about 450 nucleotides, more often at least about 600 nucleotides, more often at least about 900 nucleotides or more .

The “nucleic acid sequence identity (%)” for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide encoding nucleic acid sequences identified herein is PRO179, PRO207, PRO320, Align the PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptide coding nucleic acid sequences and introduce gaps to obtain maximum sequence identity (%), if necessary, followed by PRO179, PRO207, PRO320, It is defined as the ratio of nucleotides of the candidate sequence that is identical to the nucleotides of the PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 nucleic acid sequences. Alignment to determine percent nucleic acid sequence identity can be accomplished using various methods known in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Can be achieved. One skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to maximally align over the entire length of the sequences being compared. However, for the purposes of this disclosure, nucleic acid sequence identity values (%) can be obtained using the sequence comparison computer program ALIGN-2 as described below, the complete source code for which the ALIGN-2 program is shown in Table 1 It is described. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code shown in Table 1 is kept as a user document of the U.S. Copyright Office (20559 Washington, DC), which is registered under U.S. Copyright Registration TXU510087. It is. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be edited from the source code described in Table 1. The ALIGN-2 program can be edited and used in a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

For the purposes herein, nucleic acid sequence identity (%) of a given nucleic acid sequence C to D, or D for a given nucleic acid sequence D (constant nucleic acid sequence identity (%) to a D, or D for a given nucleic acid sequence D) Can be expressed differently by a given nucleic acid sequence C containing or comprising

W / Z × 100

Where W is the number of nucleotides recorded as identically matched by the sequence alignment program ALIGN-2 in the alignment of the sequence alignment programs ALIGN-2 of C and D, and Z is the total number of nucleotides of D. It will be appreciated that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the nucleic acid sequence identity (C) of C to D is not equal to the nucleic acid sequence identity (D) of D to C. As an example of nucleic acid sequence identity (%) calculations, Tables 2c and 2d show methods for calculating nucleic acid sequence identity (%) of nucleic acid sequences referred to as "comparative DNA" to nucleic acid sequences referred to as "PRO-DNA". .

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

When NCBI-BLAST-2 is used for nucleic acid sequence comparison, the nucleic acid sequence identity (%) of a given nucleic acid sequence C to D, or D to a given nucleic acid sequence D (for a given nucleic acid sequence D, D, or D Can be expressed differently by a given nucleic acid sequence C having or comprising a constant% nucleic acid sequence identity to

W / Z × 100

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

Nucleic acid sequence identity values (%) are also obtained using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266 : 460-80 (1996)). Most WU-BLAST-2 search parameters are set to their default values. Parameters not set by default, ie adjustable parameters, were set to the following values: overlap span = 1, overlap fraction = 0.125, word limit (T) = 11, and score matrix = BLOSUM62. To achieve the object herein, a nucleic acid sequence identity value (%) is determined by (a) a nucleic acid molecule that is compared to the nucleic acid sequence of the target PRO polypeptide encoding nucleic acid molecule having a sequence derived from a native PRO polypeptide-encoding nucleic acid (ie, target PRO The number of identical nucleotides matched with the polypeptide encoding nucleic acid molecule (which may be a PRO variant polynucleotide as a sequence) is obtained as WU-BLAST-2, followed by (b) the total nucleotides of the desired PRO polypeptide encoding nucleic acid molecule. Get divided by the number. For example, isolated nucleic acid molecule comprising nucleic acid sequence A having at least 80% nucleic acid sequence identity with nucleic acid sequence B, wherein nucleic acid sequence A is the sequence of the nucleic acid molecule of interest to be compared and nucleic acid sequence B is the PRO polypeptide. The nucleic acid sequence of the nucleic acid molecule of interest.

In other embodiments the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variant polynucleotides comprise the nucleotide sequence encoding the full length PRO179 polypeptide of FIG. 2 (SEQ ID NO: 2), FIG. Nucleotide sequence encoding the full length PRO207 polypeptide of 4 (SEQ ID NO: 7), nucleotide sequence encoding the full length PRO320 polypeptide of FIG. 6 (SEQ ID NO: 10), nucleotide sequence encoding the full length PRO219 polypeptide of FIG. 8 (SEQ ID NO: 15), FIG. Nucleotide sequence encoding the full-length PRO221 polypeptide of SEQ ID NO: 20), nucleotide sequence encoding the full-length PRO224 polypeptide of Figure 12 (SEQ ID NO: 25), nucleotide sequence encoding the full-length PRO328 polypeptide of Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35) Nucleotide sequence encoding a full-length PRO301 polypeptide of FIG. 18, a nucleus encoding the full-length PRO526 polypeptide of FIG. 18 (SEQ ID NO: 43). Nucleotide sequence encoding the full length PRO362 polypeptide of FIG. 20 (SEQ ID NO: 48), nucleotide sequence encoding the full length PRO356 polypeptide of FIG. 22 (SEQ ID NO: 55), nucleotide encoding the full length PRO509 polypeptide of FIG. 24 (SEQ ID NO: 60) The active PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, which can hybridize to the nucleotide sequence encoding the full length PRO866 polypeptide of FIG. 26 (SEQ ID NO: 62), preferably under stringent hybridization and wash conditions. , Nucleic acid molecule encoding PRO362, PRO356, PRO509, or PRO866 polypeptide, respectively. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 variant polypeptides It may be one encoded by a PRO509 or PRO866 variant polynucleotide.

With regard to amino acid sequence identity comparisons performed as described above, the term “positive” includes amino acid residues of the compared sequences having the same as well as similar characteristics. The amino acid residues having recorded the positive values of the target amino acid residues are those which are the same as the target amino acid residues or in which the target amino acid residues are preferably substituted (defined in Table 3 below).

For purposes herein, a given amino acid sequence B has a positive value (%) of B and a given amino acid sequence A for B (with a given amino acid sequence B, a constant positive value (%) for B and B). Or otherwise represented by a given amino acid sequence A, comprising:

X / Y × 100

Where X is the number of amino acid residues that record the positive value defined above in the alignment of the sequence alignment programs ALIGN-2 of A and B, and Y is the total number of amino acid residues of B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be appreciated that the positive value (%) of A for B is not equal to the positive value (%) of B for A.

"Isolated" as used to describe the various polypeptides disclosed herein means that the polypeptide has been identified, isolated and / or recovered from its natural environmental elements. Preferably, the isolated polypeptide is in an unbound state with all components bound in nature. Contaminant elements of the polypeptide's natural environment are materials that generally interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is purified to a sufficient degree to obtain (1) at least 15 residues of the N terminal or internal amino acid sequence using a spinning cup sequencer, or (2) Coomassie blue or preferably silver staining. Can be purified to show only one band in SDS-PAGE under non-reducing or reducing conditions. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 Polypeptide An isolated polypeptide is a polypeptide present in situ within recombinant cells because at least one element of the natural environment will not be present. It includes. However, normally isolated polypeptide will be obtained by one or more purification steps.

"Isolated" nucleic acid molecule or anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219 that encodes a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide , An “isolated” nucleic acid molecule encoding an anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibody is PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866-encoding nucleic acid 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- By nucleic acid molecules identified and separated from one or more contaminating nucleic acid molecules that are commonly bound in natural sources of PRO866-encoding nucleic acid. Preferably, the isolated nucleic acid is in an unbound state with all components bound in nature. Isolated PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866-encoding nucleic acid molecules or isolated 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-encoding nucleic acid molecules exist differently from the forms or states found in nature. Thus, the isolated nucleic acid molecules are PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866- present in natural cells. Coding nucleic acid molecule 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-encoding nucleic acid molecules. However, for example, it is located at a different chromosome position from natural cells and is typically PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or anti-PRO179, anti- Cells expressing PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibodies PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866-encoding nucleic acid molecules or anti-PRO179-, contained in Anti-PRO207-, anti-PRO320-, anti-PRO219-, anti-PRO221-, anti-PRO224-, anti-PRO328-, anti-PRO301-, anti-PRO526-, anti-PRO362-, anti-PRO356-, An anti-PRO509- or anti-PRO866-encoding nucleic acid molecule is an isolated nucleic acid molecule or anti-PRO179 that encodes a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. , Anti-PRO207, anti-PRO320, anti-PRO2 19, an isolated coding nucleic acid molecule encoding an anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibody.

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

Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences. For example, the DNA of a presequence or secretion leader is operably linked to the polypeptide's DNA when expressed as a proprotein, which is in the form before the polypeptide is secreted, and the promoter or enhancer affects the transcription of the polypeptide sequence. When interposed, the ribosome binding site is operably linked to the coding sequence when positioned to facilitate translation. In general, "operably linked" means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, not only are located contiguous, but also within the same reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional methods.

The term "antibody" is used in its broadest sense, specifically 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), 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 antibody compositions, single chain 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 antibodies, and anti-PRO179, anti- Fragments of PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 and anti-PRO866 antibodies Reference). As used herein, the term “monoclonal antibody” refers to an antibody obtained substantially from the population of homologous antibodies, ie the same individual antibodies that make up a population except for possible naturally occurring mutations that may be present in small amounts.

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

As defined herein, "strict conditions" or "high stringency conditions" means (1) 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfide at low ionic strength and high temperature, e.g., at washing. Conditions using pate, or (2) 50 mM sodium phosphate buffer (pH 6.5) /0.1% bovine serum albumin / 0.1 containing formamide, for example 42 ° C., 750 mM sodium chloride, 75 mM sodium citrate, upon hybridization. Conditions using a modifier such as 50% (volume / volume) formamide made from% Picol / 0.1% polyvinylpyrrolidone, (3) 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate) ), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C. 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C., and 50% forma at 55 ° C. Draw washing, and to perform a high-stringency wash with 0.1 x SSC containing EDTA at a 55 ℃.

"Moderate stringency conditions" are as described in Sambrook et al., Molecular Cloning : A Laboratory Manual, New York: Cold Spring Harbor Press (1989), and have less stringent hybridization conditions than those described above (eg, Such as temperature, ionic strength and% SDS). Examples of moderate stringency conditions include 20% formamide, 5 x SSC (150 mM sodium chloride, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt solution, 10% dextran sulfate and 20 mg The solution is incubated at 37 ° C. overnight in a solution containing crushed salmon sperm denatured DNA, followed by washing the filter with 1 × SSC at about 37-50 ° C. Those skilled in the art will know how to adjust the temperature, ionic strength, and the like necessary to match factors such as probe length and the like.

As used herein, the term “epitopically tagged” refers to a chimeric polypeptide comprising PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide fused to a “tag polypeptide”. Refers to a polypeptide. The tag polypeptide has enough residues to provide an epitope enough for the antibody to be made but short enough to not interfere with the activity of the fused polypeptide. It is desirable that the tag polypeptide is so unique that the antibody against itself does not substantially cross react with other epitopes. Suitable tag polypeptides generally have six or more amino acid residues and usually have about 8-50 amino acid residues (preferably about 10-20 amino acid residues).

As used herein, the term “immunoadhesin” refers to an antibody-like molecule that combines the effector function of an immunoglobulin constant domain and the binding specificity of a heterologous protein (“adhesin”). Structurally, immunoadhesin includes a fusion of an immunoglobulin constant domain sequence with an amino acid sequence having the desired binding specificity that is not (ie heterologous) the antigen recognition and binding site of the antibody. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in immunoadhesin may be any immune such as IgG-1, IgG-2, IgG-3, IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM Obtained from globulin.

As used herein, "active" or "active" refers to the biological and / or immunity of a naturally or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Reference is made to the form (s) of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 which possesses pharmaceutical activity. "Biological" activity here refers to the ability to induce the production of antibodies against antigenic epitopes possessed by naturally occurring or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Refers to biological function (inhibition or stimulation) by PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, other than naturally occurring or "immunological" activity Is used to refer to the ability to induce the production of antibodies against antigenic epitopes possessed by naturally occurring or naturally occurring PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. .

For antibodies or another agonist that can be identified by the screening assays disclosed herein, "biological activity" refers to the ability of the molecule to elicit one or more effects listed herein, along with the definition of "therapeutically effective amount." Used to. In certain embodiments, "biological activity" is the ability to inhibit neoplastic cell growth or proliferation. Preferred biological activity is inhibition involving slowing or complete stopping of growth of target tumor (eg cancer) cells. Another preferred biological activity is cytotoxic activity causing death of target tumor (eg cancer) cells. Another biological activity is the induction of cell death of target tumor (eg cancer) cells.

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

As used herein, "immunological cross-reactivity" refers to polyclonals in which candidate polypeptides are generated for known active PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides. It means that it is possible to competitively inhibit the quantitative biological activity of the antiserum and the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide having the above activity. The antiserum is prepared by conventional methods, for example, by subcutaneous injection of a goat or rabbit with an analog of a known activity in a complete Freund's adjuvant and boosted by intraperitoneal or subcutaneous injection in a complete Freund. The immunological cross-reactivity is preferably "specific", which corresponds to the corresponding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, of immunologically cross-reactive identified molecules (eg, antibodies). The binding affinity for the PRO362, PRO356, PRO509, or PRO866 polypeptide is significantly higher than the binding affinity of any other known molecule (preferably at least about 2 times, more preferably at least about 4 times, more preferably Is at least about 6 times, most preferably at least about 8 times).

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

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

"Treatment" means a treatment performed to prevent the occurrence of a disease or to change the pathology of a disease. Thus, "treatment" refers to both therapeutic treatment and inhibition or prophylactic measures. Those in need of treatment include those already in need of the disease as well as those in need of preventing the disease. In the treatment of tumors (eg cancer), the therapeutic agent may directly reduce the pathology of the cancer cells, or make the tumor cells more susceptible to treatment with other therapeutic agents such as radiation and / or chemotherapeutic agents. have.

The "pathology" of cancer includes all the phenomena that damage the patient's health. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, disruption of normal functioning of adjacent cells, abnormal levels of cytokines or other secretions, inhibition or exacerbation of inflammatory or immune responses, and the like.

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

A “therapeutically effective amount” in connection with the treatment of a tumor means one or more of the following effects: (1) inhibition of tumor growth to some extent, including delayed tumor growth and complete growth arrest, (2) reduction of tumor cell number, (3 ) Reduction in tumor size, (4) inhibition (ie, reduction, delay or complete stop) of tumor cell infiltration into surrounding organs, (5) inhibition (ie, reduction, delay or complete stop) of metastasis, (6) tumor Enhancement of an anti-tumor immune response that may (but need not necessarily) cause degeneration or rejection of (7) and (7) an amount that may cause some relief of one or more symptoms associated with the disease. The "therapeutically effective amount" of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist for tumor treatment can be determined empirically in conventional manner.

The "growth inhibitory amount" of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or agonist thereof may increase the growth of cells, in particular tumor cells, eg cancer cells. It is an amount that can be suppressed in vitro or in vivo. The "growth inhibition" of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists to inhibit neoplastic growth is empirically conventional. You can decide.

The "cytotoxic amount" of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or agonist thereof may be used to test for destruction of cells, in particular tumors, for example cancer cells. The amount that can be produced in vivo or in vivo. The "cytotoxic amount" of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or agonists for inhibiting neoplastic growth is empirically conventional. You can decide.

As used herein, "cytotoxic agent" refers to a substance that inhibits or inhibits the function of a cell and / or destroys a cell. The term refers to radioisotopes (eg I ¹³¹ , I ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ ), chemotherapeutic agents and toxins such as toxins or fragments thereof having enzymatic activity of bacterial, fungal, plant or animal origin. It is said to include.

A "chemotherapeutic agent" is a chemical compound useful for treating tumors, eg cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxins, taxoids, Examples include paclitaxel (Taxol ™, Bristol Myers Squibb Oncolo Co., Princeton, NJ) and docetaxel (Taxotere ™, Longplan Lorea Corporation, Antony, France), Toxotere , Methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, phosphamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carcin Minomycin, aminopterin, dactinomycin, mitomycin, esperamicin (see US Pat. No. 4,675,187), melphalan and other related nitrogen mustards. Also included in the definition of chemotherapeutic agents are hormonal agents that modulate or inhibit hormonal action on tumors such as tamoxifen and onapriston.

As used herein, "growth inhibitor" refers to a compound or composition that inhibits growth in vitro or in vivo of cells, especially tumors, eg cancer cells. Therefore, the growth inhibitory agent is a substance which greatly reduces the ratio of target cells of S phase. Examples of growth inhibitory agents include substances that block cell cycle progression (at periods other than S phase), such as those that induce G1 arrest and M arrest. Typical M blockers include vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. In addition, substances that stop G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methoxtrexate, 5-fluorouracil and ara-C also act on S phase arrest do. For further information, see Murakami et al., The Molecular Basis of Cancer , Mendelsohn and Israel, eds., "Cell cycle regulation, oncogens, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), Chapter 1, in particular page 13. It is described in.

"Cytokine" is the generic name of a protein that acts on another cell as an intercellular mediator, released by one cell population. Examples of cytokines are lymphokine, monocaine and typical polypeptide hormones. Cytokines include growth hormones such as human growth hormones, N-methionyl human growth hormones and bovine growth hormones, parathyroid hormones, thyroxine, insulin, proinsulin, rilacin, prorelaxine, vesicle stimulating hormone (FSH), Glycoprotein hormones such as thyroid stimulating hormone (TSH) and luteinizing hormone (LH), liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and -β, mullerian inhibitors, Mouse gonadotropin related peptides, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor such as NGF-β; Platelet growth factor; Transforming cell growth factors (TGF) such as TGF-α and -β, insulin-like growth factor-1 and -Π, erythropoietin (EPO), osteoinduction factors, interferons such as interferon-α, -β and -γ, colony stimulating factor (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF), interleukin (IL), for example 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 or TNF-β; And other polypeptide factors including LIF and kit ligand (KL). The term cytokine, as used herein, includes biologically active equivalents of native sequence cytokines and proteins from natural sources or from recombinant cell culture.

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

The term "agonist" is used in its broadest sense and refers to any molecule that mimics the biological activity of a native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. do. Suitable agonist molecules include specifically agonist antibodies or fragments of antibodies, fragments or amino acid sequence variants of native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, Peptides, small organic molecules, and the like. Methods for identifying agonists of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides include contacting candidate agonists with tumor cells and inhibiting tumor cell growth. It may include the step of measuring.

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

“Mammals” for therapeutic purposes are classified as mammals, including humans, livestock and breeding animals, and zoos, competitions or pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and the like. Say any animal that is being. Preferred mammals are humans.

 Administration “in combination” with one or more other therapeutic agents includes administration at the same time (simultaneously) or sequentially in any order.

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

"Natural antibodies" and "natural immunoglobulins" are generally glycoproteins of about 150,000 daltons of heterotetramers consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one disulfide covalent bond, and the number of disulfide bonds depends on the different immunoglobulin isotypes of the heavy chain. In addition, each heavy and light chain has disulfide bonds in the chain at regular intervals. Each heavy chain has at one end a variable domain (V _H ) and a number of constant domains linked to this domain. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end, the constant domain of the light chain is arranged in line with the first constant domain of the heavy chain, and the light chain variable domain is in line with the variable domain of the heavy chain. Are arranged. Particular amino acid residues are believed to form a covalent region between the light and heavy chain variable domains.

The term “variable” is contemplated that the sequences of certain portions of the variable domains differ greatly between antibodies and are used for the binding and specificity of each particular antibody to a particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions of both the light and heavy chain variable domains. The highly conserved portion of the variable domain is called the framework region (FR). The variable domains of the natural heavy and light chains comprise four framework regions that primarily select the arrangement of β-sheets by three CDRs that link the β-sheet structure or optionally form a loop that forms part of the structure. do. The CDRs in each chain are kept in close proximity to each other by the FR region and contribute to the formation of the antigen binding site of the antibody with the CDRs of the other chain (Kabat et al., NIH Publ. No. 91-3242, Vol. I). , pages 647-669 (1991). The constant domains do not directly participate in antigen binding of the antibody, but show the effector function of the antibody in several effector functions, for example antibody dependent cytotoxicity.

As used herein, "hypervariable region" refers to an amino acid residue of an antibody that functions important for antigen binding. The hypervariable region may be a residue of a "complementarity determining region" or "CDR" (ie, residues 24 to 34 (L1), 50 to 56 (L2) and 89 to 97 (L3) and residue 31 in the heavy chain variable domain in the light chain variable domain). To 35 (H1), 50 to 65 (H2) and 95 to 102 (H3) amino acid residues) [Kabat et al., Sequences of Proteins of Immunological Interests , 5th Ed. Public Health Services, National Institute of Health, Bethesda, MD. (1990)] and / or “hypervariable loops” (ie residues 26 to 32 (L1), 50 to 52 (L2) and 91 to 96 (L3) and residues 26 to heavy chain variable region in the light chain variable domain) Amino acid residues of 32 (H1), 53-55 (H2) and 96-101 (H3)) [Clothia and Lesk, J. Mol. Biol ., 196 : 901-917 (1987). "Framework" or "FR" residues are those region residues other than the hypervariable region residues defined above.

An “antibody fragment” comprises a portion of a complete antibody, preferably the antigen binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments, diabodies, linear antibodies [Zapata et al., Protein Eng . 8 (10) : 1057-1062 (1995)], multispecific antibodies formed from single chain antibody molecules and antibody fragments.

Papain digestion of the antibody produces two identical antigen binding fragments, the so-called "Fab" fragments having one antigen binding site, and the remaining "Fc" fragments, which reflect the ability to readily crystallize. Treatment of pepsin results in an F (ab ') ₂ fragment having two antigen binding sites and still capable of crosslinking to the antigen.

"Fv" is the minimum antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain that is tightly covalently bonded. In this configuration, the three CDRs of each variable donor interact to define an antigen binding site on the surface of the V _H -V _L dimer. In total, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of the Fv that contains only CDRs specific for only three antigens), although less affinity than the entire binding site, can recognize and bind to the antigen.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1). Fab fragments differ from Fab 'fragments in that several residues have been added to the carboxy terminus of the heavy chain CH1 domain comprising one or more cysteines derived from the antibody hinge region. Fab'-SH herein refers to Fab 'in which the cysteine residue (s) of the constant domains bear a free thiol group. F (ab ') ₂ antibody fragments originally were produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “light chain” of an antibody (immunoglobulin) derived from any vertebrate species can be classified into one of two distinctly different types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain. have.

Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be classified into different classes. There are five main classes of IgA, IgD, IgE, IgG and IgM in immunoglobulins, some of which are further classified into subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2. Can be.

As used herein, the term “monoclonal antibody” refers to an antibody obtained substantially from the population of homologous antibodies, ie the same individual antibodies that make up a population except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are very specific and directed against a single antigenic site. Also, in contrast to conventional antibody (polyclonal antibody) preparations, which typically include other antibodies against other determinants (epitopes), each monoclonal antibody is directed against a single determinant for the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma culture and not contaminated by other immunoglobulins. The modifier “monoclonal” refers substantially to the properties of antibodies obtained from a homogeneous group of antibodies and is not to be construed as requiring antibody production in any particular way. For example, the monoclonal antibodies used in the present invention are prepared by the hybridoma method or recombinant DNA method first described in Kohler et al., Nature , 256 : 495 (1975) [US Pat. No. 4,816,567]. Can be. In addition, “monoclonal antibodies” are described, for example, in Clackson et al., Nature , 352 : 624-628 (1991) and Marks et al., J. Mol. Biol ., 222 : 581-597 (1991) can be used to isolate from phage antibody libraries.

The monoclonal antibodies herein specifically represent a portion of the heavy and / or light chains that, if exhibiting the desired activity, is identical or different from the sequence corresponding to an antibody derived from a particular species or an antibody belonging to a particular antibody group or subclass. Homologous, and the remainder of the chain (s) comprises "chimeric" antibodies (immunoglobulins) that are identical or homologous to antibodies from another species or antibodies belonging to another antibody group or subclass or fragments of such antibodies. [US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA , 81 : 6851-6855 (1984).

A “humanized” form of a non-human (eg murine) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof comprising a minimum sequence derived from a non-human immunoglobulin (eg, Fv, Fab, Fab ', F (ab') ₂ or other antigen binding small sequence of the antibody). In most cases humanized antibodies replace residues of the recipient's complementarity determining regions (CDRs) with residues from CDRs of non-human species (donor antibodies) such as mice, rats or rabbits with the desired specificity, affinity and ability. Human immunoglobulins (receptor antibodies). In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in the awarding antibody or in the CDR or framework sequences to be introduced. Such modifications result in improved and maximized antibody performance. In general, a humanized antibody will comprise substantially all of one or more, generally two or more variable domains, where all or substantially all CDR regions correspond to regions of non-human immunoglobulin and all or substantially all FR regions Corresponds to the region of the human immunoglobulin consensus sequence. In addition, humanized antibodies will optimally comprise at least a portion of an immunoglobulin constant region (Fc), generally at least a portion of a human immunoglobulin region [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-329 (1988); And Presta, Curr. Op. Struct. Biol. 2 : 593-596 (1992). Humanized antibodies include PRIMATIZED® antibodies derived from antibodies produced by immunizing a macaque with the antigen of interest in which the antigen binding region of the antibody is desired.

“Single chain Fv” or “sFv” antibody fragments comprise the V _H and V _L domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide also comprises a polypeptide linker between the V _H and V _L domains that allow the sFv to form the structure necessary for antigen binding. For sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113 , Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to a small antibody fragment with two antigen binding sites, comprising a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) within the same polypeptide chain (V _H -V _L ). Say By using linkers that are too short to pair two domains in the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies are described, for example, in European Patent No. 404,097, International Publication No. 93/11611, and Hollinger et al., Proc. Natl. Acad. Sci. USA , 90 : 6444-6448 (1993).

 An "isolated" antibody is an antibody that has been identified, isolated and / or recovered from elements of its natural environment. Contaminant elements of its natural environment are substances that interfere with diagnostic or therapeutic use of antibodies and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) greater than 95% by weight, most preferably greater than 99% by weight of the antibody, or (2) using a spinning cup sequencer as measured by the Lowry method. Enough to obtain at least 15 residues of the N-terminal or internal amino acid, or (3) Coomassie blue, or preferably using silver staining, in one band by SDS-PAGE under reducing or non-reducing conditions. Will be 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. However, isolated antibodies will generally be purified by one or more purification steps.

"Label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to produce an "labeled" antibody. The label can be detected by itself (radioisotope label or fluorescent label) or, in the case of an enzyme label, can catalyze the chemical alteration of the detectable substrate compound or composition.

"Solid phase" means a non-aqueous matrix to which an antibody of the invention may be attached. Examples of solid phases include partially or completely formed glass (eg pore controlled glass), polysaccharides (eg agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone, and the like. In certain embodiments, depending on the content, the solid phase may comprise the wells of an assay plate, and in other embodiments the solid phase is a purification column (eg an affinity chromatography column). The term also encompasses discrete solid phases of discrete particles, such as those described in US Pat. No. 4,275,149.

A “liposome” is used to deliver a drug (eg, an antibody to a mammal, eg, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides disclosed herein) to a mammal. It is a small vesicle composed of various forms of lipids, phospholipids and / or surfactants. The components of the liposomes are typically aligned in bilayer form, similar to the lipid alignment of the biofilm.

"Small molecule" is defined herein as having a molecular weight of about 500 Daltons or less.

II. Compositions and Methods of the Invention

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

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

As described in the Examples below, cDNA clones encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356 and PRO866 polypeptides were deposited in the ATCC [clone PRO509 not deposited in ATCC] Except]. One skilled in the art can readily determine the actual nucleotide sequence of the clone by analyzing the sequence of the deposited clone using conventional methods in the art. General techniques can be used to determine the expected amino acid sequence from the nucleotide sequence. For the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides described herein, and the nucleic acids encoding them, the inventors have used the sequence information available at the time We confirmed that it is considered as a reading frame that can be confirmed well.

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

In addition to the full-length native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 polypeptides described herein, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301 It is contemplated that polypeptide variants of PRO526, PRO362, PRO356, PRO509 and PRO866 can be prepared. The PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, and PRO866 variants provide suitable nucleotide changes for the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362 , PRO356, PRO509 or PRO866 DNA and / or may be prepared by the synthesis of the desired PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Those skilled in the art will appreciate that amino acid changes, such as changes in the number or position of glycosylation sites or changes in membrane anchoring properties, can be achieved by post-translation of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 It will be appreciated that processing can be changed.

Full length native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, Variations in the various domains of PRO526, PRO362, PRO356, PRO509 or PRO866 can be prepared using, for example, the conservative and non-conservative mutation techniques and instructions disclosed in US Pat. No. 5,364,934. Variation is compared with the natural sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 when the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526 Substitution, deletion of one or more codons encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866, which changes the amino acid sequence of PRO362, PRO356, PRO509, or PRO866 Or insertion. Optionally, the mutation is generated by substituting any other amino acid for one or more amino acids in one or more domains of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Amino acid residues that can be inserted, substituted or deleted without adversely affecting the desired activity include those of the known sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. This can be determined by comparing the sequence of homologous protein molecules and minimizing the number of amino acid sequence changes generated in regions of high identity. Amino acid substitutions may be the result of substitution of one amino acid with another amino acid having similar structural and / or chemical properties, eg, replacement of leucine with serine, ie, conservative substitution of amino acids. Insertion or deletion may optionally occur at about 1-5 amino acids. Acceptable variations can be determined by systematically inserting, deleting or replacing amino acids in the sequence and testing the activity of the resulting variants represented by the full length or mature native sequence.

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

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 fragments can be prepared by any of a number of conventional techniques. Peptide fragments of interest can be synthesized chemically. Other methods include enzymatic digestion methods, for example PRO179, PRO207, PRO320, PRO219, PRO221, by treating this protein with an enzyme known to cut the protein at sites defined by specific amino acid residues or by cutting this DNA with suitable restriction enzymes. , Generating PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 fragments and isolating this purpose fragment. However, another suitable technique involves the isolation and amplification of DNA fragments encoding the desired polypeptide fragments by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are used as 5 'and 3' primers in PCR. Preferably, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 polypeptide fragments are shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), and FIG. 10 (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. 48, SEQ ID NO: 55 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 60), or each of the natural PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 Or share one or more biological and / or immunological activities with the PRO866 polypeptide.

In specific embodiments, conservative substitutions of the target are shown in Table 3 under the heading of preferred substituents. When the biological activity is changed by such substitutions, more substantial changes, which are named as substitutions in Table 3 below or described in more detail below with respect to amino acid species, were introduced and the products were screened.

Substantial modifications of the functional or immunological identity of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be achieved by (a) the structure of the polypeptide backbone at For example, by selecting substitutions that remain in sheet or helix form, (b) maintain the charge or hydrophobicity of the molecule at the target site, or (c) significantly alter its effect of retaining most of the side chains. Naturally occurring residues are divided into the following groups according to common side chain properties:

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

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

(3) acid: asp, glu,

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

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

(6) aromatic; trp, tyr, phe.

Non-conservative substitutions will replace said one type of component with another type. In addition, the residues so substituted may be introduced at conservative substitution sites or more preferably at the remaining (non-conserved) sites.

Mutations can be made using methods known in the art such as oligonucleotide mediated (positioning) mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res. , 13 : 4331 (1986); Zoller et al., Nucl. Acids Res. , 10 : 6487 (1987)], cassette mutagenesis [Wells et al., Gene , 34 : 315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA , 317 : 415 (1986)] or other well-known techniques are carried out on cloned DNA to give the present invention PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 Polypeptide variant DNA can be prepared.

Scanning amino acid assays can also be used to identify one or more amino acids along adjacent sequences. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Typically, alanine is a preferred scanning amino acid because it is less likely to remove side chains outside the beta-carbon and alter the backbone arrangement of the variant (Cunningham and Wells, Science , 244 : 1081-1085 (1989)). Alanine is also preferred because it is usually the most common amino acid. In addition, alanine is frequently found both in buried and exposed locations [Creighton, The Proteins , (WH Freeman & Co., NY); Chothia, J. Mol. Biol. , 150 : 1 (1976). If alanine substitutions do not produce adequate amounts of variants, isotopic amino acids can be used.

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

Covalent modifications of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 and PRO866 are within the scope of the present invention. One form of covalent modification is an organic derivatizing agent capable of reacting with selected side chain or N- or C-terminal residues of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. And reacting a target amino acid residue of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Derivatizations with bifunctional agents are for example anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362 PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or water-insoluble support matrix or surface for use in anti-PRO356, anti-PRO509 or anti-PRO866 antibody purification methods or It is useful for crosslinking PRO866 and vice versa. Commonly used crosslinking agents are for example 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4-azidosalicylic acid, Homo-functional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), difunctional maleimides such as bis-N-maleimido-1,8-octane and Materials such as methyl-3 [(p-azidophenyl) dithio] propioimidate.

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

Other types of covalent modifications of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides within the scope of the present invention include changes in the natural glycosylation pattern of the polypeptide. Include. "Changes in the natural glycosylation pattern" means the deletion (potential glycosylation) of one or more carbohydrate residues found in the native sequences PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866. By removing the site or deleting glycosylation by chemical and / or enzymatic methods) and / or the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or By one or more glycosylation sites that are not present in PRO866. The term also encompasses qualitative changes in glycosylation of natural proteins, including changes in the properties and proportions of the various carbohydrate residues present.

The addition of glycosylation sites to the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be achieved by changing the amino acid sequence. The change can be made, for example, by the addition or substitution of one or more serine or threonine residues in the native sequence PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 (O For linking glycosylation sites). The PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 amino acid sequences specifically produce PRO179, PRO207, PRO320, PRO219, PRO221, PRO224 to produce codons translated into the target amino acid. DNA encoding a, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide can be changed randomly through a change in DNA level by mutating at a preselected base.

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

Removal of carbohydrate residues present in a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide can be used to determine amino acid residues that function chemically or by enzymes or as glycosylation targets. It can be achieved by substitution by mutation of the coding codon. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin, et al., Arch. Biochem. Biophys. , 259 : 52 (1987) and Edge et al., Anal. Biochem. , 118 : 131 (1981). Enzymatic cleavage of carbohydrate residues on polypeptides can be accomplished using various endoglycosidases and exoglycosidases [Thotakura et al., Meth. Enzymol. , 138 : 350 (1987)].

Other types of covalent modifications of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 are described in US Pat. Nos. 4,640,835, 4,496,689, 4,301,144 and 4,670,417. To one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene, in the manner described in US Pat. No. 4,791,192 or 4,179,337, PRO221, PRO224, Linking a PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide.

In addition, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides of the present invention may be fused to other heterologous polypeptides or amino acid sequences of PRO179, PRO207, PRO320, PRO219, PRO221. And may be modified in such a way as to form chimeric molecules, including PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866.

In one embodiment, such chimeric molecules comprise 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, PRO356, PRO509 Or a fusion of the PRO866 polypeptide. Epitope tags are generally located at the amino or carboxyl terminus of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. The presence of the epitope tagged form of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide can be detected using an antibody against the tagged polypeptide. Can be. In addition, the introduction of epitope tags allows for affinity purification using anti-tag antibodies or other types of affinity matrices that bind to epitope tags, such as PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides can be readily purified. Various tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine or poly-histidine-glycine tags, flu HA tag polypeptides and antibodies thereof 12CA5 [Field et al., Mol. Cell. Biol. , 8 : 2159-2165 (1988)], c-myc tags and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology , 5 : 3610-3636 (1985)] And herpes simplex virus glycoprotein D (gD) tag and its antibodies (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)], alpha-tubulin Epitope peptides [Skinner et al., J. Biol. Chem. , 266 : 15163-15166 (1991)] and the T7 gene 10 protein tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA , 87 : 6393-6397 (1990).

In other embodiments, the chimeric molecule comprises a fusion of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with a specific region of an immunoglobulin or immunoglobulin. For the bivalent form of the chimeric molecule (also referred to as "immune adhesin"), the fusion may be the Fc region of an IgG molecule. This Ig fusion preferably comprises the site of one or more variable regions in the Ig molecule, soluble (deleted or missing) of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. In the form of an activated transmembrane domain). In particularly preferred embodiments, the immunoglobulin fusions comprise the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. For methods of producing immunoglobulin fusions, see US Pat. No. 5,428,130, published June 27, 1995.

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

Described below are PRO179 by primarily culturing cells transformed or transfected with a vector containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 nucleic acid. , PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Of course, other methods known in the art can be used to prepare PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide sequences or fragments thereof. For example, the sequences or fragments included in the above definitions of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 can be obtained by direct peptide synthesis using solid phase techniques. Stewart et al., Solid-Phase Peptide Synthesis , WH Freeman Co., San Francisco, CA (1969), Merrifield, J. Am. Chem. Soc. , 85 : 2149-2154 (1963). In vitro protein synthesis can be performed by manual or automated methods. Automated synthesis can be performed using, for example, Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Various PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide fragments can be chemically synthesized separately and combined using chemical or enzymatic methods to achieve full-length PRO179, PRO207, PRO320 , PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be prepared.

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

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 Polypeptide Coding DNA is available for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356 Can be obtained from a cDNA library prepared from tissues that are thought to possess, PRO509 or PRO866 mRNA and express it at detectable levels. Thus, human PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding DNA can be conveniently obtained from cDNA libraries prepared from human tissue as described in the Examples. Can be. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding genes are also obtained from genomic libraries or by known synthetic methods (e.g., automated nucleic acid synthesis). can do.

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

The following examples illustrate techniques for screening cDNA libraries. Oligonucleotide sequences selected as probes should be sufficiently long and sufficiently clear to minimize false positive results. Oligonucleotides are preferably labeled so that they can be detected upon hybridization to DNA in the library being screened. Labeling methods are known in the art and include the use of radiolabeled, biotinylated or enzymatic labels such as ³² P labeled ATP. Hybridization conditions, including moderate stringency and high stringency is provided in the literature (Sambrook et al., Supra).

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

Nucleic acid having protein coding sequence and the first screening a cDNA or genomic library selected using conventional methods of primer extension described in the literature (Sambrook et al., Supra) to detect the estimated amino acid sequence and the precursor, if necessary, as disclosed herein, It can be obtained by processing intermediates of mRNA that are not reverse transcribed into cDNA.

2. Selection and Transformation of Host Cells

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

Eukaryotic transfection methods and prokaryotic transformation methods such as CaCl ₂ , CaPO ₄ , liposome-mediated methods and electroporation are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques suitable for the cell. Document calcium treatment using calcium chloride or electroporation method described in (Sambrook et al., Supra), it is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is described in certain plant cells as described in Shaw et al., Gene , 23 : 315 (1983) and WO 89/05859 published June 29, 1989. Used for transformation of For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology , 52 : 456-457 (1978) can be used. General features of mammalian cell host system transfection are described in US Pat. No. 4,399,216. Transformation into yeast is generally described by Van Solingen et al., J. Bact. 130 : 949 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) , 76 : 3829 (1979)]. However, other methods of introducing DNA into cells may also be used, such as intranuclear microinjection, electroporation, fusion of circular cells with bacterial protoplasts, or polycations such as polybrene, polyornithine. For several techniques for transformation of mammalian cells, see Keown et al., Methods in Enzymology , 185 : 527-537 (1990) and Mansour et al., Nature , 336 : 348-352 (1988). do.

Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells are sedative bacteria, for example Gram negative or Gram positive organisms, for example Enterobacteriaceae, for example E. coli. E. coli, including but not limited to. A variety of teeth. E. coli strains, for example E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537). E. coli strains W3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are readily available. Other suitable prokaryotic host cells are Escherichia, such as E. coli. Coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Sererratia, for example For example Serratia marcescans and Shigella, and Bacillus, for example B. Subtilis and b. Licheniformis (e.g. B. licheniformis 41P described in DD 266,710, published April 12, 1989), Pseudomonas, for example p. Aeruginosa and Streptomyces. This example is illustrative only and is not limited thereto. Strain W3110 is a particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentation. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to result in mutation of the gene encoding the endogenous protein of the host, an example of such a host is E. coli with the complete genotype tonA . E. coli W3110 strain 1A2, E. coli with the complete genotype tonA ptr3 . E. coli W3110 strain 9E4, E. coli with the full genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r . E. coli W3110 strain 27C7 (ATCC 55,244), E. coli with the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degPOmpT rbs7 ilvG kan ^r . E. coli W3110 strain 37D6, strain 37D6 having a non-kanamycin resistant degP deletion mutation. E. coli W3110 strain 40B4 and E. coli with the Periplasm protease variant disclosed in US Pat. No. 4,946,783, issued August 7, 1990. E. coli strains. Alternatively, in vitro cloning methods such as PCR or other nucleic acid polymerase reactions are suitable.

In addition to prokaryotic cells, eukaryotic microorganisms such as fibrous fungi or yeast are suitable as cloning or expression hosts for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other microorganisms include Shichizosaccharomyces pombe (Beach and Nurse, Nature , 290: 140 [1989]; EP 139,383, published May 2, 1985); Kluyveromyces hosts (US Pat. 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. Baceriol. , 737 [1983]), k. Fragilis (ATCC 12,424), k. Bulgaricus (ATCC 16,045), k. Wickeramii (ATCC 24,178), K. Waltii (ATCC 56,500), K. Drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology , 8: 135 (1990)), K. Thermotolerans and K. Marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol. , 28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA , 76: 5259-5263 [1979]); Schwanniomyces such as, for example, occidentalis (EP 394,538, published October 31, 1990); And fibrous fungi such as neurospora, penicillium, tolypocladium (WO 91/00357, published January 10, 1991) and Aspergillus hosts, for example Listen a. Nidulans (Ballance et al, Biochem. Biophys. Res. Commun. , 112: 284-289 [1983]; Tilburn et al., Gene , 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA , 81: 1470-1474 [1984]) and A. Niger (Kelly and Hynes, EMBO J. , 4: 475-479 [1985]). Methyl nutrient-retaining yeast is suitable and consists of Hansenula, Candida, Kloeckera, Peachia, Carraomyces, Torulopsis and Rhodotorula Yeast capable of growing on methanol, selected from, but not limited to. For a list of specific species that are examples of these types of yeast, see C. Anthony, The Biochemistry of Methylotrophs , 269 (1982).

Suitable host cells for the expression of glycosylated PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding nucleic acids are from multicellular organisms. Examples of invertebrate cells are insect cells and plant cells, such as Drosophila S2 and Spodoptera Sf9. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40, human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol. , 36:59 (1997)), Chinese Hamster Ovary Cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA , 77: 4216 (1980)), Mouse Sertoli Cells ( TM4, Mather, Biol. Reprod. , 23: 243-251 (1980)), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065) and mouse breast tumors (MMT 060562, ATCC CCL51) It includes. One skilled in the art can readily select suitable host cells.

3. Selection and use of replicable vectors

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding nucleic acid (eg cDNA or genomic DNA) can be cloned for cloning (amplification of DNA) or expression Inserted into a vector Various vectors can be easily obtained. For example, the vector may be in the form of plasmids, cosmids, viral particles or phages. Suitable nucleic acid sequences can be inserted into the vector by various methods. In general, DNA is inserted into suitable restriction endonuclease site (s) using techniques known in the art. Vector components generally include, but are not limited to, signal sequences, origins of replication, one or more marker genes, enhancer components, promoters and transcription termination sequences. The preparation of suitable vectors comprising one or more such components uses standard ligation techniques known to those skilled in the art.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be produced by direct recombination methods as well as by specific cleavage sites at the N terminus of the mature protein or polypeptide. It can be produced as a fusion polypeptide with a heterologous polypeptide, which can be another polypeptide or signal sequence having. In general, the signal sequence may be a component of the vector or may be part of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding DNA inserted into the vector. The signal sequence can be for example a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leader. For yeast secretion, signal sequences are for example yeast invertase leader, α factor leader (Saccharomyces and Kluyveromyces α-factor leader (US Pat. No. 5,010,182)) or acid phosphatase Leader, mr. C. albicans glucoamylase leader (EP 362,179 published April 4, 1990) or the signal sequence described in WO 90/13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, and viral secretion leaders can be used to direct the secretion of proteins.

Both expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are known for various 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 various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are used to clone vectors in mammalian cells. useful.

Expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Representative selection genes, such as for Bacillus, the gene encoding D-alanine racemase may be (a) proteins that confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, ( b) encode proteins that complement nutritional deficiencies or (c) provide proteins that provide important nutrients that are not available from the complex medium.

Examples of selectable markers suitable for mammalian cells include the identification of cells capable of accepting PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding nucleic acid. One, for example DHFR or thymidine kinase. When wild type DHFR is used, a suitable host cell is a CHO cell line lacking DHFR activity, see Urlaub et al, Proc. Natl. Acad. Sci. USA , 77: 4216 (1980). Suitable selection genes for use in yeast are trp1 genes present in yeast plasmid YRp7 [Stinchcomb et al., Nature , 282: 39 (1979), Kingsman et al., Gene , 7: 141 (1979), Tschemper et al. , Gene , 10: 157 (1980)]. The trp1 gene provides a selection marker for variant strains of yeast lacking the ability to grow to tryptophan (eg ATCC 44076 or PEP4-1) [Jones, Genetics , 85: 12 (1977)].

Expression and cloning vectors contain a promoter operably linked to a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding nucleic acid sequence that directs mRNA synthesis. Promoters recognized by various potential host cells are known. Suitable promoters for use in prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature , 275: 615 (1978); Goeddel et al., Nature , 281: 544 (1979)], alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nucleic acid 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). In addition, promoters used in bacterial systems are Shine-Dalgano (SD) operably linked to DNA encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Will contain the sequence.

Examples of promoter sequences suitable for use in yeast hosts are described by 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem. , 255: 2073 (1980)] or other glycolysis enzymes [Hess et al., J. Adv. Enzyme Reg. , 7: 149 (1968); Holland, Biochemistry , 17: 4900 (1978)], for example enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 Promoters for phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters, which are inducible promoters with additional transcriptional benefits controlled by growth conditions, include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes involved in nitrogen metabolism, metallothionein, glyceraldehyde- Promoter region for 3-phosphate dehydrogenase and enzymes that act on maltose and galactose use. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 transcription from a vector in a mammalian host cell is a virus such as polyoma virus, poultry virus (1989 Of UK 2,211,504), adenovirus (eg, adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkey virus 40 (SV40), published July 5 From the genome, a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, is regulated by a promoter compatible with the host cell system, obtained from a heat-shock promoter.

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

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

Other methods, vectors and host cells suitable for application to the synthesis of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 in recombinant vertebrate cell cultures are described in eting et al. , Nature , 293: 620-625 (1981); Mantei et al., Nature , 281: 40-46 (1979); EP 117,060 and EP 117,058.

4. Detection of Gene Amplification / Expression

Gene amplification and / or expression may be performed using appropriately labeled probes based on the sequences provided herein, eg, conventional Southern blotting, Northern blotting to quantify transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], can be measured directly in a sample by dot blotting (DNA analysis) or in situ hybridization. It is also possible to use antibodies which can recognize specific duplexes, including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes. In other words, an antibody can be labeled and the assay can be performed by binding the duplex to the surface to detect the presence of the antibody bound to the duplex when the duplex is formed on the surface.

Gene expression can also be measured by immunological methods such as immunohistochemical staining of cell or tissue sections and direct analysis of cell culture or body fluids for direct quantification of gene products. Antibodies useful for immunohistochemical staining and / or assays of sample solutions may be monoclonal or polyclonal antibodies, and may be prepared in any mammal. Conveniently, synthetic peptides or PRO179, PRO207 based on the PPRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides of the native sequence or DNA sequences provided herein Antibodies to exogenous sequences that are fused to, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding DNA and that encode specific antibody epitopes can be prepared.

5. Purification of Polypeptides

The form of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide can be recovered from the culture medium or host cell lysate. When bound to the membrane, it can be released from the membrane using a suitable detergent solution (eg Triton-X 100) or by cleavage of the enzyme. Cells used for the expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide-encoding nucleic acids may be treated with freeze-thaw cycles, sonication, mechanical disruption or cell lysis agents. Such as by a variety of physical or chemical means.

It may be desirable to purify PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 from recombinant cell proteins or polypeptides. Examples of suitable purification methods are fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica or cation exchange resins, for example chromatography on DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, for example Sephadex Gel filtration with G-75, Protein A Sepharose column to remove contaminants such as IgG and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides Metal chelating columns for joining epitope tagged forms. Various protein purification methods can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology , 182 (1990); Scopes, Protein Purification: Principles and Practice , Springer-Verlag, New York (1982). The purification step (s) selected will depend, for example, on the production method used and the properties of the particular PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 produced. .

E. Antibodies

Some of the drug candidates for use in the compositions and methods of the present invention include antibodies that mimic the biological activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides, and Antibody fragments.

1. Polyclonal Antibodies

Methods of making polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be produced in a mammal, for example, by injecting the immunizing agent and, if necessary, an adjuvant one or more times. In general, the immunizing agent and / or adjuvant will be injected into the mammal by subcutaneous or intraperitoneal multiple injections. Immunizing agents can include PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or a fusion fragment thereof. It may be useful to conjugate an immunizing agent to a protein known to be immunogenic in a mammal to be immunized. Examples of such immunogenic proteins include keyhole limpet hemocyanin, serum albumin, bovine tyroglobulin 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 dicholinomycolate). Immunotreatment methods can be selected by one skilled in the art without unnecessary experimentation.

2. Monoclonal Antibodies

In addition, the antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods as described in Kohler and Milstein, Nature , 256 : 495 (1975). In hybridoma methods, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes capable of producing or producing antibodies that specifically bind to the immunizing agent. In addition, lymphocytes can be immunized in vitro.

Immunizing agents will typically include PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or a fusion protein thereof. Generally, peripheral blood lymphocytes (“PBLs”) are used when cells of human origin are desired, but spleen cells or lymph node cells are used when non-human mammal sources are desired. Then, a suitable fusion agent such as polyethylene glycol is used to fuse the lymphocytes with the immortalized cell line to form hybridoma cells. [Goding, Monoclonal Antibodies: Principle and Practice , Academic Press, (1986) pp. 59-103. Immortalized cell lines are largely transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. In general, rat or mouse myeloma cell lines are used. Hybridoma cells are preferably cultured in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cell lacks hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzyme, the culture medium for hybridoma typically contains hypoxanthine, aminopterin and thymidine ("HAT medium"). These substances will inhibit the growth of HGPRT-deficient cells.

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

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

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

Monoclonal antibodies secreted by the subclones are, for example, conventional immunoglobulin purification methods such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. It can be isolated and purified from the culture medium or ascites solution by.

Monoclonal antibodies can also be prepared 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 carried out using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding heavy and light chains of murine antibodies). Can be isolated and sequenced. The hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA is inserted into an expression vector and then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, to monoclonal in recombinant host cells. Raw antibodies can be synthesized. In addition, DNA, for example, to replace the coding sequence for human heavy and light chain constant domains in place of the homologous sequence of the murine or (U.S. Patent Nos. 4,816,567;. Morrison et al, supra), or a non-immunoglobulin polypeptide All or part of a coding sequence for may be modified by covalently binding to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention or for the variable domains of one antigen-binding site of the antibodies of the invention to generate chimeric bivalent antibodies.

The antibody may be a monovalent antibody. Methods of making monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light and modified heavy chains. Heavy chains are generally truncated at any point in the Fc region to prevent heavy chain crosslinking. Alternatively, related cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking.

In vitro methods are also suitable for the preparation of monovalent antibodies. Degradation of the antibody to prepare antibody fragments, particularly Fab fragments, can be carried out using techniques commonly known in the art.

3. Human and Humanized Antibodies

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

Methods of humanizing non-human antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues introduced thereto from a non-human origin. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization is essentially performed by methods such as Winter et al., Jones et al., Nature , 321 : 522-525 (1986), by substituting a rodent CDR or CDR sequence in place of the corresponding sequence of a human antibody. Riechmann et al., Nature , 332 : 323-329 (1988); Verhoeyen et al., Science , 239 : 1534-1536 (1988). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein substantially less than a fully human variable domain is substituted by the corresponding sequence from a non-human species. Indeed, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues of similar sites in rodent antibodies.

Human antibodies can also be prepared using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. , 227 : 381 (1991), Marks et al., J. Mol. Biol. , 222 : 581 (1991)]. In addition, techniques such as Coul et al. And Boerner can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985) and Boerner et al. , J. Immunol. , 147 (1) : 86-95 (1991)] Similarly, by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or wholly inactivated. Human antibodies can be prepared After challenge, human antibody production was observed, which was very similar to antibodies observed in humans in all respects, including gene rearrangements, assemblies, and antibody lists. (US Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016) and in Sciences, Marks et al., Bio / Technology 10 , 779-783 (1992), Lonberg et al., Nature 368 , 856-859 (1994), Mo rrison, Nature 368 , 812-13 (1994), Fishwild et al., Nature Biotechnology 14 , 845-51 (1996), Neuberger, Nature Biotechnology 14 , 826 (1996), Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, having binding specificities for two or more different antigens. Herein, one of the binding specificities is for PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 and the other is any other antigen, preferably cell surface For a protein, receptor or receptor subunit.

Methods of making bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain / light chain-heavy chain pairs, where the two heavy chains have different specificities [Millstein and Cuello, Nature , 305 : 537-539 (1983). Due to the random arrangement of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually carried out by an affinity chromatography step. Similar methods are described in WO 93/08829 (published May 13, 1993) and in Traunecker et al., EMBO J. , 10 : 3655-3659 (1991).

The variable domain (antibody-antigen binding site) of the antibody with the desired binding specificity is fused to an immunoglobulin constant domain sequence. Fusion is preferably in an immunoglobulin heavy chain constant domain comprising at least a portion of a hinge, CH2 and CH3 region. It is preferred that a first heavy chain constant region (CH1) comprising a site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion, and if desired, the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected with the appropriate host organism. For more details on producing bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology , 121 : 210 (1986).

According to another method disclosed in WO 96/27011, the covalent region between a pair of antibody molecules can be engineered to maximize the proportion of heterodimer recovered from recombinant cell culture. Preferred covalent regions comprise at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the covalent region of the first antibody molecule were replaced with larger side chains (eg tyrosine or tryptophan). Replacement of large amino acid side chains with small amino acid side chains (eg, alanine or threonine) created complementary "cavities" of the same or similar size to the large side chains in the covalent region of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimer to be higher than 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 generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science 229: 81 (1985) describe a method for cleaving intact antibodies by proteolysis to generate F (ab ') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize nearby dithiols and prevent the formation of intermolecular disulfide bonds. The Fab 'fragments generated were then converted to thionitrobenzoate (TNB) derivatives. Thereafter, one of the Fab'-TNB derivatives was reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of other Fab'-TNB derivatives to form bispecific antibodies. The resulting bispecific antibodies can be used as substances for the selective immobilization of enzymes.

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

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

Antibodies having a bivalent or higher valency are also considered. For example, trispecific antibodies can be made (Tutt et al., J. Immunol. 147: 60 (1991)).

Typical bispecific antibodies may bind to two different epitopes, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides described herein. Alternatively, to focus on intracellular defense mechanisms against cells expressing a particular PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, 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 ) The triggering molecule on leukocytes, such as a T-cell receptor molecule (eg CD2, CD3, CD28 or B7), or an Fc receptor (FcγR) of IgG, eg FcγRI (CD64), FcγRII (CD32) And arms that bind FcγRIII (CD16). In addition, bispecific antibodies can be used to localize cytotoxic agents to cells that express particular PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides. Such antibodies include PRO179-, PRO207-, PRO320-, PRO219-, PRO221-, PRO224-, PRO328-, PRO301-, PRO526-, PRO362-, PRO356-, PRO509- or PRO866-binding arms and cytotoxic or radionuclides It has an arm that binds to a chelating agent, for example EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds to a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide and also binds to tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also included within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies can be 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, US 92/200373, and EP 03089). Ho) proposed. It is contemplated that heterozygous antibodies can be prepared using methods known in synthetic protein chemistry, including methods using crosslinkers. For example, immunotoxins can be prepared using disulfide exchange reactions or by forming thioether bonds. Reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutyrimidate and the reagents disclosed, for example, in US Pat. No. 4,676,980.

6. Effector function operation

It may be desirable to modify the antibodies of the invention to effector function to enhance the effectiveness of the antibodies, for example in the treatment of cancer. For example, intrachain disulfide bonds in this region can be formed by introducing cysteine residue (s) into the Fc region. Homodimer antibodies thus generated enhance internalization capacity and / or increase complement-mediated cell death and antibody-dependent intracellular cytotoxicity (ADCC) [Caron et al., J. Exp Med. , 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148 : 2918-2922 (1992). See also Wolff et al. The heterodifunctional crosslinking described in Cancer Research , 53 : 2560-2565 (1993) can be used to prepare homodimer antibodies with enhanced anti-tumor activity. Alternatively, antibodies with double Fc regions can be engineered to enhance complement lytic and ADCC ability (see Stevenson et al., Anti-Cancer Drug Design , 3 : 219-230 (1989)).

7. Immunoconjugates

The invention also relates to cytotoxic agents such as chemotherapeutic agents, toxins (eg, enzymatically active bacteria, fungi, plant or animal toxins, or fragments thereof) or radioactive isotopes (ie radioconjugates). It relates to an immunoconjugate comprising an antibody bound to).

Chemotherapeutic agents useful for the production of such immunoconjugates are described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modede Renal A chain, alpha- sarsine , Aleurites fordii protein, diantine protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), bitter melon (momordica charantia) inhibitor, Curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogelin, restrictin, phenomycin, enomycin, and tricotessenes. Several radionuclides can be used to prepare radioconjugate antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Various difunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), difunctional derivatives of imidoesters (eg , Dimethyl adipimidate HCL), active esters (e.g. disuccinimidyl suverate), aldehydes (e.g. glutaraldehyde), bis-azido compounds (e.g. bis (p-a) Zidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg tolyene 2,6-diisocyanate), and bis Active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene) can be used to make conjugates of antibodies and cytotoxic substances. For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science , 238 : 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a representative chelating agent for conjugation of radionucleotides with antibodies (see WO 94/11026).

In another embodiment, antibodies and “receptors” (eg, streptavidin) used for tumor pretargeting can be conjugated, wherein the antibody-receptor conjugate is administered to the patient and then circulated using a chelating agent. The unbound conjugate is removed from and the "ligand" (eg avidin) conjugated to a cytotoxic substance (eg radionucleotide) is administered.

8. Immunoliposomes

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

Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes were extruded through filters of defined pore size to yield liposomes with the desired diameter. Martin et al., J. Biol. Chem. , 257 : 286-288 (1982), can be linked to liposomes via Fab 'fragments of the antibodies of the invention via disulfide-interchange reactions. Chemotherapeutic agents (eg, Doxorubicin) are optionally included in liposomes [Gabizon et al., J. National Cancer Inst. , 81 (19) : 1484 (1989)].

F. Identification of proteins that can inhibit neoplastic growth or proliferation

The proteins disclosed herein were analyzed in a panel of 60 tumor cell lines currently used for in vitro study drug development screening for diseases of the National Cancer Institute (NCI). The purpose of this screening is to identify molecules with cytotoxic and / or cytostatic activity against other forms of tumor. NCI has screened more than 10,000 new molecules each year. Monks et al., J. Natl Cancer Inst ., 83: 757-766 (1991), Boyd, Cancer: Princ. Pract. Oncol. Update , 3 (10) : 1-12 (1989)]. Tumor cell lines used in this study are described in Monks et al., Supra. Cell lines whose growth was significantly inhibited by the proteins of the present application are listed in the Examples.

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

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

G. Animal Models

Various well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of tumors, and to test the efficacy of candidate therapeutics, including antibodies, and other agonists of natural polypeptides, including small molecule agonists. Can be. The in vivo nature of these molecules makes it possible to predict responses in particular in human patients. The in vivo nature of the model makes the response of the therapeutic agent particularly predictable in human patients. Animal models of tumors and cancers (eg, breast cancer, colon cancer, prostate cancer, lung cancer, etc.) include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents, for example rat models. The model induces tumor cells in homogeneous mice using standard techniques, eg subcutaneous injection, tail vein injection, spleen transplantation, intraperitoneal transplantation, adrenal transplantation or orthopine transplantation, eg colon cancer cells transplanted into colon tissue. It can be prepared by PCT Publication WO97 / 33351 published September 18, 1997.

The most commonly used animals for tumor research are immunodeficient mice, especially nude mice. Nude mice with hypothyroidism have been used extensively for this purpose by the discovery that they successfully function as hosts for human tumor xenografts. The recessive autosomal nu gene is expressed in many nude mouse species of different pseudogenes, such as ASW, A / He, AKR, BALB / c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I / st, NC, NFR, NFS, NFS / N, NZB, NZC, NZW, P, RIII, and SJL. In addition to nude mice, various other animals with inherited immunological defects were also crossed and used as recipients of tumor xenografts. For further details see, eg, The Nude Mouse in Oncology Research , E. Boven and B. Winograd, eds. (CRC Press, Inc., 1991).

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

Tumor cells can be introduced into animals such as nude mice in a number of ways. The subcutaneous (sc) space of mice is well suited for tumor transplantation. Tumors can be implanted subcutaneously as solid blocks, trocar biopsy needles or cell suspensions. In the case of solid block or trocar transplantation, tumor tissue fragments of suitable size are introduced into the subcutaneous space. Cell suspensions are prepared from primary tumors or stable tumor cell lines and injected by subcutaneous transplantation. Tumor cells can also be injected into a subcutaneous graft. In this position, the inoculum is deposited between the lower part of the dermal connective tissue and the subcutaneous tissue. Animal models of breast cancer include, for example, rat neuroblastomas (cells from which neu tumor genes were first isolated) or neu transformed NIH-3T3 cells [Drebin et al., Proc. Natl. Acad. Sci. USA , 83 : 9129-9133 (1986)].

Similarly, colon cancer animal models can be used to passage colon cancer cells in animals, such as nude mice, to induce tumor expression in the animals. In situ transplantation models of 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 Rearch , 55 : 681-684 (1995). It is described. The model was based on the so-called METAMOUSE from AntiCancer Inc. (San Diego, Calif.) Commercially available.

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

Meth A, CMS4, CMS5, CMS21 and WEHI-164, for example, are chemically induced fibrosarcomas in BALB / c mouse females and provide an adjustable model system for studying the antitumor activity of various agents [Palladino et al. J. Immunol. 138 : 4023-4032 (1987)]. Briefly, tumor cells proliferate in cell culture in vitro. Cell lines are washed before animal injection and resuspended in buffer at a cell density of 10 × 10 ⁶ to 10 × 10 ⁷ cells / mL. Animals are then subcutaneously injected with 10-100 μl of cell suspension and left for 1-3 weeks to develop tumors.

In addition, Lewis lung (3LL) carcinoma of mice, the most fully studied experimental tumor, can be used as a tumor research model. The efficacy of this tumor model is associated with a beneficial effect in the treatment of human patients diagnosed with lung small cell cancer (SCLL). This tumor can be induced in normal mice by injecting tumor fragments from affected mice or cells maintained in culture, as described by Zupi et al., Br. J. Cancer 41 : suppl. 4, 309 (1980)], tumors can even begin with injection of single cells and are evidence of the survival of many of the infected tumor cells. For further information on this tumor model, see Zacharski, Haemostasis 16 : 300-320 (1986).

One method of assessing the efficacy of test compounds in animal models is to measure the size of transplanted tumors before and after treatment. Typically, the size of the implanted tumor is measured with slide calipers in two or three dimensions. Two-dimensionally defined measurements do not reflect the exact size of the tumor and are generally converted to the corresponding volume using mathematical formulas. However, the measurement of tumor size is very inaccurate. The therapeutic effect of drug candidates may be better represented by the growth retardation and specific growth retardation induced upon treatment. Another important variable indicative of tumor growth is tumor volume doubling time. Rygaard and Spang-Thomson, Proc. 6th Int. Computer programs for the calculation and display of tumor growth are available, such as those reported in Workshop on Immune-Deficient Animals , Wu and Sheng eds. (Basel, 1989, 301). However, necrosis and inflammatory responses following treatment actually lead to an increase in tumor size at least initially. Therefore, these changes should be carefully monitored in combination with morphometry and flow cytometry.

Recombinant (transgenic) animal models can be engineered by introducing the coding sites of the genes identified herein into the genome of the desired animal using standard techniques for producing transgenic animals. Targets for transgenic manipulation can be used without limitation in mice, rats, rabbits, guinea pigs, sheep, goats, pigs and non-human primates such as baboons, chimpanzees and monkeys. Known techniques for introducing transgenes into such animals include pronuclear microinjection (US Pat. No. 4,873,191), retroviral mediated gene transfer into embryos [eg, Van der Putten et al., Proc. Natl. Acad. Sci. USA , 82 : 6148-615 (1985)], gene targeting in embryonic cells [Thompson et al., Cell , 56 : 313-32 (1989)], electroporation of embryos [Lo, Mol. Cell. Biol. , 3 : 1803-1814 (1983); Sperm Mediated Gene Delivery [Lavitrano et al., Cell. 57 : 717-73 (1989). See, for example, US Pat. No. 4,736,866 for more information.

For the purposes of the present invention, transgenic animals include animals ("mosaic animals") which have a transgene in only part of the cell. The transgenes can be integrated into a single transgene or concatameric, ie head-head or hair-tail arrangement. Selective introduction of transgenes into specific cell tumors is described, for example, in Lasko et al . [ Proc. Natl. Acd. Sci. USA. 89 : 6232-636 (1992).

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

The efficacy of antibodies and other candidate drugs that specifically bind to polypeptides identified herein can also be tested in the treatment of spontaneous animal tumors. Suitable targets for such studies are cat oral squamous cell carcinoma (SCC). Feline oral SCC is the most common oral malignant tumor of cats, which accounts for 60% of the reported oral tumors in this species. Although the low incidence of metastasis only reflects the short survival of cats with this tumor, it rarely metastasizes to the primary site. Dissection of the cat's oral cavity, in general, cannot perform surgical operations on these tumors. At present, there is no effective treatment for this tumor. Prior to introduction into the study, each cat undergoes a complete clinical examination, biopsy and computed tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are excluded from the study. These tumors can paralyze the tongue, and even if the tumor is removed by treatment, the animal will not be able to eat on its own. Each cat is treated repeatedly over a long period of time. Photographs of tumors are taken daily and during subsequent review during the treatment period. After treatment, each cat is computed tomography, and every eight weeks thereafter, the computed tomography and chest radiogram are evaluated. Data are compared to controls to assess for differences in survival, response and toxicity. A positive response preferably requires evidence of tumor degeneration with improved quality of life and / or increased lifespan.

In addition, other naturally occurring animal tumors can be tested, such as fibrosarcomas, adenocarcinomas, lymphomas, pontoons, leiomyosarcomas of dogs, cats and baboons. Among these mammals, mammary gland cancer in dogs and cats is a preferred model because of their very similar form and response to humans. However, the use of these models is limited to the rare occurrence of this type of tumor in animals.

H. Screening Assays for Drug Candidates

Screening assays for drug candidates are intended to identify compounds that competitively bind to or form conjugates with receptor (s) of the polypeptides identified herein, or signals through such receptors. Such screening assays include assays capable of high throughput screening for chemical libraries, which would be particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include peptides, preferably soluble peptides, (poly) peptide-immunoglobulin fusions, and polyclonal antibodies and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotype antibodies and chimeric antibodies or such Included, but not limited to, antibodies or fragments and humanized antibodies of human antibodies and antibody fragments include synthetic organic or inorganic compounds comprising antibodies. Assays can be performed in a variety of formats including well-characterized protein-protein binding assays, biochemical screening assays, immunoassays and cell line assays.

In binding assays, the interaction is binding, and the complex formed can be isolated or detected in the reaction mixture. In certain embodiments, a receptor or drug candidate of a polypeptide encoded by a gene identified herein is immobilized to a solid phase, eg, a microtiter plate, by covalent or non-covalent attachment. Non-covalent attachment is generally achieved by coating the solid surface with a solution and drying. Alternatively, an immobilized antibody, such as a monoclonal antibody specific for an immobilized polypeptide, can be used to anchor it to a solid surface. The assay can be performed by adding an unfixed component that can be labeled with a detectable label to a coding surface comprising a fixed component, for example an anchored component. When the reaction is finished, unreacted components are removed, for example, by washing, and a binder anchored to the solid surface is detected. If the component that was not originally immobilized contained a detectable label, detection of the label immobilized on the surface indicates that a complex formation reaction occurred. If a component that is not originally immobilized does not include a label, a complexed reaction can be detected, for example, using a labeled antibody that specifically binds to the immobilized complex.

If a candidate compound interacts with, but does not bind to, a specific receptor, the interaction of the candidate compound with this polypeptide can be analyzed by well known methods for detecting protein-protein interactions. Such assays include conventional methods such as crosslinking, coimmunoprecipitation, and copurification via gradient or chromatography columns. Protein-protein interactions are also described in Fields and co-workers (Fields and Song, Nature (London) , 340: 245-246 (1989), Chien et al., Proc. Natl. Acad. Sci. USA , 88 : 9578-9582 (1991)), as described by Chevray and Nathans, Proc. Natl. Acad. Sci. USA , 89: 5789-5793 (1991)). can do. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other as a transcription-active domain. The yeast expression system described in this document (generally referred to as "2-hybrid system") takes advantage of this property and uses two hybrid proteins, one of which is that the target protein is associated with the DNA-binding domain of GAL4. The other is a candidate active protein fused with an active domain. The expression of the GAL1-lacZ reporter gene under the control of the GAL4-activation promoter depends on the reconstitution of GAL4 activity through protein-protein interactions. Colonies containing the interacting polypeptide are detected using a pigment producing substrate for β-galactosidase. A complete kit (MATCHMAKER ^™ ), which confirms protein-protein interactions between two specific proteins using 2-hybrid technology, can be purchased from Clontech. In addition, the system can be extended to map protein domains involved in specific protein interactions, as well as to accurately pinpoint the location of amino acid residues critical for such interactions.

I. Pharmaceutical Composition

Polypeptides of the invention, agonist antibodies that specifically bind to the proteins identified herein, and other molecules identified by the screening assays disclosed herein can be administered in the form of pharmaceutical compositions to treat various diseases, including cancer. .

If antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable-region sequences of an antibody, peptide molecules can be designed that can bind to the target protein sequence. Such peptides can be synthesized chemically and / or prepared by recombinant DNA techniques (eg, Marasco et al., Proc. Natl. Acad. Sci. USA , 90 : 7889-7893 (1993)). Reference).

In addition, the formulations herein may comprise one or more active compounds required for the particular indication to be treated, preferably compounds with complementary activities that do not cause side effects from each other. Alternatively, or in addition, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. The molecule is suitably present in an amount effective for the purpose intended.

Therapeutic formulations of the polypeptides identified herein may be used to store, for storage, an antibody having the desired purity in any pharmaceutically acceptable carrier, excipient or stabilizer [ Remington's Pharmaceutical Sciences , 16th edition, Osol, A. ed. (1980)] in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers, excipients or stabilizers should be nontoxic to patients at the dosages and concentrations administered, and include buffers such as buffers of phosphoric acid, citric acid and other organic acids; Antioxidants and preservatives including ascorbic acid and methionine (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethium chloride; phenol, butyl or benzyl alcohol; methyl or propyl parabens Such as alkyl parabens; catechols; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophobic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counter ions such as sodium; Metal complexes (eg, Zn-protein complexes); And / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

In addition, the formulations herein may comprise one or more active compounds required for the particular indication to be treated, preferably compounds with complementary activities that do not cause side effects from each other. Alternatively, or in addition, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. The molecule is suitably present in an amount effective for the purpose intended.

The active ingredient is for example in a colloidal drug delivery system (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example coacervation techniques or interfacial polymerization, For example, it can be enclosed in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules. This technique is disclosed in Remington's Pharmaceutical Sciences , 16th edition, Osol, A. ed. (1980).

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

Therapeutic compositions herein can generally be placed in a container having a sterile inlet, such as a vial having a stopper that can be penetrated by an intravenous solution bag or a hypodermic needle.

Sustained release formulations may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (US Pat. No. 3,773,919), L-glutamic acid Injectable microparticles consisting of a copolymer of ethyl-L-glutamate, a non-degradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer, for example LUPRON DEPOT ™ (lactic acid-glycolic acid copolymer and leuprolide acetate) Spear), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time. If the encapsulated antibody remains in the body for a long time, it may be denatured or aggregated as a result of exposure to moisture at 37 ° C. to reduce biological activity and change immunogenicity. For stabilization, rational strategies can be designed according to the mechanism involved. For example, if the aggregation mechanism is found to be intramolecular SS bond formation through thio-disulfide interchange, modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content, use of appropriate additives and specific polymers It can be stabilized by the development of the matrix composition.

J. Treatment Methods

Polypeptides of the invention and their agonists, including antibodies, peptides and small molecule agonists, can be used for the treatment of various tumors, eg cancer. Representative diseases to be treated include benign or malignant tumors (eg renal cell carcinoma, liver cancer, kidney cancer, bladder cancer, breast cancer, gastrointestinal cancer, ovarian cancer, colorectal cancer, pancreatic cancer, lung cancer, vulvar cancer, thyroid cancer, liver carcinoma, sarcoma, glioblastoma) And various types of head and neck cancer), leukemia and lymph cancer; Neurological diseases, glial diseases, astrocytic diseases, hypothalamic diseases, and other secretory gland diseases, macrophage diseases, epithelial diseases, interstitial diseases and blastocyst disease; And inflammatory diseases, angiogenic diseases and immune diseases. Antitumor agents of the invention (eg polypeptides disclosed herein and agonists, such as antibodies, peptides and small organic molecules that mimic their activity) are known methods, for example intravenous administration as bolus, or intramuscular It can be administered to mammals, preferably humans, by prolonged continuous irrigation by intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, synovial, intramedural, oral, topical or inhalation routes. Intravenous administration of the antibody is preferred.

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

It is also desirable to administer other tumor-associated antibodies such as ErbB2, EGFR, ErbB3, ErbB4 or antibodies that bind to vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies that bind to the same or two or more different antigens disclosed herein may be administered to a patient simultaneously. Sometimes, it may also be beneficial to administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein can be administered simultaneously with the growth inhibitor. For example, the anticancer agent of the present invention may be administered first after the growth inhibitory agent. However, simultaneous administration or administration of the anticancer agent of the present invention may also be considered. Suitable dosages of growth inhibitory agents are those currently used and may be lowered due to the combined action (rise) of the growth inhibitory agent and the anticancer agent herein.

For the prophylaxis or treatment of a disease, suitable dosages of the anticancer agents herein include the type of disease to be treated, the severity and course of the disease, whether the anticancer agent administered is for preventive or therapeutic purposes, prior treatments, patients The clinical history and response to the medicament, and discretion and the attending physician will vary. The formulation is suitably administered to the patient at one time or through a series of treatments. Animal testing provides reliable guidance in determining effective amounts for human therapies. The ratio adjustment of effective amounts between species is described in Mordenti, J and Chappell, W. "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Uacobi et al., Eds., Pergamon Press, New York 1989, pp 42-. 96].

For example, depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg 0.1 to 20 mg / kg) of antibody may be administered to a patient by one or more separate administrations or by continuous irrigation. Initial candidate dose for administration of. Typical daily dosages are about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days, depending on the condition, treatment continues until the desired signs of disease are suppressed. However, other dosages may be effective. The progress of this therapy can be easily monitored by conventional techniques and assays. Guidance and delivery methods for specific dosages are provided in the literature (see, eg, US Pat. No. 4,657,760, 5,206,344 or 5,225,212). It is anticipated that other agents may be effective for other therapeutic compounds and other diseases, and administrations that target one organ or tissue may require, for example, a method of delivery that is different from another organ or tissue.

K. Product

In another embodiment of the invention, there is provided a product containing a substance useful for the diagnosis or treatment of the diseases described above. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container may be formed from various materials such as glass or plastic. The container contains a composition that is effective for diagnosing or treating a disease and may have a sterile inlet (eg, the container may be an intravenous sachet or a vial with a stopper through which a hypodermic syringe needle can penetrate). . The active substance of the composition is the antitumor agent of the present invention. The label attached to or on the container indicates that the composition is used for the diagnosis or treatment of the selected disease. The product may also include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. In addition, the product may include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

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

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

Commercial reagents mentioned in the examples were used according to the manufacturer's instructions unless otherwise indicated. The source of cells identified by the ATCC accession number throughout the following examples and specification is the American Type Culture Collection (Manassas, VA).

Example 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNAs Encoding It

The EST database was examined using extracellular domain (ECD) sequences (including secretion signal sequences, if present) from about 950 known secretory proteins from the Swiss-Prot public database. . The EST database includes public databases (e.g. Dayhoff, Jenbank) and private databases (e.g. LIFESEQ ™, Incyte Pharmaceuticals, Palo Alto, Calif.). Included. Investigations were performed using the computer programs BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)) as a comparison of the ECD protein sequences to the six frame translations of the EST sequences. Comparatives with BLAST scores of 70 (or in some cases 90) or greater that do not encode known proteins are clustered and consensus DNA sequences using the program "phrap" (Phil Green, University of Washington, Seattle, WA) Assembled.

Using this extracellular domain homology screening, consensus DNA sequences were assembled for other EST sequences identified using phrap. In addition, consensus DNA sequences obtained are often (but not always) stretched using a repeat cycle of BLAST or BLAST-2 and phrap to stretch the consensus sequence as much as possible using the sources of EST sequences discussed above. .

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

The cDNA library used to isolate the cDNA clones was constructed by standard methods using commercially available reagents such as reagents from Invitrogen (San Diego, CA, USA). The cDNA is primed with oligo dT with a NotI site, ligated to the hemikinase treated SalI adapter, cleaved with NotI, appropriately sized by gel electrophoresis, and appropriate cloning vector (e.g., pRKB or pRKD; pRK5B is a precursor of pRK5D without the SfiI site; see Holmes et al., Science , 253 : 1278-1280 (1991) and cloned in a defined direction to the only XhoI and NotI sites.

Example 2: Isolation of cDNA Clone Encoding Human PRO179

CDNA clones encoding the native human PRO179 polypeptide (DNA16451-1078) were identified using yeast screening in a human fetal liver library that predominantly represents the 5 'end of the primary cDNA clone.

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

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

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

Clone DNA16451-1078 contains a single open reading frame with an explicit translation initiation site at nucleotide positions 37 to 39 and an apparent stop codon at nucleotide positions 1417 to 1419 (FIG. 1, SEQ ID NO: 1). The expected polypeptide precursor is 460 amino acids long. Full length PRO179 protein is shown in Figure 2 (SEQ ID NO: 2).

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

Clone DNA16451-1078 was deposited with ATCC on September 18, 1997 and was assigned accession number 209281. The estimated molecular weight of the full-length PRO179 protein shown in FIG. 2 is about 53,637 Daltons and the estimated pI is about 6.61.

Analysis of the dayhof database (version 35.45 Swiss Prot 35) of the full length sequence shown in FIG. 2 (SEQ ID NO: 2) was performed with two known human ligands of the TIE-2 receptor (h-TIE-2L1 and h-TIE-2L2). The presence of fibrinogen-like domains exhibiting high sequence homology was demonstrated. Abbreviation "TIE" is an acronym indicating a tyrosine kinase having a "Ig and EGF homology domains (t yrosine kinase containing I g and E GF homology domains), is coined to refer to a new family of receptor tyrosine kinases. Accordingly, PRO179 Has been identified as a new member of the TIE ligand family.

Example 3: Isolation of cDNA Clone Encoding Human PRO207

Expressed sequence tag (EST) DNA databases (e.g., LIFESEQ® (Incyte Pharmaceuticals, Palo Alto, Calif.)) Were used to examine ESTs exhibiting homology with human Apo-2 ligands. Next, human fetal kidney cDNA libraries were screened cDNA libraries were prepared using mRNA isolated from human fetal kidney tissue (Clontech, Inc.) Using this RNA, Life Technologies, Gaithersbug MD) reagent and protocol (superscript plasmid system) were used to generate an oligo dT primed cDNA library in vector pRK5D In this procedure, the size of the double stranded cDNA was greater than 1000 bp and the SalI / NotI linked cDNA was XhoI. Cloned into a vector cleaved by / NotI pRK5D was followed by the sp6 transcription initiation site followed by the SfiI restriction enzyme site. Cloning vector with XhoI / NotI cDNA cloning site This library was screened based on EST by hybridizing with synthetic oligonucleotide probe 5'-CCAGCCCTCTGCGCTACAACCGCCAGATCGGGGAGTTTATAGTCACCCGG-3 '(SEQ ID NO: 8).

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

Analysis of the sequence of full-length PRO207 shown in FIG. 4 (SEQ ID NO: 7) demonstrates the presence of several important polypeptide domains as shown in FIG. 4, with the positions for these important polypeptide domains being approximately as described above. Analysis of the full-length PRO207 sequence (FIG. 4, SEQ ID NO: 7) revealed a signal peptide of about amino acid 1 to about amino acid 40, an N-glycosylation site of about amino acid 139 to about amino acid 143, about amino acid 27 to about amino acid 33, about amino acid 29 to about amino acid 35, about amino acid 36 to about amino acid 42, about amino acid 45 to about amino acid 51, about amino acid 118 to about amino acid 124, about amino acid 121 to about amino acid 127, about amino acid 125 to about amino acid 131, and about amino acid 128 to Demonstrates the presence of an N-myristoylation site of about amino acid 134, an amidation site of about amino acid 10 to about amino acid 14 and about amino acid 97 to about amino acid 101, and a prokaryotic lipoprotein lipid attachment site of about amino acid 24 to about amino acid 35 . Clone DNA30879-1152 was deposited with ATCC on October 10, 1997 and assigned accession number 209358. The estimated molecular weight of the full-length PRO207 protein shown in FIG. 4 is about 27,216 Daltons and the estimated pI is about 9.61.

Based on BLAST and FastA sequence alignment analysis of the full length PRO207 sequence shown in FIG. 4 (SEQ ID NO: 7), PRO207 is a member of the TNF cytokine family, in particular human lymphotoxin-beta (23.4%) and human CD40 ligand (19.8%). ) And amino acid sequence identity.

Example 4: Isolation of cDNA Clone Encoding Human PRO320

Consensus DNA sequences were assembled for other EST sequences using phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA28739. Based on the DNA28739 consensus sequence, oligonucleotides were synthesized for use as 1) 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.

PCR primer pairs (forward and reverse) were synthesized:

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

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

In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA28739.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO320 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from lung tissue (LIB025) of human fetus.

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

The entire coding sequence of DNA32284-1307 is shown in Figure 5 (SEQ ID NO: 9). Clones DNA32284-1307 contain a single open reading frame with an explicit translation initiation site at nucleotide positions 135-137 and an apparent stop codon at nucleotide positions 1149-1151. The expected polypeptide precursor is 338 amino acids long. Analysis of the sequence of full-length PRO320 shown in FIG. 6 (SEQ ID NO: 10) demonstrates the presence of several important polypeptide domains, with the positions for these important polypeptide domains being approximately as described above. As a result of analyzing the full length PRO320 polypeptide shown in FIG. 6, a signal peptide of about amino acid 1 to about amino acid 21, an amidation site of about amino acid 330 to about amino acid 334, about amino acid 109 to about amino acid 121, about amino acid 191 to about amino acid 203 And aspartic acid and asparagine hydroxylation sites of about amino acids 236 to about 248, EGF-like domain cysteine form signals of about amino acids 80 to about amino acids 91, about amino acids 103 to about amino acids 125, about amino acids 230 to about amino acids 252, and about amino acids 185 To the presence of a calcium binding EGF-like domain of about amino acid 207. Clone DNA32284-1307 was deposited with ATCC on March 11, 1998 and was assigned accession number 209670. The estimated molecular weight of the full-length PRO320 protein shown in FIG. 6 is about 37,143 Daltons, and the estimated pI is about 8.92.

Example 5: Isolation of cDNA Clone Encoding Human PRO219

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA28729. Based on the DNA28729 consensus sequence, oligonucleotides were synthesized for use as 1) a cDNA library comprising the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO219.

PCR primer pairs (forward and reverse) were synthesized:

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

Reverse PCR Primer 5'-ACAGCCATGGTCTATAGCTTGG-3 '(SEQ ID NO: 17)

In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA28729.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, clones encoding the PRO219 gene were isolated with one of the probe oligonucleotides and PCR primers by using a positive library. RNA for preparing cDNA library was isolated from kidney tissue of human fetus.

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

The entire coding sequence of DNA32290-1164 is shown in FIG. 7 (SEQ ID NO: 14). Clone DNA32290-1164 contains a single open reading frame with an explicit translation initiation site at nucleotide positions 204-206 and an apparent stop codon at nucleotide positions 2949-2951. The expected polypeptide precursor is 1005 amino acids long. Analysis of the sequence of full length PRO219 shown in FIG. 8 (SEQ ID NO: 15) demonstrates the presence of several important polypeptide domains, with the positions for these important polypeptide domains being approximately as described above. As a result of analyzing the full length PRO219 polypeptide shown in FIG. 8, a signal peptide of about amino acid 1 to about amino acid 23, an N-glycosylation site of about amino acid 221 to about amino acid 225, about amino acid 115 to about amino acid 119, about amino acid 606 to about CAMP and cGMP dependent protein kinase phosphorylation sites of amino acids 610 and about amino acids 892 to about amino acids 896, about amino acids 133 to about amino acids 139, about amino acids 258 to about amino acids 264, about amino acids 299 to about amino acids 305, about amino acids 340 to about amino acids 346, about amino acid 453 to about amino acid 459, about amino acid 494 to about amino acid 500, about amino acid 639 to about amino acid 645, about amino acid 690 to about amino acid 694, about amino acid 752 to about amino acid 758 and about amino acid 792 to about amino acid 798 N-myristoylation site, about amino acid 314 to about Amino acid 318, about amino acid 560 to about amino acid 564 and about amino acid 601 to about amino acid 605, the amidation site, about amino acid 253 to about amino acid 265, about amino acid 294 to about amino acid 306, about amino acid 335 to about amino acid 347, about amino acid 376 To the presence of aspartic acid and asparagine hydroxide sites of from about amino acid 388, about amino acid 417 to about amino acid 429, about amino acid 458 to about amino acid 470, about amino acid 540 to about amino acid 552, and about amino acid 581 to about amino acid 593. Clone DNA32290-1164 was deposited with ATCC on October 17, 1997 and was assigned accession number 209384. The estimated molecular weight of the full-length PRO219 protein shown in FIG. 8 is about 102,233 Daltons and the estimated pI is about 6.02.

Analysis of the full-length PRO219 sequence shown in FIG. 8 (SEQ ID NO: 15) showed some homology with mouse and human matyllin-2 precursor polypeptides.

Example 6: Isolation of cDNA Clone Encoding Human PRO221

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA28756. Based on the DNA28756 consensus sequence, oligonucleotides were synthesized for use as 1) 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.

PCR primer pairs (forward and reverse) were synthesized:

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

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

In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA28756.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO221 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from lung tissue of a human fetus.

DNA sequence analysis of the clones isolated as described above yielded the full length DNA sequence of DNA33089-1132 (FIG. 9, SEQ ID NO: 19) and the protein sequence of PRO221 derived therefrom.

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

Analysis of the full-length PRO221 sequence shown in FIG. 10 (SEQ ID NO: 20) showed homology with the members of the leucine-rich repeat protein superfamily, including the SLIT protein.

Example 7: Isolation of cDNA Clone Encoding Human PRO224

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA30845. Based on the DNA30845 consensus sequence, oligonucleotides were synthesized for use as 1) a cDNA library comprising the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO224.

PCR primer pairs (forward and reverse) were synthesized:

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

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

In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA30845.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO224 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from liver tissue of a human fetus.

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

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

Analysis of the full-length PRO224 sequence shown in FIG. 12 (SEQ ID NO: 25) showed homology with the lowest density lipoprotein receptor, apolipoprotein E receptor, and chicken oocyte receptor P95. Based on BLAST and FastA sequence alignment analysis of the full length sequence, PRO224 had sequence identity in the range of 28% to 45% with some of these proteins and 33% to 39% in identity with all of these proteins.

Example 8: Isolation of cDNA Clone Encoding Human PRO328

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA35615. Based on the DNA35615 consensus sequence, oligonucleotides were synthesized for use as 1) 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 PRO328.

PCR primer pairs (forward and reverse) were synthesized:

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

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

In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA35165.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, clones encoding the PRO328 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from kidney tissue of human fetus.

DNA sequencing of the clones isolated as described above yielded the full length DNA sequence of DNA40587-1231 (FIG. 13, SEQ ID NO: 29) and the protein sequence of PRO328 derived therefrom.

The entire coding sequence of DNA40587-1231 is shown in FIG. 13 (SEQ ID NO: 29). Clone DNA40587-1231 contains a single open reading frame with an explicit translation initiation site at nucleotide positions 15-17 and an apparent stop codon at nucleotide positions 1404-1406. The expected polypeptide precursor is 463 amino acids long. Analysis of the sequence of full length PRO328 shown in FIG. 14 (SEQ ID NO: 30) demonstrates the presence of several important polypeptide domains, with the positions for these important polypeptide domains being approximately as described above. Analysis of the full-length PRO328 polypeptide shown in FIG. 14 showed N-glycos of the signal peptide of about amino acid 1 to about amino acid 22, about amino acid 114 to about amino acid 118, about amino acid 403 to about amino acid 407, and about amino acid 409 to about amino acid 413. Misfire site, glycosaminoglycan attachment site of about amino acid 439 to about amino acid 443, about amino acid 123 to about amino acid 129, about amino acid 143 to about amino acid 149, about amino acid 152 to about amino acid 158, about amino acid 169 to about amino acid 175 Amidation of the N-myristoylation site of about amino acid 180 to about amino acid 186, about amino acid 231 to about amino acid 237 and about amino acid 250 to about amino acid 256, about amino acid 82 to about amino acid 88 and about amino acid 172 to about amino acid 176 Site, peroxidase contiguous heme of about amino acid 287 to about amino acid 298 Extracellular protein SCP / Tpx-1 / Ag5 / PR-1 / Sc7 signal 1 domain of ligand signal, about amino acid 127 to about amino acid 138, extracellular protein SCP / Tpx-1 / Ag5 of about amino acid 160 to about amino acid 172 / PR-1 / Sc7 signal 2 demonstrates the presence of domain. The clone DNA40587-1231 was deposited with ATCC on November 7, 1997 and was assigned accession number 209438. The estimated molecular weight of the full-length PRO328 protein shown in FIG. 14 is about 49,471 Daltons, and the estimated pI is about 5.36.

As a result of analysis of the full-length PRO328 sequence shown in FIG. 14 (SEQ ID NO: 30), part of it showed considerable homology with human glioblastoma protein and cysteine-rich secretory protein, whereby PRO328 is a novel glioblastoma protein or cysteine-rich secretion. It can be a protein.

Example 9: Isolation of cDNA Clone Encoding Human PRO301

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA35936. Based on the DNA35936 consensus sequence, oligonucleotides were synthesized for use as 1) a cDNA library comprising the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of PRO301.

Oligonucleotides used in this procedure are as follows:

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

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

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

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

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

Hybridization Probe 1

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO301 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from kidney tissue of human fetus.

DNA sequence analysis of clones isolated as described above yielded the full length DNA sequence of DNA40628-1216 (FIG. 15, SEQ ID NO: 34) and the protein sequence of PRO301 derived therefrom.

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

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

Example 10 Isolation of cDNA Clones Encoding Human PRO526

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This starting consensus sequence is referred to herein as DNA39626.init. In addition, repeated cycles of BLAST and phrap were used to elongate the start consensus DNA sequence as long as the source of EST sequences discussed above could be used. The assembled consensus sequence is referred to herein as <consen01>. Based on the <consen01> consensus sequence, oligonucleotides were synthesized for use as 1) 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 PRO526.

PCR primer pairs (forward and reverse) were synthesized:

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

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

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

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO526 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from liver tissue of human fetus (LIB228).

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

The entire coding sequence of DNA44184-1319 is shown in FIG. 17 (SEQ ID NO: 42). Clone DNA44184-1319 contains a single open reading frame with an explicit translation initiation site at nucleotide positions 514-516 and an apparent stop codon at nucleotide positions 1933-1935. The expected polypeptide precursor is 473 amino acids long. Analysis of the sequence of full length PRO526 shown in FIG. 18 (SEQ ID NO: 43) demonstrates the presence of several important polypeptide domains, with the positions for these important polypeptide domains being approximately as described above. Analysis of the full-length PRO526 polypeptide shown in FIG. 18 revealed glycosaminoglycan attachment of the signal peptide of about amino acids 1 to about amino acid 26, the leucine zipper form of about amino acids 135 to about amino acid 157, and the amino acids 436 to about amino acid 440. Site, about amino acid 82 to about amino acid 86, about amino acid 179 to about amino acid 183, about amino acid 237 to about amino acid 241, about amino acid 372 to about amino acid 376 and about amino acid 423 to about amino acid 427, the N-glycosylation site, about amino acid 411 to about amino acid 427 demonstrates the presence of Willbrand factor (VWF) type C domains. Clone DNA44184-1319 was deposited with ATCC on March 26, 1998 and was assigned accession number 209704. The estimated molecular weight of the full-length PRO526 protein shown in FIG. 18 is about 50,708 Daltons, and the estimated pI is about 9.28.

Analysis of the full-length PRO526 sequence shown in FIG. 18 (SEQ ID NO: 43) showed some of its homology to proteins rich in leucine repeats, including human ALS, SLIT, carboxypeptidase and platelet glycoprotein V, This indicated that PRO526 is a novel protein involved in protein-protein interactions.

Example 11: Isolation of cDNA Clone Encoding Human PRO362

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA42257. Based on the DNA42257 consensus sequence, oligonucleotides were synthesized for use as 1) 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.

PCR primer pairs (forward and reverse) were synthesized:

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

Omnidirectional 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, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA42257.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO362 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from brain tissue of human fetus (LIB153).

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

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

Analysis of the full length PRO362 sequence shown in FIG. 20 (SEQ ID NO: 48) showed significant similarity with the A33 antigen protein and HCAR protein. More specifically, analysis of the Deihof database (version 35.45 Swiss Prot 35) revealed significant differences between the PRO362 amino acid sequence and the DA-Hope sequences of AB002341_1, HSU55258_1, HSC7NRCAM_1, RNU81037_1, A33_HUMAN, P_W14158, NMNCAMRI_1, HSTITINN2_1, S71824_1 and HSU63041_1. Proved to have the same sex.

Example 12 Isolation of cDNA Clone Encoding Human PRO356

The expressed sequence tag (EST) DNA database (e.g., LIFESEQ®, Insight Pharmaceuticals, Palo Alto, Calif.) Was examined to determine DNA from DNA16451-1078 (FIG. 1, EST (# 2939340) showing homology to the same) was identified. To clone PRO356, a human fetal lung library prepared from mRNA purchased from Clontech (Palo Alto, CA) was used according to the manufacturer's instructions.

The cDNA used to isolate the cDNA clone encoding human PRO356 was constructed by standard methods using commercially available reagents such as, for example, Invitrogen, San Diego, California. Prime cDNA with oligo dT with NotI site, connect blunt ends to hemikinase treated SalI adapters, cleave with NotI, classify according to size by gel electrophoresis, and select appropriate cloning vectors (e.g. pRKB or pRKD; pRK5B is a precursor of pRK5D that does not include a SfiI site; see Holmes et al., Science , 253 : 1278-1280 (1991), and cloned in a predetermined direction to the only XhoI and NotI sites .

Then, based on the EST sequences described above, oligonucleotide probes for use as 1) a cDNA library comprising the sequence of interest by PCR, and 2) as a probe to isolate a clone of the full-length coding sequence of the PRO356 polypeptide Was synthesized. Forward and reverse PCR primers generally consist of 20 to 30 nucleotides and are often designed to provide a PCR product about 100 to 1000 bp in length. Probe sequences are typically 40 to 55 bp in length. To screen several libraries for full length clones, DNAs from these libraries were screened by PCR amplification using PCR primer pairs according to Ausubel et al., Current Protocols in Molecular Biology, supra. A positive library was then used to isolate clones encoding the gene of interest using one of the probe oligonucleotide and primer pairs.

Oligonucleotides used were as follows:

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

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

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

The entire cDNA clone was identified and sequenced. The total 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 with a clear translation initiation site at nucleotide positions 215-217 and a stop codon at nucleotide positions 1253-1255 (FIG. 21, SEQ ID NO: 54). The expected polypeptide precursor is 346 amino acids long, estimated molecular weight is about 40,018 daltons, and estimated pi is about 8.19. Full length PRO356 protein is shown in Figure 22 (SEQ ID NO: 55).

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

Clone DNA47470-1130-P1 was deposited with ATCC on October 28, 1997 and was assigned accession number 209422. This deposited clone is understood to have a practically accurate sequence rather than a representative provided herein.

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

Example 13: Isolation of cDNA Clone Encoding Human PRO509

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

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

The total nucleotide sequence of DNA50148-1068 is shown in FIG. 23 (SEQ ID NO: 59). The cDNA contained one open reading frame with a translation initiation site designated by the ATG codon at nucleotide positions 82-84. This open reading frame was terminated at the stop codon TGA at nucleotide positions 931-933.

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

Analysis of the full length PRO509 sequence shown in FIG. 24 (SEQ ID NO: 60) demonstrates the presence of several important polypeptide domains, as shown in FIG. 24, with the positions for these important polypeptide domains being approximately as described above. Analysis of the full length PRO509 sequence (FIG. 24, SEQ ID NO: 60) shows a signal peptide of about amino acid 1 to about amino acid 36, the transmembrane domain of about amino acid 205 to about amino acid 221, about amino acid 110 to about amino acid 114 and about amino acid 173 to An N-glycosylation site of about amino acid 177, about amino acid 81 to about amino acid 87, about amino acid 89 to about amino acid 95, about amino acid 104 to about amino acid 110, about amino acid 120 to about amino acid 126, about amino acid 153 to about amino acid 159, The presence of the N-myristoylation site of about amino acids 193 to about amino acid 199, about amino acids 195 to about amino acid 201 and about amino acids 220 to about amino acid 226, and about cell attachment sequences of about amino acids 231 to about amino acid 234.

Aligning the 58 amino acid length cytoplasmic region of PRO509 with other known members of the human TNF receptor family (using the ALIGN ™ computer program) confirmed that some sequences were similar, in particular CD40 (12 identical) and LT- Similar to beta receptors (11 identical).

Example 14 Isolation of cDNA Clone Encoding Human PRO866

Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA44708. Based on the DNA44708 consensus sequence, oligonucleotides were synthesized for use as 1) a cDNA library comprising the sequence of interest by PCR and 2) as a probe to isolate a clone of the full-length coding sequence of PRO866.

PCR primers (forward and reverse) were synthesized:

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

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

Omnidirectional 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, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were prepared from consensus sequence DNA44708.

Hybridization probe

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

In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO866 gene with one of the probe oligonucleotides and PCR primers were isolated by using a positive library. RNA for preparing cDNA library was isolated from kidney tissue of human fetus (LIB228).

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

The entire coding sequence of DNA53971-1359 is shown in FIG. 25 (SEQ ID NO: 61). Clone DNA53971-1359 contains a single open reading frame with an explicit translation initiation site at nucleotide positions 275-277 and an apparent stop codon at nucleotide positions 1268-1270. The expected polypeptide precursor is 331 amino acids long. Analysis of the sequence of full length PRO866 shown in FIG. 26 (SEQ ID NO: 62) demonstrates the presence of several important polypeptide domains, with the positions for these important polypeptide domains being approximately as described above. Analysis of the full-length PRO866 polypeptide shown in FIG. 26 revealed a signal peptide of about amino acids 1 to about amino acid 26, a glycosaminoglycan attachment site of about amino acids 131 to about amino acid 135, cAMP and cGMP of about amino acids 144 to about amino acid 148 Dependent protein kinase phosphorylation site, about amino acid 26 to about amino acid 32, about amino acid 74 to about amino acid 80, about amino acid 132 to about amino acid 138, about amino acid 134 to about amino acid 140, about amino acid 190 to about amino acid 196, about amino acid 287 to The presence of the N-myristoylation site of about amino acid 293 and about amino acid 290 to about amino acid 296 is demonstrated. The clone DNA53971-1359 was deposited with the ATCC on April 7, 1998, assigned accession number 209750. The estimated molecular weight of the full-length PRO866 protein shown in FIG. 26 is about 35,844 Daltons and the estimated pI is about 5.45.

Analysis of the full-length PRO866 sequence shown in FIG. 26 (SEQ ID NO: 62) showed that it was quite similar to the proteins of the mindin / spondin family, whereby PRO866 could be a novel mindine homologue. More specifically, analysis of the Deihof database (version 35.45 Swiss Prot 35) revealed significant differences between the PRO866 amino acid sequence and the Dayhof sequences of AB006085_1, AB006084_1, AB006086_1, AF017267_1, CWU42213_1, AC004160_1, CPMICRP_1, S49108, A48569 and 146687. Proved to have the same sex.

Example 15 In situ Hybridization

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

In situ hybridization was performed using ³³ P-labeled riboprobes generated by PCR with an optimal version of the protocol according to Lu and Gillett, Cell Vision 1: 169-176 (1994). Briefly, human tissues immobilized with formalin and cleaved in paraffin are cleaved, paraffin is removed, and proteins are removed with proteinase K (20 g / ml) at 37 ° C. for 15 minutes, as described in Lu and Gillett, Further treatment was performed for in situ hybridization as described above. ( ³³ P) UTP-labeled antisense riboprobe generated as a PCR product was hybridized overnight at 55 ° C. The slides were immersed in Kodak NTB2 ™ nuclear track emulsion and exposed for 4 weeks.

³³ P-riboprobe synthesis

6.0 μl (125 mCi) of ³³ P-UTP (Amersham BF 1002, SA <2000 Ci / mMol) was dried at high speed vac. The following ingredients were added to each tube of dried ³³ P-UTP:

2.0 μl 5x transcription buffer

1.0 μl DTT (100 mM)

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

1.0 μl UTP (50 μM)

1.0 μl RNasin

1.0 μl DNA template (l μg)

1.0 μl H ₂ 0

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

The tube was incubated for 1 hour at 37 ° C. 1.0 μl of RQ1 DNase was added and then incubated at 37 ° C. for 15 minutes. 90 μl of TE (10 mM Tris, pH 7.6) / l mM EDTA, pH 8.0) was added and the mixture was pipetted into DE81 paper. The Microcon-50 ™ ultrafiltration unit was loaded with residual solution and spun into program 10 for 6 minutes. The filtration unit was transferred to the second tube and spun with program 2 for 3 minutes. After the last recovery spinning, a total of 100 μl of TE was added, then 1 μl of the final product was pipetted onto DE81 paper and counted with 6 ml Bioflour II (tradename).

Probes were electrophoresed on TBE / urea gel. To 3 μl of loading buffer a total of 1-3 μl of probe or 5 μl of RNA Mrk III was added. After heating for 3 minutes in a 95 ° C heat block, the gel was immediately transferred to ice. After washing the wells of the gel, the samples were loaded and electrophoresed for 45 minutes at 180-250 volts. The gel was wrapped in a saran wrap and exposed to XAR film on an enhancement screen for 1 hour to overnight in a freezer at −70 ° C.

³³ P-hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer and placed in an aluminum tray and melted for 5 minutes at room temperature. To reduce condensation, the trays were placed in a 55 ° C. incubator for 5 minutes. Slides were fixed for 10 minutes in ice 4% paraformaldehyde in a fume hood and washed for 5 minutes at 0.5x SSC (25 ml 20x SSC + 975 ml SQ H ₂ O) at room temperature. Protein was removed for 10 minutes at 37 ° C. with 0.5 μg / ml proteinase K (12.5 μl of 10 mg / ml stock solution in 250 ml of RNase preheated RNase buffer), followed by 0.5 × SSC for 10 minutes at room temperature. Sections were washed. Sections were dehydrated for 2 minutes with 70%, 95% and 100% ethanol, respectively.

B. Pretreatment of sections buried in paraffin

The paraffins on the slides were removed and placed in SQ H ₂ O and rinsed twice with 2 × SSC for 5 minutes at room temperature. 20 μl / ml proteinase K (10 mg / ml in 250 ml RNase buffer without RNase, 500 μl, 37 ° C., 15 min) (human embryos), or 8 × proteinase K (250 ml RNase buffer) 100 μl, 37 ° C., 30 min) (formalin tissue) to remove the proteins of the sections. Then rinsed with 0.5 × SSC and dehydrated as described above.

C. Prehybridization

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

D. Hybridization

1.0 x 10 ⁶ cpm probe and 1.0 μl of tRNA (50 mg / ml stock) were added per slide and heated at 95 ° C. for 3 minutes. After ice cooling, 48 μl of hybridization buffer was added per slide. After vortexing, 50 μl of ³³ P mixture was added to 50 μl of prehybridization sample on the slide. This slide was incubated at 55 ° C. overnight.

E. Wash

After washing 2x SSC, EDTA twice for 10 minutes at room temperature (400 ml 20x SSC + 16 ml 0.25 M EDTA, final volume = 4 L), treated with RNase A for 30 minutes at 37 ° C. (RNase buffer 250 10 mg / ml in ml, 500 μl = 20 μg / ml). Slides were washed twice with 2 × SSC, EDTA for 10 minutes at room temperature. Stringent washing conditions are as follows: 2 hours at 55 ° C. with 0.1 × SSC and EDTA (20 ml 20 × SSC + 16 ml EDTA, final volume = 4 L).

F. Oligonucleotides

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

(1) DNA30879-1152 (PRO207)

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

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

(2) DNA33089-1132 (PRO221)

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

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

(3) DNA33221-1133 (PRO224)

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

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

(4) DNA40628-1216 (PRO301)

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

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

(5) DNA45416-1251 (PRO362)

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

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

G. Results

In situ analysis was performed on the five DNA sequences disclosed herein. The analysis results are as follows.

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

It was expressed at low levels in cartilage sarcoma and other soft tissue sarcomas. All other organizations were negative.

Human fetal tissues examined (E12-E16 weeks) include placenta, umbilical cord, liver, kidney, adrenal gland, thyroid gland, lung, heart, major blood vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eyes, spinal cord, Body wall, pelvis and lower extremity.

Adult tissues examined include kidneys (normal and late kidneys), adrenal glands, myocardium, spleen, lymph nodes, pancreas, lungs, skin, eyes (including the retina), bladder and liver (normal liver, sclerotic liver, acute liver failure liver). Included.

Non-human primate tissues examined include:

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

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

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

It was specifically expressed in fetal cerebral gray matter, as well as in spinal cord neurons. The probe appears to cross react with the rat. It was expressed at low levels in the cerebellar neurons of the mature rhesus monkey brain. All other organizations were negative.

Fetal tissues examined (weeks E12-E16) include placenta, umbilical cord, liver, kidney, adrenal gland, thyroid gland, lung, heart, major blood vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eyes, spinal cord, and body wall , Pelvis and lower extremities.

Adult tissues examined include liver, kidney, adrenal gland, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm), hippocampal tissue (rm), cerebellum (rm), penis, eyes, bladder and stomach , Gastric cancer, colon, colon cancer and cartilage sarcoma, as well as liver damage and cirrhosis induced by acetoaminophen.

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

It was expressed only in the vascular endothelial tissues of fetal spleen, adult spleen, fetal liver, adult thyroid gland and adult lymph nodes (chimpanzees). Additional site of expression was observed in growing spinal ganglia. All other organizations were negative.

Human fetal tissues examined (E12-E16 weeks) include placenta, umbilical cord, liver, kidney, adrenal gland, thyroid gland, lung, heart, major blood vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eyes, spinal cord, Body wall, pelvis and lower extremity.

Adult tissues examined include kidneys (normal and late kidneys), adrenal glands, myocardium, aorta, spleen, lymph nodes, pancreas, lungs, skin, eyes (including retina), bladder and liver (normal liver, sclerosis liver, acute liver failure liver) ) Is included.

Non-human primate tissues examined include:

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

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

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

Expression in human inflammatory tissue (psoriasis, IBD, inflammatory kidneys, inflammatory lungs, hepatitis, normal tonsils, adult and chimpanzee multiblock):

Expression was predominant in human inflammatory tissues compared to several normal human and non-human primate tissues. Including mucosal epithelium of the colon, epithelium of the airway of the bronchus, oral mucosa (tongue), tonsils and mucosa, placental mucosa, prostate mucosa, gastric gland mucosa, epithelial cells of the gastrointestinal thymus, hepatocytes, bile epithelium and placental epithelium , Expressed in all epithelial structures. The only evidence that it was expressed outside the epithelial structure is a weak low level of irregular expression in the embryonic center of the focllicle in the tonsils with reactive hyperplasia.

The following non-human primate tissues were observed:

Chimpanzee tissue: Weak diffuse expression was observed in the epidermis of the tongue epithelium. In the thymus, a weak specific expression was observed in the thymic epithelium of the islet body. In the stomach, mild levels of diffuse expression were observed in the epithelium of the gastric mucosa.

In human tissue: liver (multiblock including chronic cholangitis, lobular hyperplasia, acetominophene toxicity), low to moderate diffuse expression was observed in hepatocytes and bile epithelium. It was most predominantly expressed in the lobular / peripheral hepatocytes. This was most prevalent in the bile epithelium of the liver with chronic sclerosis cholangitis. Not all samples were expressed, which may reflect the quality of the sample rather than the expression variability.

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

In lungs with chronic interstitial pneumonia and chronic bronchitis, low levels of diffuse expression were observed in airway mucosal epithelium and weak diffuse expression in alveolar epithelium. It was not expressed at all in the epithelium of the submucosal glands of the bronchial (s).

In the placenta, moderate levels of diffusion were expressed in the placental epithelium.

In the prostate, low levels of diffuse expression were observed in the prostate epithelium.

In the gallbladder, moderate diffuse expression was observed in the mucosal epithelium.

In tonsils with reactive hyperplasia, highly diffuse expression was observed in the tonsil mucosa and epithelial tonsils, and the signal was highest in mucosal cells along the tonsils. Weak irregular diffuse expression was observed in the embryonic center of the cortical vesicles (B lymphocyte site), but not at all in B lymphocytes in lymphoid structures or other tissues assessed with lymphocyte inflammation.

In colons with inflammatory bowel disease and polyp / adenomatous changes, low levels of expression were observed in the mucosal epithelium and peaked at the villi tip. In one specimen with polyps, there was no evidence of increased expression in the dysplasia of the polyps compared to the adjacent mucosa. There was no apparent expression in reactive mucosal lymphoid tissues present at various sites.

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

Expression in human inflammatory tissue (psoriasis, IBD, inflammatory kidneys, inflammatory lungs, hepatitis, normal tonsils, adult and chimpanzee multiblock):

Expression of novel proteins in several human and non-human primate tissues was evaluated and found to be highly limited. It was expressed only in pulmonary alveolar macrophages and hepatic isovascular Cooper cells. Expression in these cells was significantly increased when each of these cell populations was activated. Although two subpopulations of these tissue macrophages are located in different organs, they have similar biological functions. Both of these types of phagocytic cells act as biological filters to remove substances from the bloodstream or airways, including pathogens, geriatric cells, and proteins, both of which can secrete a wide range of important preinflammatory cytokines.

In inflammatory lungs (7 patient samples), expression was predominant in the reactive alveolar macrophage population, defined as large, thin, often phobic cells present alone or in groups in the alveoli, and normal, non-reactive macrophages (normal sized alone). Cells scattered with) were weakly expressed or negative. Expression in alveolar macrophages was increased during inflammatory reactions in which both the number and size of these cells increased (activated). Despite the presence of histocytes at the site of interstitial inflammation and peribronchial lymphoid hyperplasia of these tissues, expression was limited to alveolar macrophages. Several inflammatory lungs also had some purulent inflammation and were not expressed in neutrophil granulocytes.

In the liver, it was strongly expressed in responsive / activated Cooper cells of the liver with acute mesolobular necrosis (acetominophene toxicity) or pronounced periportal inflammation. However, they were weakly expressed or not expressed in Cooper cells in normal liver or liver with mild inflammation or mild to moderate leaflet hyperplasia / low formation. Thus, in the lung, expression in activated / reactive cells was increased.

This molecule was not expressed in tissue cells / macrophages present in inflammatory bowel, hyperplastic / reactive tonsils or normal lymphocytes. All expression in these tissues, including histocyte inflammation or resident macrophage populations, supports the limited expression in a unique macrophage subpopulation defined as alveolar macrophages and hepatic Cooper cells.

Human tissues evaluated with no detectable expression include inflammatory bowel disease tissue (7 patient samples with moderate to severe disease), tonsil tissue with reactive hyperplasia, peripheral lymphocytes, psoriasis skin tissue (mild to moderate disease). 2 patient samples), heart and peripheral nerves.

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

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

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

Homologous DNA encoding human homologous DNA (e.g., PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866) in the human tissue cDNA library or human tissue genome library Full length or mature PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 (SEQ ID NOS: 1, 6, 9, 14, 19, 24, respectively) , 29, 34, 42, 47, 54, 59 and 61, respectively, as shown in FIGS. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25, respectively. DNA or fragments thereof are used.

Hybridization and washing of filters comprising these library DNAs is performed under the following high stringency conditions. Hybridization of a radiolabeled probe derived from a gene encoding a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide to a filter at 42 ° C. with 50% formamide , 5 × SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 × Denhardt's solution and 10% dextran sulfate solution for 20 hours. Washing of the filter is carried out at 42 ° C. in an aqueous solution of 0.1 × SSC and 0.1% SDS.

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

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

In this embodiment, Recombinant expression in E. coli represents a method for producing aglycosylated forms of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866.

Initially, DNA sequences encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were amplified using selected PCR primers. The primer must include a restriction enzyme site corresponding to the restriction enzyme site on the selected expression vector. Several expression vectors can be used. Examples of suitable vectors include pBR322, which includes ampicillin and tetracycline resistance genes. From coli; Bolivar et al., Gene 2 : 95 (1977). The vector was digested with restriction enzymes and dephosphorylated. PCR amplified sequences were then ligated to the vector. The vector is preferably an antibiotic resistance gene, trp promoter, poly-His leader (including the first six STII codons, poly-His sequences and enterokinase cleavage sites), PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, Sequences encoding the PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding sites, lambda transcription terminators and argU genes.

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

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

After culturing the cells for several hours or more, the cells can be recovered by centrifugation. Cell pellets obtained by centrifugation were lysed using various reagents known in the art, and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 proteins can be purified.

PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be obtained using the following procedure. It has been successfully expressed in E. coli with poly-His tags. DNAs encoding PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were initially amplified using the selected primers. Primers included restriction enzyme sites corresponding to restriction enzyme sites on selected expression vectors and other useful sequences that provide efficient and reliable translation initiation, rapid purification on metal chelate columns, and proteolytic removal with enterokinase. The PCR amplified, poly-His tagged sequence was then ligated into the expression vector and then transformed into E. coli host based on strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq). The transformants were first shaken in LB medium containing 50 mg / ml carbenicillin at 30 ° C. until the OD ₆₀₀ reached 3-5. 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, and in 500 ml of water, and Prepared by mixing 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO ₄ ) 50 to 100-fold and shake incubated at 30 ° C. for about 20 to 30 hours. Confirm expression by SDS-PAGE assay and centrifuge the bulk culture to pellet the cells (C) it was kept frozen until purification and refolding to the cell pellet.

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

The protein was refolded by slow dilution of the sample in freshly prepared refolding buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. The refolding volume was set to a final protein concentration of 50-100 μg / ml. The refolding solution was slowly stirred at 4 ° C. for 12-36 hours. The refolding reaction was stopped by adding TFA to a final concentration of 0.4% (about pH 3). Before further purification of the protein, the solution was filtered through a 0.22 μm filter and acetonitrile was added to a final concentration of 2-10%. Refolded proteins were analyzed in Poros R1 / H reversed phase column chromatography using a transfer buffer of 0.1% TFA, eluting with acetonitrile concentration gradient of 10-80%. Aliquots of fractions showing A ₂₈₀ absorbance were analyzed on an SDS polyacrylamide gel to collect fractions containing homogeneously refolded protein. In general, properly refolded proteins form most tightly to the hydrophobic inner portion that is protected from interaction with the reverse phase resin, so in most proteins the properly refolded protein form elutes at the lowest concentration of acetonitrile. Aggregated proteins are usually eluted at high acetonitrile concentrations. The reverse phase step not only separates the misfolded form of protein from the desired form of protein but also removes endotoxins from the sample.

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

PRO207, PRO224, and PRO301 are E. coli with poly-His tag by the above procedure. Successfully expressed in E. coli.

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

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

Vector pRK5 (see EP 307,247 published March 15, 1989) was used as the expression vector. Optionally, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 DNA were selected using the restriction enzymes described by Sambrook et al. (Supra). pRK5 was ligated to insert PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 DNA. The generated vectors were pRK5-PRO179, pRK5-PRO207, pRK5-PRO320, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO301, pRK5-PRO526, pRK5-PRO362, pRK5-PRO356, pRK5- It was named PRO509 or pRK5-PRO866.

In an embodiment, 293 cells can be selected as host cells. Human 293 cells (ATCC CCL 1573) were cultured to be confluent in tissue culture plates in medium such as fetal calf serum and optionally DMEM supplemented with nutrients and / or antibiotics. About 10 μg pRK5-PRO179, pRK5-PRO207, pRK5-PRO320, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO301, pRK5-PRO526, pRK5-PRO362, pRK5-PRO356, pRK5-PRO509 Or pRK5-PRO866 DNA with about 1 μg of DNA encoding the VA RNA gene (Thimmappaya et al., Cell, 31: 543 (1982)), 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA and 0.227 M Dissolved in CaCl ₂ . 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added dropwise to the mixture to form a precipitate at 25 ° C. for 10 minutes. The precipitate was suspended and added to 293 cells and left at about 4 hours at 37 ° C. The medium was aspirated off and 2 ml of 20% glycerol in PBS was added for 30 seconds. 293 cells were then washed with serum free medium, fresh medium was added and the cells were cultured for about 5 days.

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

Alternatively, Somparyrac et al., Proc. Natl. Acad. Sci. USA , 12 : 7575 (1981)] using the dextran sulfate method described above, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding genes were transferred to 293 cells. It could be introduced temporarily. 293 cells were cultured to the highest density in a rotating flask and 700 μg of pRK5-PRO179, pRK5-PRO207, pRK5-PRO320, pRK5-PRO219, pRK5-PRO221, pRK5-PRO224, pRK5-PRO328, pRK5-PRO301, pRK5-PRO526 , pRK5-PRO362, pRK5-PRO356, pRK5-PRO509 or pRK5-PRO866 DNA were added. Cells were first centrifuged, concentrated from a rotary flask and washed with PBS. DNA-dextran precipitates were incubated on cell pellets for 4 hours. Cells were treated with 20% glycerol for 90 seconds and washed with tissue culture medium and then placed in a rotating flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about 4 days the conditioned media was centrifuged and filtered to remove cells and debris. Concentrate the sample comprising expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 and select any selected method such as dialysis and / or column chromatography Purification with

In other embodiments, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 coding genes 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-PRO356, pRK5-PRO509 or pRK5- PRO866 can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE dextran. As mentioned above, the cell cultures were incubated and the cultures replaced with medium only or with a medium containing radiolabels such as ³⁵ S-methionine. After confirming the presence of expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, the culture medium was replaced with serum free medium. Preferably, the cultures were incubated for about 6 days and the conditioned medium was recovered. The medium containing the expressed PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide was concentrated and purified by any chosen method.

In addition, epitope tagged PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be expressed in CHO host cells. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be subcloned from the pRK5 vector. Insertion of the subclones can be fused to baculovirus expression vectors in the form with selected epitope tags, such as poly-His tags, by PCR. PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 inserts with poly-His tags include an SV40 with a selection marker such as DHFR to select stable clones. Can be subcloned into an induction vector. Finally, CHO cells can be transfected with an SV40 induction vector (as above). It can be labeled in the same manner as above to confirm expression. Subsequently, the culture medium comprising PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with the expressed poly-His tag is concentrated to give Ni ²⁺ -chelate affinity. It may be purified by a selected method such as chromatograph.

In addition, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be expressed in CHO and / or COS cells by transient expression methods, or by another stable expression method. It was expressed in CHO cells.

The following experimental procedure was used to stably express in CHO cells. The coding sequence of each protein's soluble form (eg, extracellular domain) is expressed as an IgG construct (immunoadhesine) fused to an IgG1 constant region sequence comprising a hinge, CH2 and CH2 domains, and / or poly-His tags. The protein was expressed in the attached form.

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

Using a commercially available transfection reagent, Superfect, Qiagen, Dosper or Fugene, Boehringer Mannheim, approximately 12 μg of the desired plasmid DNA was obtained. It was introduced into 10 million CHO cells. Cells were cultured according to the method described by Lucas et al., Supra. About 3 × 10 ⁻⁷ cells were frozen in ampoules according to the method described below for later culturing and producing cells.

Ampoules containing plasmid DNA were dissolved in a water bath and vortexed and mixed. 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 off and the cells resuspended in 10 ml selection medium (PS20 filtered through 0.2 μm filter paper, containing 5% bovine serum of 0.2 μm diafiltration filtered). Cells were aliquoted into a 100 ml rotating flask containing 90 ml of selection medium. After 1-2 days, cells were transferred to a 250 ml rotating flask filled with 150 ml of selection medium and incubated at 37 ° C. After 2-3 days 250 ml, 500 ml and 2000 ml rotating flasks were seeded with 3 × 10 ⁵ cells per ml. Cell cultures were centrifuged, exchanged with fresh medium and resuspended in production medium. Any suitable CHO medium may be used, but the production medium described in US Pat. No. 5,122,469 (June 16, 1992) was actually used. 3 L production rotary flasks were seeded at 1.2 × 10 ⁶ cells / ml. On day 0, cell number and pH were measured. On day 1 samples were taken from a rotating flask to initiate sparging of filtered air. On day 2, take a sample from a rotating flask and change the temperature to 33 ° C. and 30 ml of 500 g / L-glucose and 0.6 ml of 10% defoamer (eg 35% polydimethyl siloxane emulsion, Dow Corning 365 medicinal grade emulsion) ) Was added. During the entire production period, the pH was adjusted to maintain about 7.2 as needed. Cell cultures were recovered by centrifugation after 10 days or when viability dropped below 70% and filtered through a 0.22 μm filter. The filtrate was stored at 4 ° C. or immediately loaded onto a preparative column.

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

The immunoadhesin (including Fc) constructs from the conditioned media were purified as follows. Conditioned media was injected into 5 ml of Protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 6.8). After loading, the column was washed with equilibration buffer and eluted with 100 mM citric acid, pH 3.5. The eluted protein was recovered in 1 ml fractions in a tube containing 275 μl of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed to salt in storage buffer as described above for the case of poly-His tagged proteins. N-terminal amino acid sequencing performed by SDS-polyacrylamide gel and Edman digestion confirmed the homogeneity of the protein.

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

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

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

First, yeast expression vectors for intracellular production or secretion of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 were prepared from the ADH2 / GAPDH promoter. For direct intracellular expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526 , PRO362, PRO356, PRO509 or PRO866 coding DNA and promoter were inserted at the appropriate restriction enzyme sites of the selected plasmid. For secretion, for the expression of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 DNA, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301 DNA encoding PRO526, PRO362, PRO356, PRO509 or PRO866, ADH2 / GAPDH promoter, native PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 signal peptide or other The mammalian signal peptides can be cloned into selected plasmids with DNA encoding yeast alpha-factor or invertase secretion signal / leader sequence and linker sequence (if required).

Yeast cells, for example yeast strain AB110, can be transformed with the expression plasmids described above and cultured in selected fermentation medium. Transformed yeast supernatants can be precipitated with 10% trichloroacetic acid, separated by SDS-PAGE and analyzed by staining the gel with Coomassie Blue.

Subsequently, the yeast cells are recovered from the fermentation medium by centrifugation, and the medium is concentrated using a selected cartridge filter, and the recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be isolated and purified. Concentrates containing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 can be further purified using selected column chromatography resins.

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

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

The coding sequence of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 was fused upstream of the epitope tag included in the 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 (Novagen). Briefly, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 Coding Sequence or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362 The desired portion of the PRO356, PRO509 or PRO866 coding sequence (e.g., the extracellular domain of the transmembrane protein or the sequence encoding the mature protein if it is an extracellular protein) by PCR using primers complementary to the 5 'and 3' regions. Amplified. The 5 'primer may comprise contiguous (selected) restriction enzyme sites. The product was then cleaved with the selected restriction enzyme and subcloned into the expression vector.

Recombinant baculoviruses utilize the lipofectin (gibbo-BRL) to plasmid and BaculoGold ™ viral DNA (Pharmingen) from Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711). And transfection at the same time. After 4-5 days of incubation at 28 ° C., the released virus was recovered and used for subsequent amplification. Viral infection and protein expression were performed according to O'Reilley et al., Baculovirus Expression vectors: A laboratory Manual , Oxford: Oxford University Press (1994).

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

Alternatively, purification of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 with IgG tagged (or with Fc tag) can be performed using known chromatography techniques, For example, using Protein A or Protein G column chromatography.

Subsequent PCR amplification of each coding sequence was then subcloned into baculovirus expression vectors (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins) and lipofectin (GIBCO-BRL) was used to simultaneously transfect vectors and BaculoGold ™ baculovirus DNA (Pharmingen) into 105 Spodoptera prugiferda (Sf9) cells (ATCC CRL 1711). pb.PH.IgG and pb.PH.His are modifications of the commercial baculovirus expression vector pVL1393 (Pharmingen) comprising a modified polylinker site comprising a His sequence or an Fc tag sequence. The cells were cultured in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). Cells were incubated at 28 ° C. for 5 days. The supernatant was recovered and subsequently infected with Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS so that the fold of infection (MOI) was about 10 and used for first virus amplification. Cells were incubated at 28 ° C. for 3 days. The supernatant was recovered and placed in a 1 mL supernatant in 25 mL Ni ²⁺ -NTA beads (QIAGEN) for histidine tagged protein or protein A Sepharose CL-4B beads (Pharmacia) for IgG added protein. After binding, an SDS-PAGE analysis was performed to measure the expression of the constructs in the baculovirus expression vectors as compared to the standard protein at a concentration already known by Coomassie blue staining.

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

Conditioned medium of transfected cells (0.5-3 L) was centrifuged to remove cells and filtered with a 0.22 μm filter. Poly-His tagged structures were purified using a Ni ²⁺ -NTA column (Qiagen). Imidazole was added to the conditioned medium to 5 mM concentration before purification. The conditioned medium was loaded into a 6 ml Ni ²⁺ -NTA column equilibrated with 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml / min at 4 ° C. After loading the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed with salt in a storage buffer (pH6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol with 25 ml G25 Superfine (Pharmacia) column and stored at -80 ° C.

The immunoadhesin (including Fc) constructs of the protein 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, it was extensively washed with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in a 1 ml fraction into a tube containing 275 ml of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed from the storage buffer as in the case of the poly-His tagged protein described above. The homogeneity of the protein was confirmed by N-terminal amino acid sequence analysis performed by SDS-PAGE and Edman digestion.

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

Alternatively, the modified baculovirus method can be used on high-5 cells. In this method, DNA encoding the desired sequence can be amplified using a suitable system, such as Pfu (Stratagene), or can be fused with the upstream (5 ') of the epitope tag contained in the baculovirus expression vector. have. Such epitope tags include poly-His tags and immunoglobulin tags (such as the Fc region of IgG). Various plasmids can be used, including commercially available plasmids, for example plasmids derived from pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors are designed to constitutively express recombinant proteins from baculovirus ie1 promoters in stably transformed insect cells (1). Both plasmids differ only in the direction of the multiple cloning site and include all promoter sequences and hr5 enhancer components known to be important for ie1 mediated gene expression of uninfected insect cells. pIE1-1 and pIE1-2 include translation initiation sites and can be used to produce fusion proteins. Briefly, a sequence that encodes the desired sequence or desired portion of the sequence, eg, the extracellular domain of the transmembrane protein, was amplified by PCR using primers complementary to the 5 'and 3' regions. The 5 'primer may comprise contiguous (selected) restriction enzyme sites. The product was then cleaved with the selected restriction enzyme and subcloned into the expression vector. For example, derivatives of pEI1-1 may comprise an Fc region or 8 histitin (pb.PH.His) tags of human IgG (pb.PH.IgG) downstream (3 ′) of the desired sequence. Preferably, the vector constructs are sequenced to identify.

High-5 cells were cultured with 50% confluency under conditions of 27 ° C. without CO ₂ , NO pen / strep. For each 150 mm plate, a PIE substrate vector containing 30 μg of sequence was added with 1 ml of Ex-Cell medium (Ex-Cell 401 + 1/100 L-Glu JRH Biosciences # 14401-78P, noteworthy). 100 μl of Celfectin (CellFECTIN, purchased from GIBCO-BRL # 10362-010, vortex mixed) was mixed with 1 ml of Ex-Cell medium in separate test tubes. Both solutions were mixed and incubated for 15 minutes at room temperature. 8 ml of Ex-Cell medium was added to 2 ml of DNA / Secfectin mixture and covered on high-5 cells washed once with Ex-Cell medium. The plates were then incubated in the dark for 1 hour at room temperature. The DNA / Selfectin mixture was then removed with an aspirator and the cells washed once with Ex-Cell to remove excess Celfectin. 30 ml of fresh Ex-Cell medium was added and cells were incubated at 28 ° C. for 3 days. The supernatant was recovered and placed in a 1 mL supernatant in 25 mL Ni ²⁺ -NTA beads (QIAGEN) for histidine tagged protein or protein A Sepharose CL-4B beads (Pharmacia) for IgG added protein. After binding, SDS-PAGE analysis was performed to determine the expression of sequences in baculovirus expression vectors as compared to standard proteins of concentrations already known by Coomassie blue staining.

Conditioned medium of transfected cells (0.5-3 L) was centrifuged to remove cells and filtered with a 0.22 μm filter. Poly-His tagged structures were purified using a Ni ²⁺ -NTA column (Qiagen) containing the sequence. Imidazole was added to the conditioned medium to 5 mM concentration before purification. The conditioned medium was loaded into a 6 ml Ni ²⁺ -NTA column equilibrated with 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml / min at 48 ° C. After loading the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed with salt in a storage buffer (pH6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol with 25 ml G25 Superfine (Pharmacia) column and stored at -80 ° C.

The immunoadhesin (including Fc) constructs of the protein 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, it was extensively washed with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in a 1 ml fraction into a tube containing 275 ml of 1 M Tris buffer (pH 9) and immediately neutralized. The highly purified protein was then removed from the storage buffer as in the case of the poly-His tagged protein described above. The homogeneity of the protein was confirmed by N-terminal amino acid sequence analysis performed by SDS polyacrylamide gel and Edman digestion and other analytical methods if desired or if necessary.

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

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

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

Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used include purified PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328 Expresses recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 on the fusion protein and cell surface, including PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 Cell. The skilled person can select an immunogen without unnecessary experimentation.

Subcutaneous or intraperitoneal of mice, e.g., PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 immunogens emulsified in complete supplements with Balb / c I was immunized by my injection. Alternatively, the immunogens were immunized by emulsification in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the hind paw of the animal. Immunized mice were then boosted with additional immunogen emulsified in selected adjuvant after 10-12 days. Afterwards, the mice can be boosted with further immunization for several weeks. Serum samples were taken periodically from mice by reverse bleeding and tested for ELISA assay to determine anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti- PRO301, anti-PRO526, anti-PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 antibodies were detected.

Once a suitable antibody titer was detected, the animals that were "positive" for the antibody were given a final intravenous injection of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. After 3-4 days, the mice are sacrificed to recover the spleen cells. The spleen cells were then fused with P3X63AgU.1 available from selected murine myeloma cell lines, eg ATCC No. CRL 1597 (using 35% polyethylene glycol). The fusion produced hybridoma cells, which were then played on 96 well tissue culture plates containing HAT (Hypoxanthine, aminopterin and thymidine) media that inhibit the proliferation of non-fusion cells, myeloma hybrids and splenocyte hybrids. Can be set.

Hybridoma cells can be screened by ELISA for responsiveness to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. Methods of determining "positive" hybridoma cells secreting the desired monoclonal antibody against PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 It is well known to those skilled in the art.

Positive hybridoma cells are syngeneic Balb / c mice with anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti It may be injected intraperitoneally to produce ascites comprising -PRO362, anti-PRO356, anti-PRO509 or anti-PRO866 monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in tissue culture flasks or roller bottles. Monoclonal antibodies generated in the ascites can be purified using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on protein A or protein G binding of the antibody may be employed.

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

Natural or recombinant PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides can be purified by various standard techniques in the field of protein purification. 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 PRO221, 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-PRO221, pre-PRO224, pre-PRO328, pre-PRO301, pre-PRO526, pre-PRO362, pre-PRO356, pre-PRO509 Or pre-PRO866 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide of interest. Generally, immuno-affinity columns are anti-PRO179, anti-PRO207, anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301, anti-PRO526, anti-PRO362, anti- It is made by covalently coupling a PRO356, anti-PRO509 or anti-PRO866 polypeptide antibody to an activated chromatography resin.

Polyclonal immunoglobulins were prepared by precipitation from ammonium serum with ammonium sulfate or by purification on immobilized protein A (Pharmacia LKB Biotechnology, Fitzkataway, NJ). Similarly, monoclonal antibodies were prepared by ammonium sulphate precipitation or chromatography on immobilized Protein A from mouse ascites. Partially purified immunoglobulin was covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE ™ (Pharmacia LKB Biotechnology). The antibody was coupled to the resin, the resin blocked and the derived resin was washed according to the manufacturer's instructions.

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

A formulation containing a soluble PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide is passed over an immunoaffinity column and the column is passed through the PRO179, PRO207, PRO320, PRO219, PRO221 Washed under conditions that allow for differential absorption of, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides (eg high ion concentration buffer in the presence of detergent). The column was then antibody / PRO179, antibody / PRO207, antibody / PRO320, antibody / PRO219, antibody / PRO221, antibody / PRO224, antibody / PRO328, antibody / PRO301, antibody / PRO526, antibody / PRO362, antibody / PRO356, antibody / Elute under conditions that disrupt PRO509 or antibody / PRO866 polypeptide binding (e.g., a low pH buffer such as about pH 2-3 or a high concentration of chaotrope, such as urea or thiocyanate ions), and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides were recovered.

Example 23 Drug Screening

The present invention is particularly useful for screening compounds with any of various drug screening techniques using PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or binding fragments thereof. . The PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides or fragments used in these tests are free in solution, adhered to a solid support, or retained at the cell surface Or may be located within the cell. One method of drug screening involves eukaryotic or prokaryotic transformed stably with recombinant nucleic acids expressing PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or fragment. Use host cells. Drugs were screened for such transformed cells in a competitive binding assay. Such cells, either live or fixed, can be used for standard binding assays. For example, complex formation between a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or fragment and the substance being tested can be measured. Alternatively, the reduction of complex formation between PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides caused by the substance being tested can be achieved. Can be checked

Accordingly, the present invention relates to a drug or any other active substance that may affect PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide-related diseases or disorders. Provided are methods for screening. These methods contact such active substances with PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or fragments thereof, and are known in the art as (i) The presence of a complex between the substance and PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or fragment or (ii) PRO179, PRO207, PRO320, PRO219, PRO221, PRO224 Assaying for the presence of a polypeptide or fragment and a complex between a cell and a PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866. In this competitive binding assay, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides or fragments are usually labeled. After suitable incubation, the glass PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or fragments are separated from those present in bound form and the glass or non-complexed label The amount of the specific substance can bind to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, It is a measure of the ability to inhibit PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide / cell complex.

Other drug screening techniques provide high throughput screening for compounds with binding affinity suitable for polypeptides and are described in detail in WO84 / 03564 (published Sep. 13, 1984). In brief, many different small peptide test compounds are synthesized on solid substrates such as plastic pins or some other surface. Peptide test compounds are applied to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides to provide peptide test compounds React and wash with 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 a plate for use in the aforementioned drug screening techniques. Non-neutralized antibodies can also be used to capture peptides and immobilize them on solid support.

The present invention also provides a neutralizing antibody capable of binding to PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide. Consider the use of competitive drug screening assays that specifically compete with the test compound for binding to a PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide or fragment thereof. In this manner, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide. Can be.

Example 24 Rational Drug Design

The goal of rational drug design is to target the biologically active polypeptides of interest (i.e., PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptides) or small molecules with which they interact, for example For example, to produce structural homologs of agonists, antagonists or inhibitors. Any of these examples include a drug that is a more active or more stable form of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide, or in vivo PRO179, PRO207, It can be used to form drugs that enhance or inhibit the function of PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides (Hodgson, Bio / Technology , 9 : 1921 (1991)).

In one study, PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide or PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362 The three-dimensional structure of the, PRO356, PRO509 or PRO866 polypeptide-inhibitor complex is determined by x-ray crystallography, computer modeling or most typically a combination of the two methods. Both the form and the charge of the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides should be identified to elucidate the structure of the molecule and determine the active site (s). Although less frequent, useful information about the structure of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide can be obtained by modeling the structure of homologous proteins. . In both cases, relevant structural information is used to design cognate PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide-like molecules or identify valid inhibitors. Useful examples of rational drug design include molecules that enhance activity or stability, as shown in Braxton and Wells, Biochemistry, 31 : 7796-7801 (1992), or Athauda. ), J. Biochem. , 113 : 742-746 (1993), may include molecules that act as inhibitors, agonists or antagonists of natural peptides.

It is also possible to isolate target-specific antibodies, select them by functional analysis as described above, and then interpret their crystal structure. In this way, a pharmacore can be obtained, in principle, on which subsequent drug design can be based. Protein Determination It is also possible to avoid all protein determination methods by producing anti-genetic antibodies (anti-ids) against functional pharmacologically active antibodies. As a mirror image, the binding site of the anti-id will be expected to be the homologue of the original receptor. Anti-ids may then be used to identify and isolate peptides from chemically or biologically produced peptide banks. The isolated peptide will then act as a Pharmacore.

According to the present invention, sufficient amounts of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides are available for carrying out analytical studies such as x-ray crystallography. can do. In addition, the PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509, or PRO866 polypeptide amino acid sequence knowledge provided herein provides instructions for using computer modeling techniques instead of or in addition to x-ray determination. Will provide.

Example 25 In Vitro Antitumor Assay

Antiproliferative activity of PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptides is described by Skehan et al., J. Natl. In Vitro Anticancer Research Drug Discovery Assay for Diseases of the National Cancer Institute (NCI), using the sulforhodamine B (SRB) dye binding assay described essentially in Cancer Inst ., 82 : 1107-1112 (1990). Measured by. The 60 tumor cell lines ("NCI panels") used in this study and the maintenance and in vitro culture methods of these cell lines are described in Monks et al., J. Natl. Cancer Inst. , 83 : 757-766 (1991). The purpose of this screening is to initially assess the cytotoxic and / or cytostatic activity of test compounds against different tumor types [Monks et al., Boyd, Cancer: Princ. Pract. Oncol. Update , 3 (10) : 1-12 (1989).

Cells of approximately 60 human tumor cell lines were incubated with trypsin / EDTA (Gibco), washed once and resuspended in IMEM to determine viability. Cell suspensions were pipetted into each 96 well microtiter plate (100 μl volume). After 6 days incubation, hyperproliferation was inhibited so that the cell density was lower than after 2 days incubation. Preincubation at 37 ° C. for 24 hours to stabilize the inoculum. Two desired test concentration dilutions were added to the microtiter plate at 0:00 in 100 μl aliquots of the wells (1: 2 dilution). Test compounds were evaluated at 5 1/2 log dilution concentrations (1000 to 100,000 fold). 2 and 6 days incubation in 5% CO ₂ atmosphere and 100% humidity.

After incubation, the medium was removed and the cells were fixed in 0.1 ml of 10% trichloroacetic acid at 40 ° C. The plates were washed five times with deionized water, dried and stained with 0.1 ml of 0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsed four times with 1% acetic acid to remove unbound dyes, After drying, the dyeing was extracted with 0.1 ml of 10 mM Tris base [tris (hydroxymethyl) aminomethane] (pH 10.5) for 5 minutes. The absorbance (OD) of sulforhodamine B at 492 nm was measured using a computer controlled 96 well microphone titer plate reader.

Test samples were considered positive if they exhibited at least 40% growth inhibitory effect at one or more concentrations. The results are shown in Table 4 below. In the abbreviations of tumor cell types, NSCL represents non-small cell lung cancer and CNS represents the central nervous system.

Deposit of matter

The following materials were deposited in the American Type Culture Collection (ATCC), Manassas University Boulevard 10801, Virginia, USA: 20110-2209.

These deposits were made under the provisions of the Budapest Treaty and its rules (Budapest Treaty) on the international approval of microbial deposits under the patent procedure. This ensures maintenance of the viable culture of the deposit for 30 years from the date of deposit. The deposits will be distributed from ATCC under the Convention of the Budapest Treaty under the agreement between Genentech Inc and ATCC, upon the granting of the relevant U.S. patent or the publication of a U.S. or foreign patent application (when it is first). To ensure the permanent and non-limiting distribution of the deposit progeny, and to the regulations of the US Patent and Trademark Commissioner, 35 USC §122 and the rules of the US Patent and Trademark Commissioner (including 37 CFR §1.14, in particular 886 OG 638). Progenie's sale is assured to those who decide to have rights under

The assignee of the present application agrees that upon incubation under appropriate conditions, if the culture of the deposited material is killed, lost or damaged, the material will be replaced immediately with another identical material upon notification. The sale of deposited materials should not be construed as an authorization of the governments to carry out the invention in violation of the rights granted under the patent law.

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

Sequence Listing <110> Genentech, Inc. Ashkenazi, Avi, J. Goddard, Audrey Godowski, Paul, J. Gurney, Austin, L. Marsters, Scot, A. Napier, Mary, A. Pitti, Robert, M. Wood, William, I. <120> METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL GROWTH <130> P2834R1PCT <140> PCT / US99 / 28565 <141> 1999-12-02 <150> US 60 / 113,296 <151> 1998-12-22 <150> PCT / US99 / 05028 <151> 1999-03-08 <150> US 60 / 130,232 <151> 1999-04-21 <150> US 60 / 131,445 <151> 1999-04-28 <150> US 60 / 134,287 <151> 1999-05-14 <150> US 60 / 144,758 <151> 1999-07-20 <150> US 60 / 145,698 <151> 1999-07-26 <150> PCT / US99 / 21090 <151> 1999-09-15 <150> PCT / US99 / 21547 <151> 1999-09-15 <160> 79 <210> 1 <211> 2042 <212> DNA <213> Homo Sapien <400> 1 gcggacgcgt gggtgaaatt gaaaatcaag ataaaaatgt tcacaattaa 50 gctccttctt tttattgttc ctctagttat ttcctccaga attgatcaag 100 acaattcatc atttgattct ctatctccag agccaaaatc aagatttgct 150 atgttagacg atgtaaaaat tttagccaat ggcctccttc agttgggaca 200 tggtcttaaa gactttgtcc ataagacgaa gggccaaatt aatgacatat 250 ttcaaaaact caacatattt gatcagtctt tttatgatct atcgctgcaa 300 accagtgaaa tcaaagaaga agaaaaggaa ctgagaagaa ctacatataa 350 actacaagtc aaaaatgaag aggtaaagaa tatgtcactt gaactcaact 400 caaaacttga aagcctccta gaagaaaaaa ttctacttca acaaaaagtg 450 aaatatttag aagagcaact aactaactta attcaaaatc aacctgaaac 500 tccagaacac ccagaagtaa cttcacttaa aacttttgta gaaaaacaag 550 ataatagcat caaagacctt ctccagaccg tggaagacca atataaacaa 600 ttaaaccaac agcatagtca aataaaagaa atagaaaatc agctcagaag 650 gactagtatt caagaaccca cagaaatttc tctatcttcc aagccaagag 700 caccaagaac tactcccttt cttcagttga atgaaataag aaatgtaaaa 750 catgatggca ttcctgctga atgtaccacc atttataaca gaggtgaaca 800 tacaagtggc atgtatgcca tcagacccag caactctcaa gtttttcatg 850 tctactgtga tgttatatca ggtagtccat ggacattaat tcaacatcga 900 atagatggat cacaaaactt caatgaaacg tgggagaact acaaatatgg 950 ttttgggagg cttgatggag aattttggtt gggcctagag aagatatact 1000 ccatagtgaa gcaatctaat tatgttttac gaattgagtt ggaagactgg 1050 aaagacaaca aacattatat tgaatattct ttttacttgg gaaatcacga 1100 aaccaactat acgctacatc tagttgcgat tactggcaat gtccccaatg 1150 caatcccgga aaacaaagat ttggtgtttt ctacttggga tcacaaagca 1200 aaaggacact tcaactgtcc agagggttat tcaggaggct ggtggtggca 1250 tgatgagtgt ggagaaaaca acctaaatgg taaatataac aaaccaagag 1300 caaaatctaa gccagagagg agaagaggat tatcttggaa gtctcaaaat 1350 ggaaggttat actctataaa atcaaccaaa atgttgatcc atccaacaga 1400 ttcagaaagc tttgaatgaa ctgaggcaat ttaaaggcat atttaaccat 1450 taactcattc caagttaatg tggtctaata atctggtata aatccttaag 1500 agaaagcttg agaaatagat tttttttatc ttaaagtcac tgtctattta 1550 agattaaaca tacaatcaca taaccttaaa gaataccgtt tacatttctc 1600 aatcaaaatt cttataatac tatttgtttt aaattttgtg atgtgggaat 1650 caattttaga tggtcacaat ctagattata atcaataggt gaacttatta 1700 aataactttt ctaaataaaa aatttagaga cttttatttt aaaaggcatc 1750 atatgagcta atatcacaac tttcccagtt taaaaaacta gtactcttgt 1800 taaaactcta aacttgacta aatacagagg actggtaatt gtacagttct 1850 taaatgttgt agtattaatt tcaaaactaa aaatcgtcag cacagagtat 1900 gtgtaaaaat ctgtaataca aatttttaaa ctgatgcttc attttgctac 1950 aaaataattt ggagtaaatg tttgatatga tttatttatg aaacctaatg 2000 aagcagaatt aaatactgta ttaaaataag ttcgctgtct tt 2042 <210> 2 <211> 460 <212> PRT <213> Homo Sapien <400> 2 Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile 1 5 10 15 Ser Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser 20 25 30 Pro Glu Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile 35 40 45 Leu Ala Asn Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe 50 55 60 Val His Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu 65 70 75 Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser 80 85 90 Glu Ile Lys Glu Glu Glu Lys Glu Leu Arg Arg Thr Thr Tyr Lys 95 100 105 Leu Gln Val Lys Asn Glu Glu Val Lys Asn Met Ser Leu Glu Leu 110 115 120 Asn Ser Lys Leu Glu Ser Leu Leu Glu Glu Lys Ile Leu Leu Gln 125 130 135 Gln Lys Val Lys Tyr Leu Glu Glu Gln Leu Thr Asn Leu Ile Gln 140 145 150 Asn Gln Pro Glu Thr Pro Glu His Pro Glu Val Thr Ser Leu Lys 155 160 165 Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys Asp Leu Leu Gln 170 175 180 Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln His Ser Gln 185 190 195 Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile Gln Glu 200 205 210 Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg Thr 215 220 225 Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp 230 235 240 Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His 245 250 255 Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe 260 265 270 His Val Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile 275 280 285 Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu 290 295 300 Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu 305 310 315 Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val 320 325 330 Leu Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr Ile 335 340 345 Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr Asn Tyr Thr Leu 350 355 360 His Leu Val Ala Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu 365 370 375 Asn Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly 380 385 390 His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His 395 400 405 Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro 410 415 420 Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys 425 430 435 Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu 440 445 450 Ile His Pro Thr Asp Ser Glu Ser Phe Glu 455 460 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 3 ccacgttggc ttgaaattga 20 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Olgonucleotide Probe <400> 4 cctttagaat tgatcaagac aattcatgat ttgattctct atctccagag 50 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 5 tcgtctaaca tagcaaatc 19 <210> 6 <211> 1353 <212> DNA <213> Homo Sapien <400> 6 cgatccctcg ggtcccggga tgggggggcg gtgaggcagg cacagccccc 50 cgcccccatg gccgcccgtc ggagccagag gcggaggggg cgccgggggg 100 agccgggcac cgccctgctg gtcccgctcg cgctgggcct gggcctggcg 150 ctggcctgcc tcggcctcct gctggccgtg gtcagtttgg ggagccgggc 200 atcgctgtcc gcccaggagc ctgcccagga ggagctggtg gcagaggagg 250 accaggaccc gtcggaactg aatccccaga cagaagaaag ccaggatcct 300 gcgcctttcc tgaaccgact agttcggcct cgcagaagtg cacctaaagg 350 ccggaaaaca cgggctcgaa gagcgatcgc agcccattat gaagttcatc 400 cacgacctgg acaggacgga gcgcaggcag gtgtggacgg gacagtgagt 450 ggctgggagg aagccagaat caacagctcc agccctctgc gctacaaccg 500 ccagatcggg gagtttatag tcacccgggc tgggctctac tacctgtact 550 gtcaggtgca ctttgatgag gggaaggctg tctacctgaa gctggacttg 600 ctggtggatg gtgtgctggc cctgcgctgc ctggaggaat tctcagccac 650 tgcggcgagt tccctcgggc cccagctccg cctctgccag gtgtctgggc 700 tgttggccct gcggccaggg tcctccctgc ggatccgcac cctcccctgg 750 gcccatctca aggctgcccc cttcctcacc tacttcggac tcttccaggt 800 tcactgaggg gccctggtct ccccgcagtc gtcccaggct gccggctccc 850 ctcgacagct ctctgggcac ccggtcccct ctgccccacc ctcagccgct 900 ctttgctcca gacctgcccc tccctctaga ggctgcctgg gcctgttcac 950 gtgttttcca tcccacataa atacagtatt cccactctta tcttacaact 1000 cccccaccgc ccactctcca cctcactagc tccccaatcc ctgacccttt 1050 gaggccccca gtgatctcga ctcccccctg gccacagacc cccaggtcat 1100 tgtgttcact gtactctgtg ggcaaggatg ggtccagaag accccacttc 1150 aggcactaag aggggctgga cctggcggca ggaagccaaa gagactgggc 1200 ctaggccagg agttcccaaa tgtgaggggc gagaaacaag acaagctcct 1250 cccttgagaa ttccctgtgg atttttaaaa cagatattat ttttattatt 1300 attgtgacaa aatgttgata aatggatatt aaatagaata agtcataaaa 1350 aaa 1353 <210> 7 <211> 249 <212> PRT <213> Homo Sapien <400> 7 Met Ala Ala Arg Arg Ser Gln Arg Arg Arg Gly Arg Arg Gly Glu 1 5 10 15 Pro Gly Thr Ala Leu Leu Val Pro Leu Ala Leu Gly Leu Gly Leu 20 25 30 Ala Leu Ala Cys Leu Gly Leu Leu Leu Ala Val Val Ser Leu Gly 35 40 45 Ser Arg Ala Ser Leu Ser Ala Gln Glu Pro Ala Gln Glu Glu Leu 50 55 60 Val Ala Glu Glu Asp Gln Asp Pro Ser Glu Leu Asn Pro Gln Thr 65 70 75 Glu Glu Ser Gln Asp Pro Ala Pro Phe Leu Asn Arg Leu Val Arg 80 85 90 Pro Arg Arg Ser Ala Pro Lys Gly Arg Lys Thr Arg Ala Arg Arg 95 100 105 Ala Ile Ala Ala His Tyr Glu Val His Pro Arg Pro Gly Gln Asp 110 115 120 Gly Ala Gln Ala Gly Val Asp Gly Thr Val Ser Gly Trp Glu Glu 125 130 135 Ala Arg Ile Asn Ser Ser Ser Pro Leu Arg Tyr Asn Arg Gln Ile 140 145 150 Gly Glu Phe Ile Val Thr Arg Ala Gly Leu Tyr Tyr Leu Tyr Cys 155 160 165 Gln Val His Phe Asp Glu Gly Lys Ala Val Tyr Leu Lys Leu Asp 170 175 180 Leu Leu Val Asp Gly Val Leu Ala Leu Arg Cys Leu Glu Glu Phe 185 190 195 Ser Ala Thr Ala Ala Ser Ser Leu Gly Pro Gln Leu Arg Leu Cys 200 205 210 Gln Val Ser Gly Leu Leu Ala Leu Arg Pro Gly Ser Ser Leu Arg 215 220 225 Ile Arg Thr Leu Pro Trp Ala His Leu Lys Ala Ala Pro Phe Leu 230 235 240 Thr Tyr Phe Gly Leu Phe Gln Val His 245 249 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 8 ccagccctct gcgctacaac cgccagatcg gggagtttat agtcacccgg 50 <210> 9 <211> 2260 <212> DNA <213> Homo Sapien <220> <221> unsure <222> 2009, 2026, 2033, 2055, 2074, 2078, 2086 <223> unknown base <400> 9 cggacgcgtg ggtgcgagtg gagcggagga cccgagcggc tgaggagaga 50 ggaggcggcg gcttagctgc tacggggtcc ggccggcgcc ctcccgaggg 100 gggctcagga ggaggaagga ggacccgtgc gagaatgcct ctgccctgga 150 gccttgcgct cccgctgctg ctctcctggg tggcaggtgg tttcgggaac 200 gcggccagtg caaggcatca cgggttgtta gcatcggcac gtcagcctgg 250 ggtctgtcac tatggaacta aactggcctg ctgctacggc tggagaagaa 300 acagcaaggg agtctgtgaa gctacatgcg aacctggatg taagtttggt 350 gagtgcgtgg gaccaaacaa atgcagatgc tttccaggat acaccgggaa 400 aacctgcagt caagatgtga atgagtgtgg aatgaaaccc cggccatgcc 450 aacacagatg tgtgaataca cacggaagct acaagtgctt ttgcctcagt 500 ggccacatgc tcatgccaga tgctacgtgt gtgaactcta ggacatgtgc 550 catgataaac tgtcagtaca gctgtgaaga cacagaagaa gggccacagt 600 gcctgtgtcc atcctcagga ctccgcctgg ccccaaatgg aagagactgt 650 ctagatattg atgaatgtgc ctctggtaaa gtcatctgtc cctacaatcg 700 aagatgtgtg aacacatttg gaagctacta ctgcaaatgt cacattggtt 750 tcgaactgca atatatcagt ggacgatatg actgtataga tataaatgaa 800 tgtactatgg atagccatac gtgcagccac catgccaatt gcttcaatac 850 ccaagggtcc ttcaagtgta aatgcaagca gggatataaa ggcaatggac 900 ttcggtgttc tgctatccct gaaaattctg tgaaggaagt cctcagagca 950 cctggtacca tcaaagacag aatcaagaag ttgcttgctc acaaaaacag 1000 catgaaaaag aaggcaaaaa ttaaaaatgt taccccagaa cccaccagga 1050 ctcctacccc taaggtgaac ttgcagccct tcaactatga agagatagtt 1100 tccagaggcg ggaactctca tggaggtaaa aaagggaatg aagagaaatg 1150 aaagaggggc ttgaggatga gaaaagagaa gagaaagccc tgaagaatga 1200 catagaggag cgaagcctgc gaggagatgt gtttttccct aaggtgaatg 1250 aagcaggtga attcggcctg attctggtcc aaaggaaagc gctaacttcc 1300 aaactggaac ataaagattt aaatatctcg gttgactgca gcttcaatca 1350 tgggatctgt gactggaaac aggatagaga agatgatttt gactggaatc 1400 ctgctgatcg agataatgct attggcttct atatggcagt tccggccttg 1450 gcaggtcaca agaaagacat tggccgattg aaacttctcc tacctgacct 1500 gcaaccccaa agcaacttct gtttgctctt tgattaccgg ctggccggag 1550 acaaagtcgg gaaacttcga gtgtttgtga aaaacagtaa caatgccctg 1600 gcatgggaga agaccacgag tgaggatgaa aagtggaaga cagggaaaat 1650 tcagttgtat caaggaactg atgctaccaa aagcatcatt tttgaagcag 1700 aacgtggcaa gggcaaaacc ggcgaaatcg cagtggatgg cgtcttgctt 1750 gtttcaggct tatgtccaga tagcctttta tctgtggatg actgaatgtt 1800 actatcttta tatttgactt tgtatgtcag ttccctggtt tttttgatat 1850 tgcatcatag gacctctggc attttagaat tactagctga aaaattgtaa 1900 tgtaccaaca gaaatattat tgtaagatgc ctttcttgta taagatatgc 1950 caatatttgc tttaaatatc atatcactgt atcttctcag tcatttctga 2000 atctttccnc attatattat aaaatntgga aangtcagtt tatctcccct 2050 cctcngtata tctgatttgt atangtangt tgatgngctt ctctctacaa 2100 catttctaga aaatagaaaa aaaagcacag agaaatgttt aactgtttga 2150 ctcttatgat acttcttgga aactatgaca tcaaagatag acttttgcct 2200 aagtggctta gctgggtctt tcatagccaa acttgtatat ttaattcttt 2250 gtaataataa 2260 <210> 10 <211> 338 <212> PRT <213> Homo Sapien <400> 10 Met Pro Leu Pro Trp Ser Leu Ala Leu Pro Leu Leu Leu Ser Trp 1 5 10 15 Val Ala Gly Gly Phe Gly Asn Ala Ala Ser Ala Arg His His Gly 20 25 30 Leu Leu Ala Ser Ala Arg Gln Pro Gly Val Cys His Tyr Gly Thr 35 40 45 Lys Leu Ala Cys Cys Tyr Gly Trp Arg Arg Asn Ser Lys Gly Val 50 55 60 Cys Glu Ala Thr Cys Glu Pro Gly Cys Lys Phe Gly Glu Cys Val 65 70 75 Gly Pro Asn Lys Cys Arg Cys Phe Pro Gly Tyr Thr Gly Lys Thr 80 85 90 Cys Ser Gln Asp Val Asn Glu Cys Gly Met Lys Pro Arg Pro Cys 95 100 105 Gln His Arg Cys Val Asn Thr His Gly Ser Tyr Lys Cys Phe Cys 110 115 120 Leu Ser Gly His Met Leu Met Pro Asp Ala Thr Cys Val Asn Ser 125 130 135 Arg Thr Cys Ala Met Ile Asn Cys Gln Tyr Ser Cys Glu Asp Thr 140 145 150 Glu Glu Gly Pro Gln Cys Leu Cys Pro Ser Ser Gly Leu Arg Leu 155 160 165 Ala Pro Asn Gly Arg Asp Cys Leu Asp Ile Asp Glu Cys Ala Ser 170 175 180 Gly Lys Val Ile Cys Pro Tyr Asn Arg Arg Cys Val Asn Thr Phe 185 190 195 Gly Ser Tyr Tyr Cys Lys Cys His Ile Gly Phe Glu Leu Gln Tyr 200 205 210 Ile Ser Gly Arg Tyr Asp Cys Ile Asp Ile Asn Glu Cys Thr Met 215 220 225 Asp Ser His Thr Cys Ser His His Ala Asn Cys Phe Asn Thr Gln 230 235 240 Gly Ser Phe Lys Cys Lys Cys Lys Gln Gly Tyr Lys Gly Asn Gly 245 250 255 Leu Arg Cys Ser Ala Ile Pro Glu Asn Ser Val Lys Glu Val Leu 260 265 270 Arg Ala Pro Gly Thr Ile Lys Asp Arg Ile Lys Lys Leu Leu Ala 275 280 285 His Lys Asn Ser Met Lys Lys Lys Ala Lys Ile Lys Asn Val Thr 290 295 300 Pro Glu Pro Thr Arg Thr Pro Thr Pro Lys Val Asn Leu Gln Pro 305 310 315 Phe Asn Tyr Glu Glu Ile Val Ser Arg Gly Gly Asn Ser His Gly 320 325 330 Gly Lys Lys Gly Asn Glu Glu Lys 335 338 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 11 cctcagtggc cacatgctca tg 22 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 12 ggctgcacgt atggctatcc atag 24 <210> 13 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 13 gataaactgt cagtacagct gtgaagacac agaagaaggg ccacagtgcc 50 <210> 14 <211> 3449 <212> DNA <213> Homo Sapien <400> 14 acttggagca agcggcggcg gcggagacag aggcagaggc agaagctggg 50 gctccgtcct cgcctcccac gagcgatccc cgaggagagc cgcggccctc 100 ggcgaggcga agaggccgac gaggaagacc cgggtggctg cgcccctgcc 150 tcgcttccca ggcgccggcg gctgcagcct tgcccctctt gctcgccttg 200 aaaatggaaa agatgctcgc aggctgcttt ctgctgatcc tcggacagat 250 cgtcctcctc cctgccgagg ccagggagcg gtcacgtggg aggtccatct 300 ctaggggcag acacgctcgg acccacccgc agacggccct tctggagagt 350 tcctgtgaga acaagcgggc agacctggtt ttcatcattg acagctctcg 400 cagtgtcaac acccatgact atgcaaaggt caaggagttc atcgtggaca 450 tcttgcaatt cttggacatt ggtcctgatg tcacccgagt gggcctgctc 500 caatatggca gcactgtcaa gaatgagttc tccctcaaga ccttcaagag 550 gaagtccgag gtggagcgtg ctgtcaagag gatgcggcat ctgtccacgg 600 gcaccatgac tgggctggcc atccagtatg ccctgaacat cgcattctca 650 gaagcagagg gggcccggcc cctgagggag aatgtgccac gggtcataat 700 gatcgtgaca gatgggagac ctcaggactc cgtggccgag gtggctgcta 750 aggcacggga cacgggcatc ctaatctttg ccattggtgt gggccaggta 800 gacttcaaca ccttgaagtc cattgggagt gagccccatg aggaccatgt 850 cttccttgtg gccaatttca gccagattga gacgctgacc tccgtgttcc 900 agaagaagtt gtgcacggcc cacatgtgca gcaccctgga gcataactgt 950 gcccacttct gcatcaacat ccctggctca tacgtctgca ggtgcaaaca 1000 aggctacatt ctcaactcgg atcagacgac ttgcagaatc caggatctgt 1050 gtgccatgga ggaccacaac tgtgagcagc tctgtgtgaa tgtgccgggc 1100 tccttcgtct gccagtgcta cagtggctac gccctggctg aggatgggaa 1150 gaggtgtgtg gctgtggact actgtgcctc agaaaaccac ggatgtgaac 1200 atgagtgtgt aaatgctgat ggctcctacc tttgccagtg ccatgaagga 1250 tttgctctta acccagatga aaaaacgtgc acaaggatca actactgtgc 1300 actgaacaaa ccgggctgtg agcatgagtg cgtcaacatg gaggagagct 1350 actactgccg ctgccaccgt ggctacactc tggaccccaa tggcaaaacc 1400 tgcagccgag tggaccactg tgcacagcag gaccatggct gtgagcagct 1450 gtgtctgaac acggaggatt ccttcgtctg ccagtgctca gaaggcttcc 1500 tcatcaacga ggacctcaag acctgctccc gggtggatta ctgcctgctg 1550 agtgaccatg gttgtgaata ctcctgtgtc aacatggaca gatcctttgc 1600 ctgtcagtgt cctgagggac acgtgctccg cagcgatggg aagacgtgtg 1650 caaaattgga ctcttgtgct ctgggggacc acggttgtga acattcgtgt 1700 gtaagcagtg aagattcgtt tgtgtgccag tgctttgaag gttatatact 1750 ccgtgaagat ggaaaaacct gcagaaggaa agatgtctgc caagctatag 1800 accatggctg tgaacacatt tgtgtgaaca gtgacgactc atacacgtgc 1850 gagtgcttgg agggattccg gctcgctgag gatgggaaac gctgccgaag 1900 gaaggatgtc tgcaaatcaa cccaccatgg ctgcgaacac atttgtgtta 1950 ataatgggaa ttcctacatc tgcaaatgct cagagggatt tgttctagct 2000 gaggacggaa gacggtgcaa gaaatgcact gaaggcccaa ttgacctggt 2050 ctttgtgatc gatggatcca agagtcttgg agaagagaat tttgaggtcg 2100 tgaagcagtt tgtcactgga attatagatt ccttgacaat ttcccccaaa 2150 gccgctcgag tggggctgct ccagtattcc acacaggtcc acacagagtt 2200 cactctgaga aacttcaact cagccaaaga catgaaaaaa gccgtggccc 2250 acatgaaata catgggaaag ggctctatga ctgggctggc cctgaaacac 2300 atgtttgaga gaagttttac ccaaggagaa ggggccaggc ccctttccac 2350 aagggtgccc agagcagcca ttgtgttcac cgacggacgg gctcaggatg 2400 acgtctccga gtgggccagt aaagccaagg ccaatggtat cactatgtat 2450 gctgttgggg taggaaaagc cattgaggag gaactacaag agattgcctc 2500 tgagcccaca aacaagcatc tcttctatgc cgaagacttc agcacaatgg 2550 atgagataag tgaaaaactc aagaaaggca tctgtgaagc tctagaagac 2600 tccgatggaa gacaggactc tccagcaggg gaactgccaa aaacggtcca 2650 acagccaaca gaatctgagc cagtcaccat aaatatccaa gacctacttt 2700 cctgttctaa ttttgcagtg caacacagat atctgtttga agaagacaat 2750 cttttacggt ctacacaaaa gctttcccat tcaacaaaac cttcaggaag 2800 ccctttggaa gaaaaacacg atcaatgcaa atgtgaaaac cttataatgt 2850 tccagaacct tgcaaacgaa gaagtaagaa aattaacaca gcgcttagaa 2900 gaaatgacac agagaatgga agccctggaa aatcgcctga gatacagatg 2950 aagattagaa atcgcgacac atttgtagtc attgtatcac ggattacaat 3000 gaacgcagtg cagagcccca aagctcaggc tattgttaaa tcaataatgt 3050 tgtgaagtaa aacaatcagt actgagaaac ctggtttgcc acagaacaaa 3100 gacaagaagt atacactaac ttgtataaat ttatctagga aaaaaatcct 3150 tcagaattct aagatgaatt taccaggtga gaatgaataa gctatgcaag 3200 gtattttgta atatactgtg gacacaactt gcttctgcct catcctgcct 3250 tagtgtgcaa tctcatttga ctatacgata aagtttgcac agtcttactt 3300 ctgtagaaca ctggccatag gaaatgctgt ttttttgtac tggactttac 3350 cttgatatat gtatatggat gtatgcataa aatcatagga catatgtact 3400 tgtggaacaa gttggatttt ttatacaata ttaaaattca ccacttcag 3449 <210> 15 <211> 915 <212> PRT <213> Homo Sapien <400> 15 Met Glu Lys Met Leu Ala Gly Cys Phe Leu Leu Ile Leu Gly Gln 1 5 10 15 Ile Val Leu Leu Pro Ala Glu Ala Arg Glu Arg Ser Arg Gly Arg 20 25 30 Ser Ile Ser Arg Gly Arg His Ala Arg Thr His Pro Gln Thr Ala 35 40 45 Leu Leu Glu Ser Ser Cys Glu Asn Lys Arg Ala Asp Leu Val Phe 50 55 60 Ile Ile Asp Ser Ser Arg Ser Val Asn Thr His Asp Tyr Ala Lys 65 70 75 Val Lys Glu Phe Ile Val Asp Ile Leu Gln Phe Leu Asp Ile Gly 80 85 90 Pro Asp Val Thr Arg Val Gly Leu Leu Gln Tyr Gly Ser Thr Val 95 100 105 Lys Asn Glu Phe Ser Leu Lys Thr Phe Lys Arg Lys Ser Glu Val 110 115 120 Glu Arg Ala Val Lys Arg Met Arg His Leu Ser Thr Gly Thr Met 125 130 135 Thr Gly Leu Ala Ile Gln Tyr Ala Leu Asn Ile Ala Phe Ser Glu 140 145 150 Ala Glu Gly Ala Arg Pro Leu Arg Glu Asn Val Pro Arg Val Ile 155 160 165 Met Ile Val Thr Asp Gly Arg Pro Gln Asp Ser Val Ala Glu Val 170 175 180 Ala Ala Lys Ala Arg Asp Thr Gly Ile Leu Ile Phe Ala Ile Gly 185 190 195 Val Gly Gln Val Asp Phe Asn Thr Leu Lys Ser Ile Gly Ser Glu 200 205 210 Pro His Glu Asp His Val Phe Leu Val Ala Asn Phe Ser Gln Ile 215 220 225 Glu Thr Leu Thr Ser Val Phe Gln Lys Lys Leu Cys Thr Ala His 230 235 240 Met Cys Ser Thr Leu Glu His Asn Cys Ala His Phe Cys Ile Asn 245 250 255 Ile Pro Gly Ser Tyr Val Cys Arg Cys Lys Gln Gly Tyr Ile Leu 260 265 270 Asn Ser Asp Gln Thr Thr Cys Arg Ile Gln Asp Leu Cys Ala Met 275 280 285 Glu Asp His Asn Cys Glu Gln Leu Cys Val Asn Val Pro Gly Ser 290 295 300 Phe Val Cys Gln Cys Tyr Ser Gly Tyr Ala Leu Ala Glu Asp Gly 305 310 315 Lys Arg Cys Val Ala Val Asp Tyr Cys Ala Ser Glu Asn His Gly 320 325 330 Cys Glu His Glu Cys Val Asn Ala Asp Gly Ser Tyr Leu Cys Gln 335 340 345 Cys His Glu Gly Phe Ala Leu Asn Pro Asp Glu Lys Thr Cys Thr 350 355 360 Arg Ile Asn Tyr Cys Ala Leu Asn Lys Pro Gly Cys Glu His Glu 365 370 375 Cys Val Asn Met Glu Glu Ser Tyr Tyr Cys Arg Cys His Arg Gly 380 385 390 Tyr Thr Leu Asp Pro Asn Gly Lys Thr Cys Ser Arg Val Asp His 395 400 405 Cys Ala Gln Gln Asp His Gly Cys Glu Gln Leu Cys Leu Asn Thr 410 415 420 Glu Asp Ser Phe Val Cys Gln Cys Ser Glu Gly Phe Leu Ile Asn 425 430 435 Glu Asp Leu Lys Thr Cys Ser Arg Val Asp Tyr Cys Leu Leu Ser 440 445 450 Asp His Gly Cys Glu Tyr Ser Cys Val Asn Met Asp Arg Ser Phe 455 460 465 Ala Cys Gln Cys Pro Glu Gly His Val Leu Arg Ser Asp Gly Lys 470 475 480 Thr Cys Ala Lys Leu Asp Ser Cys Ala Leu Gly Asp His Gly Cys 485 490 495 Glu His Ser Cys Val Ser Ser Glu Asp Ser Phe Val Cys Gln Cys 500 505 510 Phe Glu Gly Tyr Ile Leu Arg Glu Asp Gly Lys Thr Cys Arg Arg 515 520 525 Lys Asp Val Cys Gln Ala Ile Asp His Gly Cys Glu His Ile Cys 530 535 540 Val Asn Ser Asp Asp Ser Tyr Thr Cys Glu Cys Leu Glu Gly Phe 545 550 555 Arg Leu Ala Glu Asp Gly Lys Arg Cys Arg Arg Lys Asp Val Cys 560 565 570 Lys Ser Thr His His Gly Cys Glu His Ile Cys Val Asn Asn Gly 575 580 585 Asn Ser Tyr Ile Cys Lys Cys Ser Glu Gly Phe Val Leu Ala Glu 590 595 600 Asp Gly Arg Arg Cys Lys Lys Cys Thr Glu Gly Pro Ile Asp Leu 605 610 615 Val Phe Val Ile Asp Gly Ser Lys Ser Leu Gly Glu Glu Asn Phe 620 625 630 Glu Val Val Lys Gln Phe Val Thr Gly Ile Ile Asp Ser Leu Thr 635 640 645 Ile Ser Pro Lys Ala Ala Arg Val Gly Leu Leu Gln Tyr Ser Thr 650 655 660 Gln Val His Thr Glu Phe Thr Leu Arg Asn Phe Asn Ser Ala Lys 665 670 675 Asp Met Lys Lys Ala Val Ala His Met Lys Tyr Met Gly Lys Gly 680 685 690 Ser Met Thr Gly Leu Ala Leu Lys His Met Phe Glu Arg Ser Phe 695 700 705 Thr Gln Gly Glu Gly Ala Arg Pro Leu Ser Thr Arg Val Pro Arg 710 715 720 Ala Ala Ile Val Phe Thr Asp Gly Arg Ala Gln Asp Asp Val Ser 725 730 735 Glu Trp Ala Ser Lys Ala Lys Ala Asn Gly Ile Thr Met Tyr Ala 740 745 750 Val Gly Val Gly Lys Ala Ile Glu Glu Glu Leu Gln Glu Ile Ala 755 760 765 Ser Glu Pro Thr Asn Lys His Leu Phe Tyr Ala Glu Asp Phe Ser 770 775 780 Thr Met Asp Glu Ile Ser Glu Lys Leu Lys Lys Gly Ile Cys Glu 785 790 795 Ala Leu Glu Asp Ser Asp Gly Arg Gln Asp Ser Pro Ala Gly Glu 800 805 810 Leu Pro Lys Thr Val Gln Gln Pro Thr Glu Ser Glu Pro Val Thr 815 820 825 Ile Asn Ile Gln Asp Leu Leu Ser Cys Ser Asn Phe Ala Val Gln 830 835 840 His Arg Tyr Leu Phe Glu Glu Asp Asn Leu Leu Arg Ser Thr Gln 845 850 855 Lys Leu Ser His Ser Thr Lys Pro Ser Gly Ser Pro Leu Glu Glu 860 865 870 Lys His Asp Gln Cys Lys Cys Glu Asn Leu Ile Met Phe Gln Asn 875 880 885 Leu Ala Asn Glu Glu Val Arg Lys Leu Thr Gln Arg Leu Glu Glu 890 895 900 Met Thr Gln Arg Met Glu Ala Leu Glu Asn Arg Leu Arg Tyr Arg 905 910 915 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 16 gtgaccctgg ttgtgaatac tcc 23 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 17 acagccatgg tctatagctt gg 22 <210> 18 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 18 gcctgtcagt gtcctgaggg acacgtgctc cgcagcgatg ggaag 45 <210> 19 <211> 1305 <212> DNA <213> Homo Sapien <400> 19 gcccgggact ggcgcaaggt gcccaagcaa ggaaagaaat aatgaagaga 50 cacatgtgtt agctgcagcc ttttgaaaca cgcaagaagg aaatcaatag 100 tgtggacagg gctggaacct ttaccacgct tgttggagta gatgaggaat 150 gggctcgtga ttatgctgac attccagcat gaatctggta gacctgtggt 200 taacccgttc cctctccatg tgtctcctcc tacaaagttt tgttcttatg 250 atactgtgct ttcattctgc cagtatgtgt cccaagggct gtctttgttc 300 ttcctctggg ggtttaaatg tcacctgtag caatgcaaat ctcaaggaaa 350 tacctagaga tcttcctcct gaaacagtct tactgtatct ggactccaat 400 cagatcacat ctattcccaa tgaaattttt aaggacctcc atcaactgag 450 agttctcaac ctgtccaaaa atggcattga gtttatcgat gagcatgcct 500 tcaaaggagt agctgaaacc ttgcagactc tggacttgtc cgacaatcgg 550 attcaaagtg tgcacaaaaa tgccttcaat aacctgaagg ccagggccag 600 aattgccaac aacccctggc actgcgactg tactctacag caagttctga 650 ggagcatggc gtccaatcat gagacagccc acaacgtgat ctgtaaaacg 700 tccgtgttgg atgaacatgc tggcagacca ttcctcaatg ctgccaacga 750 cgctgacctt tgtaacctcc ctaaaaaaac taccgattat gccatgctgg 800 tcaccatgtt tggctggttc actatggtga tctcatatgt ggtatattat 850 gtgaggcaaa atcaggagga tgcccggaga cacctcgaat acttgaaatc 900 cctgccaagc aggcagaaga aagcagatga acctgatgat attagcactg 950 tggtatagtg tccaaactga ctgtcattga gaaagaaaga aagtagtttg 1000 cgattgcagt agaaataagt ggtttacttc tcccatccat tgtaaacatt 1050 tgaaactttg tatttcagtt ttttttgaat tatgccactg ctgaactttt 1100 aacaaacact acaacataaa taatttgagt ttaggtgatc caccccttaa 1150 ttgtaccccc gatggtatat ttctgagtaa gctactatct gaacattagt 1200 tagatccatc tcactattta ataatgaaat ttattttttt aatttaaaag 1250 caaataaaag cttaactttg aaccatggga aaaaaaaaaa aaaaaaaaaa 1300 aaaca 1305 <210> 20 <211> 259 <212> PRT <213> Homo Sapien <400> 20 Met Asn Leu Val Asp Leu Trp Leu Thr Arg Ser Leu Ser Met Cys 1 5 10 15 Leu Leu Leu Gln Ser Phe Val Leu Met Ile Leu Cys Phe His Ser 20 25 30 Ala Ser Met Cys Pro Lys Gly Cys Leu Cys Ser Ser Ser Gly Gly 35 40 45 Leu Asn Val Thr Cys Ser Asn Ala Asn Leu Lys Glu Ile Pro Arg 50 55 60 Asp Leu Pro Pro Glu Thr Val Leu Leu Tyr Leu Asp Ser Asn Gln 65 70 75 Ile Thr Ser Ile Pro Asn Glu Ile Phe Lys Asp Leu His Gln Leu 80 85 90 Arg Val Leu Asn Leu Ser Lys Asn Gly Ile Glu Phe Ile Asp Glu 95 100 105 His Ala Phe Lys Gly Val Ala Glu Thr Leu Gln Thr Leu Asp Leu 110 115 120 Ser Asp Asn Arg Ile Gln Ser Val His Lys Asn Ala Phe Asn Asn 125 130 135 Leu Lys Ala Arg Ala Arg Ile Ala Asn Asn Pro Trp His Cys Asp 140 145 150 Cys Thr Leu Gln Gln Val Leu Arg Ser Met Ala Ser Asn His Glu 155 160 165 Thr Ala His Asn Val Ile Cys Lys Thr Ser Val Leu Asp Glu His 170 175 180 Ala Gly Arg Pro Phe Leu Asn Ala Ala Asn Asp Ala Asp Leu Cys 185 190 195 Asn Leu Pro Lys Lys Thr Thr Asp Tyr Ala Met Leu Val Thr Met 200 205 210 Phe Gly Trp Phe Thr Met Val Ile Ser Tyr Val Val Tyr Tyr Val 215 220 225 Arg Gln Asn Gln Glu Asp Ala Arg Arg His Leu Glu Tyr Leu Lys 230 235 240 Ser Leu Pro Ser Arg Gln Lys Lys Ala Asp Glu Pro Asp Asp Ile 245 250 255 Ser Thr Val Val 259 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 21 ccatgtgtct cctcctacaa ag 22 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 22 gggaatagat gtgatctgat tgg 23 <210> 23 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 23 cacctgtagc aatgcaaatc tcaaggaaat acctagagat cttcctcctg 50 <210> 24 <211> 1210 <212> DNA <213> Homo Sapien <400> 24 cagcgcgtgg ccggcgccgc tgtggggaca gcatgagcgg cggttggatg 50 gcgcaggttg gagcgtggcg aacaggggct ctgggcctgg cgctgctgct 100 gctgctcggc ctcggactag gcctggaggc cgccgcgagc ccgctttcca 150 ccccgacctc tgcccaggcc gcaggcccca gctcaggctc gtgcccaccc 200 accaagttcc agtgccgcac cagtggctta tgcgtgcccc tcacctggcg 250 ctgcgacagg gacttggact gcagcgatgg cagcgatgag gaggagtgca 300 ggattgagcc atgtacccag aaagggcaat gcccaccgcc ccctggcctc 350 ccctgcccct gcaccggcgt cagtgactgc tctgggggaa ctgacaagaa 400 actgcgcaac tgcagccgcc tggcctgcct agcaggcgag ctccgttgca 450 cgctgagcga tgactgcatt ccactcacgt ggcgctgcga cggccaccca 500 gactgtcccg actccagcga cgagctcggc tgtggaacca atgagatcct 550 cccggaaggg gatgccacaa ccatggggcc ccctgtgacc ctggagagtg 600 tcacctctct caggaatgcc acaaccatgg ggccccctgt gaccctggag 650 agtgtcccct ctgtcgggaa tgccacatcc tcctctgccg gagaccagtc 700 tggaagccca actgcctatg gggttattgc agctgctgcg gtgctcagtg 750 caagcctggt caccgccacc ctcctccttt tgtcctggct ccgagcccag 800 gagcgcctcc gcccactggg gttactggtg gccatgaagg agtccctgct 850 gctgtcagaa cagaagacct cgctgccctg aggacaagca cttgccacca 900 ccgtcactca gccctgggcg tagccggaca ggaggagagc agtgatgcgg 950 atgggtaccc gggcacacca gccctcagag acctgagttc ttctggccac 1000 gtggaacctc gaacccgagc tcctgcagaa gtggccctgg agattgaggg 1050 tccctggaca ctccctatgg agatccgggg agctaggatg gggaacctgc 1100 cacagccaga actgaggggc tggccccagg cagctcccag ggggtagaac 1150 ggccctgtgc ttaagacact ccctgctgcc ccgtctgagg gtggcgatta 1200 aagttgcttc 1210 <210> 25 <211> 282 <212> PRT <213> Homo Sapien <400> 25 Met Ser Gly Gly Trp Met Ala Gln Val Gly Ala Trp Arg Thr Gly 1 5 10 15 Ala Leu Gly Leu Ala Leu Leu Leu Leu Leu Gly Leu Gly Leu Gly 20 25 30 Leu Glu Ala Ala Ala Ser Pro Leu Ser Thr Pro Thr Ser Ala Gln 35 40 45 Ala Ala Gly Pro Ser Ser Gly Ser Cys Pro Pro Thr Lys Phe Gln 50 55 60 Cys Arg Thr Ser Gly Leu Cys Val Pro Leu Thr Trp Arg Cys Asp 65 70 75 Arg Asp Leu Asp Cys Ser Asp Gly Ser Asp Glu Glu Glu Cys Arg 80 85 90 Ile Glu Pro Cys Thr Gln Lys Gly Gln Cys Pro Pro Pro Gly 95 100 105 Leu Pro Cys Pro Cys Thr Gly Val Ser Asp Cys Ser Gly Gly Thr 110 115 120 Asp Lys Lys Leu Arg Asn Cys Ser Arg Leu Ala Cys Leu Ala Gly 125 130 135 Glu Leu Arg Cys Thr Leu Ser Asp Asp Cys Ile Pro Leu Thr Trp 140 145 150 Arg Cys Asp Gly His Pro Asp Cys Pro Asp Ser Ser Asp Glu Leu 155 160 165 Gly Cys Gly Thr Asn Glu Ile Leu Pro Glu Gly Asp Ala Thr Thr 170 175 180 Met Gly Pro Pro Val Thr Leu Glu Ser Val Thr Ser Leu Arg Asn 185 190 195 Ala Thr Thr Met Gly Pro Pro Val Thr Leu Glu Ser Val Pro Ser 200 205 210 Val Gly Asn Ala Thr Ser Ser Ser Ala Gly Asp Gln Ser Gly Ser 215 220 225 Pro Thr Ala Tyr Gly Val Ile Ala Ala Ala Ala Val Leu Ser Ala 230 235 240 Ser Leu Val Thr Ala Thr Leu Leu Leu Leu Ser Trp Leu Arg Ala 245 250 255 Gln Glu Arg Leu Arg Pro Leu Gly Leu Leu Val Ala Met Lys Glu 260 265 270 Ser Leu Leu Leu Ser Glu Gln Lys Thr Ser Leu Pro 275 280 282 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 26 aagttccagt gccgcaccag tggc 24 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 27 ttggttccac agccgagctc gtcg 24 <210> 28 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 28 gaggaggagt gcaggattga gccatgtacc cagaaagggc aatgcccacc 50 <210> 29 <211> 1875 <212> DNA <213> Homo Sapien <400> 29 gacggctggc caccatgcac ggctcctgca gtttcctgat gcttctgctg 50 ccgctactgc tactgctggt ggccaccaca ggccccgttg gagccctcac 100 agatgaggag aaacgtttga tggtggagct gcacaacctc taccgggccc 150 aggtatcccc gacggcctca gacatgctgc acatgagatg ggacgaggag 200 ctggccgcct tcgccaaggc ctacgcacgg cagtgcgtgt ggggccacaa 250 caaggagcgc gggcgccgcg gcgagaatct gttcgccatc acagacgagg 300 gcatggacgt gccgctggcc atggaggagt ggcaccacga gcgtgagcac 350 tacaacctca gcgccgccac ctgcagccca ggccagatgt gcggccacta 400 cacgcaggtg gtatgggcca agacagagag gatcggctgt ggttcccact 450 tctgtgagaa gctccagggt gttgaggaga ccaacatcga attactggtg 500 tgcaactatg agcctccggg gaacgtgaag gggaaacggc cctaccagga 550 ggggactccg tgctcccaat gtccctctgg ctaccactgc aagaactccc 600 tctgtgaacc catcggaagc ccggaagatg ctcaggattt gccttacctg 650 gtaactgagg ccccatcctt ccgggcgact gaagcatcag actctaggaa 700 aatgggtact ccttcttccc tagcaacggg gattccggct ttcttggtaa 750 cagaggtctc aggctccctg gcaaccaagg ctctgcctgc tgtggaaacc 800 caggccccaa cttccttagc aacgaaagac ccgccctcca tggcaacaga 850 ggctccacct tgcgtaacaa ctgaggtccc ttccattttg gcagctcaca 900 gcctgccctc cttggatgag gagccagtta ccttccccaa atcgacccat 950 gttcctatcc caaaatcagc agacaaagtg acagacaaaa caaaagtgcc 1000 ctctaggagc ccagagaact ctctggaccc caagatgtcc ctgacagggg 1050 caagggaact cctaccccat gcccaggagg aggctgaggc tgaggctgag 1100 ttgcctcctt ccagtgaggt cttggcctca gtttttccag cccaggacaa 1150 gccaggtgag ctgcaggcca cactggacca cacggggcac acctcctcca 1200 agtccctgcc caatttcccc aatacctctg ccaccgctaa tgccacgggt 1250 gggcgtgccc tggctctgca gtcgtccttg ccaggtgcag agggccctga 1300 caagcctagc gttgtgtcag ggctgaactc gggccctggt catgtgtggg 1350 gccctctcct gggactactg ctcctgcctc ctctggtgtt ggctggaatc 1400 ttctgaatgg gataccactc aaagggtgaa gaggtcagct gtcctcctgt 1450 catcttcccc accctgtccc cagcccctaa acaagatact tcttggttaa 1500 ggccctccgg aagggaaagg ctacggggca tgtgcctcat cacaccatcc 1550 atcctggagg cacaaggcct ggctggctgc gagctcagga ggccgcctga 1600 ggactgcaca ccgggcccac acctctcctg cccctccctc ctgagtcctg 1650 ggggtgggag gatttgaggg agctcactgc ctacctggcc tggggctgtc 1700 tgcccacaca gcatgtgcgc tctccctgag tgcctgtgta gctggggatg 1750 gggattccta ggggcagatg aaggacaagc cccactggag tggggttctt 1800 tgagtggggg aggcagggac gagggaagga aagtaactcc tgactctcca 1850 ataaaaacct gtccaacctg tgaaa 1875 <210> 30 <211> 463 <212> PRT <213> Homo Sapien <400> 30 Met His Gly Ser Cys Ser Phe Leu Met Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Val Ala Thr Thr Gly Pro Val Gly Ala Leu Thr Asp 20 25 30 Glu Glu Lys Arg Leu Met Val Glu Leu His Asn Leu Tyr Arg Ala 35 40 45 Gln Val Ser Pro Thr Ala Ser Asp Met Leu His Met Arg Trp Asp 50 55 60 Glu Glu Leu Ala Ala Phe Ala Lys Ala Tyr Ala Arg Gln Cys Val 65 70 75 Trp Gly His Asn Lys Glu Arg Gly Arg Arg Gly Glu Asn Leu Phe 80 85 90 Ala Ile Thr Asp Glu Gly Met Asp Val Pro Leu Ala Met Glu Glu 95 100 105 Trp His His Glu Arg Glu His Tyr Asn Leu Ser Ala Ala Thr Cys 110 115 120 Ser Pro Gly Gln Met Cys Gly His Tyr Thr Gln Val Val Trp Ala 125 130 135 Lys Thr Glu Arg Ile Gly Cys Gly Ser His Phe Cys Glu Lys Leu 140 145 150 Gln Gly Val Glu Glu Thr Asn Ile Glu Leu Leu Val Cys Asn Tyr 155 160 165 Glu Pro Pro Gly Asn Val Lys Gly Lys Arg Pro Tyr Gln Glu Gly 170 175 180 Thr Pro Cys Ser Gln Cys Pro Ser Gly Tyr His Cys Lys Asn Ser 185 190 195 Leu Cys Glu Pro Ile Gly Ser Pro Glu Asp Ala Gln Asp Leu Pro 200 205 210 Tyr Leu Val Thr Glu Ala Pro Ser Phe Arg Ala Thr Glu Ala Ser 215 220 225 Asp Ser Arg Lys Met Gly Thr Pro Ser Ser Leu Ala Thr Gly Ile 230 235 240 Pro Ala Phe Leu Val Thr Glu Val Ser Gly Ser Leu Ala Thr Lys 245 250 255 Ala Leu Pro Ala Val Glu Thr Gln Ala Pro Thr Ser Leu Ala Thr 260 265 270 Lys Asp Pro Pro Ser Met Ala Thr Glu Ala Pro Pro Cys Val Thr 275 280 285 Thr Glu Val Pro Ser Ile Leu Ala Ala His Ser Leu Pro Ser Leu 290 295 300 Asp Glu Glu Pro Val Thr Phe Pro Lys Ser Thr His Val Pro Ile 305 310 315 Pro Lys Ser Ala Asp Lys Val Thr Asp Lys Thr Lys Val Pro Ser 320 325 330 Arg Ser Pro Glu Asn Ser Leu Asp Pro Lys Met Ser Leu Thr Gly 335 340 345 Ala Arg Glu Leu Leu Pro His Ala Gln Glu Glu Ala Glu Ala Glu 350 355 360 Ala Glu Leu Pro Pro Ser Ser Glu Val Leu Ala Ser Val Phe Pro 365 370 375 Ala Gln Asp Lys Pro Gly Glu Leu Gln Ala Thr Leu Asp His Thr 380 385 390 Gly His Thr Ser Ser Lys Ser Leu Pro Asn Phe Pro Asn Thr Ser 395 400 405 Ala Thr Ala Asn Ala Thr Gly Gly Arg Ala Leu Ala Leu Gln Ser 410 415 420 Ser Leu Pro Gly Ala Glu Gly Pro Asp Lys Pro Ser Val Val Ser 425 430 435 Gly Leu Asn Ser Gly Pro Gly His Val Trp Gly Pro Leu Leu Gly 440 445 450 Leu Leu Leu Leu Pro Pro Leu Val Leu Ala Gly Ile Phe 455 460 463 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 31 tcctgcagtt tcctgatgc 19 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 32 ctcatattgc acaccagtaa ttcg 24 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 33 atgaggagaa acgtttgatg gtggagctgc acaacctcta ccggg 45 <210> 34 <211> 1857 <212> DNA <213> Homo Sapien <400> 34 gtctgttccc aggagtcctt cggcggctgt tgtgtcagtg gcctgatcgc 50 gatggggaca aaggcgcaag tcgagaggaa actgttgtgc ctcttcatat 100 tggcgatcct gttgtgctcc ctggcattgg gcagtgttac agtgcactct 150 tctgaacctg aagtcagaat tcctgagaat aatcctgtga agttgtcctg 200 tgcctactcg ggcttttctt ctccccgtgt ggagtggaag tttgaccaag 250 gagacaccac cagactcgtt tgctataata acaagatcac agcttcctat 300 gaggaccggg tgaccttctt gccaactggt atcaccttca agtccgtgac 350 acgggaagac actgggacat acacttgtat ggtctctgag gaaggcggca 400 acagctatgg ggaggtcaag gtcaagctca tcgtgcttgt gcctccatcc 450 aagcctacag ttaacatccc ctcctctgcc accattggga accgggcagt 500 gctgacatgc tcagaacaag atggttcccc accttctgaa tacacctggt 550 tcaaagatgg gatagtgatg cctacgaatc ccaaaagcac ccgtgccttc 600 agcaactctt cctatgtcct gaatcccaca acaggagagc tggtctttga 650 tcccctgtca gcctctgata ctggagaata cagctgtgag gcacggaatg 700 ggtatgggac acccatgact tcaaatgctg tgcgcatgga agctgtggag 750 cggaatgtgg gggtcatcgt ggcagccgtc cttgtaaccc tgattctcct 800 gggaatcttg gtttttggca tctggtttgc ctatagccga ggccactttg 850 acagaacaaa gaaagggact tcgagtaaga aggtgattta cagccagcct 900 agtgcccgaa gtgaaggaga attcaaacag acctcgtcat tcctggtgtg 950 agcctggtcg gctcaccgcc tatcatctgc atttgcctta ctcaggtgct 1000 accggactct ggcccctgat gtctgtagtt tcacaggatg ccttatttgt 1050 cttctacacc ccacagggcc ccctacttct tcggatgtgt ttttaataat 1100 gtcagctatg tgccccatcc tccttcatgc cctccctccc tttcctacca 1150 ctgctgagtg gcctggaact tgtttaaagt gtttattccc catttctttg 1200 agggatcagg aaggaatcct gggtatgcca ttgacttccc ttctaagtag 1250 acagcaaaaa tggcgggggt cgcaggaatc tgcactcaac tgcccacctg 1300 gctggcaggg atctttgaat aggtatcttg agcttggttc tgggctcttt 1350 ccttgtgtac tgacgaccag ggccagctgt tctagagcgg gaattagagg 1400 ctagagcggc tgaaatggtt gtttggtgat gacactgggg tccttccatc 1450 tctggggccc actctcttct gtcttcccat gggaagtgcc actgggatcc 1500 ctctgccctg tcctcctgaa tacaagctga ctgacattga ctgtgtctgt 1550 ggaaaatggg agctcttgtt gtggagagca tagtaaattt tcagagaact 1600 tgaagccaaa aggatttaaa accgctgctc taaagaaaag aaaactggag 1650 gctgggcgca gtggctcacg cctgtaatcc cagaggctga ggcaggcgga 1700 tcacctgagg tcgggagttc gggatcagcc tgaccaacat ggagaaaccc 1750 tactggaaat acaaagttag ccaggcatgg tggtgcatgc ctgtagtccc 1800 agctgctcag gagcctggca acaagagcaa aactccagct caaaaaaaaa 1850 aaaaaaa 1857 <210> 35 <211> 299 <212> PRT <213> Homo Sapien <400> 35 Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe 1 5 10 15 Ile Leu Ala Ile Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr 20 25 30 Val His Ser Ser Glu Pro Glu Val Arg Ile Pro Glu Asn Asn Pro 35 40 45 Val Lys Leu Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val 50 55 60 Glu Trp Lys Phe Asp Gln Gly Asp Thr Thr Arg Leu Val Cys Tyr 65 70 75 Asn Asn Lys Ile Thr Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu 80 85 90 Pro Thr Gly Ile Thr Phe Lys Ser Val Thr Arg Glu Asp Thr Gly 95 100 105 Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly 110 115 120 Glu Val Lys Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro 125 130 135 Thr Val Asn Ile Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val 140 145 150 Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr 155 160 165 Trp Phe Lys Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr 170 175 180 Arg Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly 185 190 195 Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr 200 205 210 Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn 215 220 225 Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val 230 235 240 Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu Val Phe 245 250 255 Gly Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp Arg Thr Lys 260 265 270 Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser Ala 275 280 285 Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val 290 295 299 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 36 tcgcggagct gtgttctgtt tccc 24 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 37 acacctggtt caaagatggg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 38 ttgccttact caggtgctac 20 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 39 taggaagagt tgctgaaggc acgg 24 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 40 actcagcagt ggtaggaaag 20 <210> 41 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 41 tgatcgcgat ggggacaaag gcgcaagctc gagaggaaac tgttgtgcct 50 <210> 42 <211> 2236 <212> DNA <213> Homo Sapien <400> 42 ggcgccggtg caccgggcgg gctgagcgcc tcctgcggcc cggcctgcgc 50 gccccggccc gccgcgccgc ccacgcccca accccggccc gcgcccccta 100 gcccccgccc gggcccgcgc ccgcgcccgc gcccaggtga gcgctccgcc 150 cgccgcgagg ccccgccccg gcccgccccc gccccgcccc ggccggcggg 200 ggaaccgggc ggattcctcg cgcgtcaaac cacctgatcc cataaaacat 250 tcatcctccc ggcggcccgc gctgcgagcg ccccgccagt ccgcgccgcc 300 gccgccctcg ccctgtgcgc cctgcgcgcc ctgcgcaccc gcggcccgag 350 cccagccaga gccgggcgga gcggagcgcg ccgagcctcg tcccgcggcc 400 gggccggggc cgggccgtag cggcggcgcc tggatgcgga cccggccgcg 450 gggagacggg cgcccgcccc gaaacgactt tcagtccccg acgcgccccg 500 cccaacccct acgatgaaga gggcgtccgc tggagggagc cggctgctgg 550 catgggtgct gtggctgcag gcctggcagg tggcagcccc atgcccaggt 600 gcctgcgtat gctacaatga gcccaaggtg acgacaagct gcccccagca 650 gggcctgcag gctgtgcccg tgggcatccc tgctgccagc cagcgcatct 700 tcctgcacgg caaccgcatc tcgcatgtgc cagctgccag cttccgtgcc 750 tgccgcaacc tcaccatcct gtggctgcac tcgaatgtgc tggcccgaat 800 tgatgcggct gccttcactg gcctggccct cctggagcag ctggacctca 850 gcgataatgc acagctccgg tctgtggacc ctgccacatt ccacggcctg 900 ggccgcctac acacgctgca cctggaccgc tgcggcctgc aggagctggg 950 cccggggctg ttccgcggcc tggctgccct gcagtacctc tacctgcagg 1000 acaacgcgct gcaggcactg cctgatgaca ccttccgcga cctgggcaac 1050 ctcacacacc tcttcctgca cggcaaccgc atctccagcg tgcccgagcg 1100 cgccttccgt gggctgcaca gcctcgaccg tctcctactg caccagaacc 1150 gcgtggccca tgtgcacccg catgccttcc gtgaccttgg ccgcctcatg 1200 acactctatc tgtttgccaa caatctatca gcgctgccca ctgaggccct 1250 ggcccccctg cgtgccctgc agtacctgag gctcaacgac aacccctggg 1300 tgtgtgactg ccgggcacgc ccactctggg cctggctgca gaagttccgc 1350 ggctcctcct ccgaggtgcc ctgcagcctc ccgcaacgcc tggctggccg 1400 tgacctcaaa cgcctagctg ccaatgacct gcagggctgc gctgtggcca 1450 ccggccctta ccatcccatc tggaccggca gggccaccga tgaggagccg 1500 ctggggcttc ccaagtgctg ccagccagat gccgctgaca aggcctcagt 1550 actggagcct ggaagaccag cttcggcagg caatgcgctg aagggacgcg 1600 tgccgcccgg tgacagcccg ccgggcaacg gctctggccc acggcacatc 1650 aatgactcac cctttgggac tctgcctggc tctgctgagc ccccgctcac 1700 tgcagtgcgg cccgagggct ccgagccacc agggttcccc acctcgggcc 1750 ctcgccggag gccaggctgt tcacgcaaga accgcacccg cagccactgc 1800 cgtctgggcc aggcaggcag cgggggtggc gggactggtg actcagaagg 1850 ctcaggtgcc ctacccagcc tcacctgcag cctcaccccc ctgggcctgg 1900 cgctggtgct gtggacagtg cttgggccct gctgaccccc agcggacaca 1950 agagcgtgct cagcagccag gtgtgtgtac atacggggtc tctctccacg 2000 ccgccaagcc agccgggcgg ccgacccgtg gggcaggcca ggccaggtcc 2050 tccctgatgg acgcctgccg cccgccaccc ccatctccac cccatcatgt 2100 ttacagggtt cggcggcagc gtttgttcca gaacgccgcc tcccacccag 2150 atcgcggtat atagagatat gcattttatt ttacttgtgt aaaaatatcg 2200 gacgacgtgg aataaagagc tcttttctta aaaaaa 2236 <210> 43 <211> 473 <212> PRT <213> Homo Sapien <400> 43 Met Lys Arg Ala Ser Ala Gly Gly Ser Arg Leu Leu Ala Trp Val 1 5 10 15 Leu Trp Leu Gln Ala Trp Gln Val Ala Ala Pro Cys Pro Gly Ala 20 25 30 Cys Val Cys Tyr Asn Glu Pro Lys Val Thr Thr Ser Ser Cys Pro Gln 35 40 45 Gln Gly Leu Gln Ala Val Pro Val Gly Ile Pro Ala Ala Ser Gln 50 55 60 Arg Ile Phe Leu His Gly Asn Arg Ile Ser His Val Pro Ala Ala 65 70 75 Ser Phe Arg Ala Cys Arg Asn Leu Thr Ile Leu Trp Leu His Ser 80 85 90 Asn Val Leu Ala Arg Ile Asp Ala Ala Ala Phe Thr Gly Leu Ala 95 100 105 Leu Leu Glu Gln Leu Asp Leu Ser Asp Asn Ala Gln Leu Arg Ser 110 115 120 Val Asp Pro Ala Thr Phe His Gly Leu Gly Arg Leu His Thr Leu 125 130 135 His Leu Asp Arg Cys Gly Leu Gln Glu Leu Gly Pro Gly Leu Phe 140 145 150 Arg Gly Leu Ala Ala Leu Gln Tyr Leu Tyr Leu Gln Asp Asn Ala 155 160 165 Leu Gln Ala Leu Pro Asp Asp Thr Phe Arg Asp Leu Gly Asn Leu 170 175 180 Thr His Leu Phe Leu His Gly Asn Arg Ile Ser Ser Val Pro Glu 185 190 195 Arg Ala Phe Arg Gly Leu His Ser Leu Asp Arg Leu Leu Leu His 200 205 210 Gln Asn Arg Val Ala His Val His Pro His Ala Phe Arg Asp Leu 215 220 225 Gly Arg Leu Met Thr Leu Tyr Leu Phe Ala Asn Asn Leu Ser Ala 230 235 240 Leu Pro Thr Glu Ala Leu Ala Pro Leu Arg Ala Leu Gln Tyr Leu 245 250 255 Arg Leu Asn Asp Asn Pro Trp Val Cys Asp Cys Arg Ala Arg Pro 260 265 270 Leu Trp Ala Trp Leu Gln Lys Phe Arg Gly Ser Ser Ser Glu Val 275 280 285 Pro Cys Ser Leu Pro Gln Arg Leu Ala Gly Arg Asp Leu Lys Arg 290 295 300 Leu Ala Ala Asn Asp Leu Gln Gly Cys Ala Val Ala Thr Gly Pro 305 310 315 Tyr His Pro Ile Trp Thr Gly Arg Ala Thr Asp Glu Glu Pro Leu 320 325 330 Gly Leu Pro Lys Cys Cys Gln Pro Asp Ala Ala Asp Lys Ala Ser 335 340 345 Val Leu Glu Pro Gly Arg Pro Ala Ser Ala Gly Asn Ala Leu Lys 350 355 360 Gly Arg Val Pro Pro Gly Asp Ser Pro Pro Gly Asn Gly Ser Gly 365 370 375 Pro Arg His Ile Asn Asp Ser Pro Phe Gly Thr Leu Pro Gly Ser 380 385 390 Ala Glu Pro Pro Leu Thr Ala Val Arg Pro Glu Gly Ser Glu Pro 395 400 405 Pro Gly Phe Pro Thr Ser Gly Pro Arg Arg Arg Pro Gly Cys Ser 410 415 420 Arg Lys Asn Arg Thr Arg Ser His Cys Arg Leu Gly Gln Ala Gly 425 430 435 Ser Gly Gly Gly Gly Thr Gly Asp Ser Glu Gly Ser Gly Ala Leu 440 445 450 Pro Ser Leu Thr Cys Ser Leu Thr Pro Leu Gly Leu Ala Leu Val 455 460 465 Leu Trp Thr Val Leu Gly Pro Cys 470 473 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 44 tggctgccct gcagtacctc tacc 24 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 45 ccctgcaggt cattggcagc tagg 24 <210> 46 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 46 aggcactgcc tgatgacacc ttccgcgacc tgggcaacct cacac 45 <210> 47 <211> 2181 <212> DNA <213> Homo Sapien <400> 47 cccacgcgtc cgcccacgcg tccgcccacg ggtccgccca cgcgtccggg 50 ccaccagaag tttgagcctc tttggtagca ggaggctgga agaaaggaca 100 gaagtagctc tggctgtgat ggggatctta ctgggcctgc tactcctggg 150 gcacctaaca gtggacactt atggccgtcc catcctggaa gtgccagaga 200 gtgtaacagg accttggaaa ggggatgtga atcttccctg cacctatgac 250 cccctgcaag gctacaccca agtcttggtg aagtggctgg tacaacgtgg 300 ctcagaccct gtcaccatct ttctacgtga ctcttctgga gaccatatcc 350 agcaggcaaa gtaccagggc cgcctgcatg tgagccacaa ggttccagga 400 gatgtatccc tccaattgag caccctggag atggatgacc ggagccacta 450 cacgtgtgaa gtcacctggc agactcctga tggcaaccaa gtcgtgagag 500 ataagattac tgagctccgt gtccagaaac tctctgtctc caagcccaca 550 gtgacaactg gcagcggtta tggcttcacg gtgccccagg gaatgaggat 600 tagccttcaa tgccaggctc ggggttctcc tcccatcagt tatatttggt 650 ataagcaaca gactaataac caggaaccca tcaaagtagc aaccctaagt 700 accttactct tcaagcctgc ggtgatagcc gactcaggct cctatttctg 750 cactgccaag ggccaggttg gctctgagca gcacagcgac attgtgaagt 800 ttgtggtcaa agactcctca aagctactca agaccaagac tgaggcacct 850 acaaccatga catacccctt gaaagcaaca tctacagtga agcagtcctg 900 ggactggacc actgacatgg atggctacct tggagagacc agtgctgggc 950 caggaaagag cctgcctgtc tttgccatca tcctcatcat ctccttgtgc 1000 tgtatggtgg tttttaccat ggcctatatc atgctctgtc ggaagacatc 1050 ccaacaagag catgtctacg aagcagccag gtaagaaagt ctctcctctt 1100 ccatttttga ccccgtccct gccctcaatt ttgattactg gcaggaaatg 1150 tggaggaagg ggggtgtggc acagacccaa tcctaaggcc ggaggccttc 1200 agggtcagga catagctgcc ttccctctct caggcacctt ctgaggttgt 1250 tttggccctc tgaacacaaa ggataattta gatccatctg ccttctgctt 1300 ccagaatccc tgggtggtag gatcctgata attaattggc aagaattgag 1350 gcagaagggt gggaaaccag gaccacagcc ccaagtccct tcttatgggt 1400 ggtgggctct tgggccatag ggcacatgcc agagaggcca acgactctgg 1450 agaaaccatg agggtggcca tcttcgcaag tggctgctcc agtgatgagc 1500 caacttccca gaatctgggc aacaactact ctgatgagcc ctgcatagga 1550 caggagtacc agatcatcgc ccagatcaat ggcaactacg cccgcctgct 1600 ggacacagtt cctctggatt atgagtttct ggccactgag ggcaaaagtg 1650 tctgttaaaa atgccccatt aggccaggat ctgctgacat aattgcctag 1700 tcagtccttg ccttctgcat ggccttcttc cctgctacct ctcttcctgg 1750 atagcccaaa gtgtccgcct accaacactg gagccgctgg gagtcactgg 1800 ctttgccctg gaatttgcca gatgcatctc aagtaagcca gctgctggat 1850 ttggctctgg gcccttctag tatctctgcc gggggcttct ggtactcctc 1900 tctaaatacc agagggaaga tgcccatagc actaggactt ggtcatcatg 1950 cctacagaca ctattcaact ttggcatctt gccaccagaa gacccgaggg 2000 aggctcagct ctgccagctc agaggaccag ctatatccag gatcatttct 2050 ctttcttcag ggccagacag cttttaattg aaattgttat ttcacaggcc 2100 agggttcagt tctgctcctc cactataagt ctaatgttct gactctctcc 2150 tggtgctcaa taaatatcta atcataacag c 2181 <210> 48 <211> 321 <212> PRT <213> Homo Sapien <400> 48 Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val 1 5 10 15 Asp Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr 20 25 30 Gly Pro Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro 35 40 45 Leu Gln Gly Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg 50 55 60 Gly Ser Asp Pro Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp 65 70 75 His Ile Gln Gln Ala Lys Tyr Gln Gly Arg Leu His Val Ser His 80 85 90 Lys Val Pro Gly Asp Val Ser Leu Gln Leu Ser Thr Leu Glu Met 95 100 105 Asp Asp Arg Ser His Tyr Thr Cys Glu Val Thr Trp Gln Thr Pro 110 115 120 Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr Glu Leu Arg Val 125 130 135 Gln Lys Leu Ser Val Ser Lys Pro Thr Val Thr Thr Gly Ser Gly 140 145 150 Tyr Gly Phe Thr Val Pro Gln Gly Met Arg Ile Ser Leu Gln Cys 155 160 165 Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile Trp Tyr Lys Gln 170 175 180 Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr Leu Ser Thr 185 190 195 Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser Tyr Phe 200 205 210 Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp Ile 215 220 225 Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys 230 235 240 Thr Glu Ala Pro Thr Thr Met Met Tyr Pro Leu Lys Ala Thr Ser 245 250 255 Thr Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr 260 265 270 Leu Gly Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe 275 280 285 Ala Ile Ile Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr 290 295 300 Met Ala Tyr Ile Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His 305 310 315 Val Tyr Glu Ala Ala Arg 320 321 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 49 tatccctcca attgagcacc ctgg 24 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 50 gtcggaagac atcccaacaa g 21 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 51 cttcacaatg tcgctgtgct gctc 24 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 52 agccaaatcc agcagctggc ttac 24 <210> 53 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 53 tggatgaccg gagccactac acgtgtgaag tcacctggca gactcctgat 50 <210> 54 <211> 2212 <212> DNA <213> Homo Sapien <400> 54 gaaagctata ggctacccat tcagctcccc tgtcagagac tcaagctttg 50 agaaaggcta gcaaagagca aggaaagaga gaaaacaaca aagtggcgag 100 gccctcagag tgaaagcgta aggttcagtc agcctgctgc agctttgcag 150 acctcagctg ggcatctcca gactcccctg aaggaagagc cttcctcacc 200 caaacccaca aaagatgctg aaaaagcctc tctcagctgt gacctggctc 250 tgcattttca tcgtggcctt tgtcagccac ccagcgtggc tgcagaagct 300 ctctaagcac aagacaccag cacagccaca gctcaaagcg gccaactgct 350 gtgaggaggt gaaggagctc aaggcccaag ttgccaacct tagcagcctg 400 ctgagtgaac tgaacaagaa gcaggagagg gactgggtca gcgtggtcat 450 gcaggtgatg gagctggaga gcaacagcaa gcgcatggag tcgcggctca 500 cagatgctga gagcaagtac tccgagatga acaaccaaat tgacatcatg 550 cagctgcagg cagcacagac ggtcactcag acctccgcag atgccatcta 600 cgactgctct tccctctacc agaagaacta ccgcatctct ggagtgtata 650 agcttcctcc tgatgacttc ctgggcagcc ctgaactgga ggtgttctgt 700 gacatggaga cttcaggcgg aggctggacc atcatccaga gacgaaaaag 750 tggccttgtc tccttctacc gggactggaa gcagtacaag cagggctttg 800 gcagcatccg tggggacttc tggctgggga acgaacacat ccaccggctc 850 tccagacagc caacccggct gcgtgtagag atggaggact gggagggcaa 900 cctgcgctac gctgagtata gccactttgt tttgggcaat gaactcaaca 950 gctatcgcct cttcctgggg aactacactg gcaatgtggg gaacgacgcc 1000 ctccagtatc ataacaacac agccttcagc accaaggaca aggacaatga 1050 caactgcttg gacaagtgtg cacagctccg caaaggtggc tactggtaca 1100 actgctgcac agactccaac ctcaatggag tgtactaccg cctgggtgag 1150 cacaataagc acctggatgg catcacctgg tatggctggc atggatctac 1200 ctactccctc aaacgggtgg agatgaaaat ccgcccagaa gacttcaagc 1250 cttaaaagga ggctgccgtg gagcacggat acagaaactg agacacgtgg 1300 agactggatg agggcagatg aggacaggaa gagagtgtta gaaagggtag 1350 gactgagaaa cagcctataa tctccaaaga aagaataagt ctccaaggag 1400 cacaaaaaaa tcatatgtac caaggatgtt acagtaaaca ggatgaacta 1450 tttaaaccca ctgggtcctg ccacatcctt ctcaaggtgg tagactgagt 1500 ggggtctctc tgcccaagat ccctgacata gcagtagctt gtcttttcca 1550 catgatttgt ctgtgaaaga aaataatttt gagatcgttt tatctatttt 1600 ctctacggct taggctatgt gagggcaaaa cacaaatccc tttgctaaaa 1650 agaaccatat tattttgatt ctcaaaggat aggcctttga gtgttagaga 1700 aaggagtgaa ggaggcaggt gggaaatggt atttctattt ttaaatccag 1750 tgaaattatc ttgagtctac acattatttt taaaacacaa aaattgttcg 1800 gctggaactg acccaggctg gacttgcggg gaggaaactc cagggcactg 1850 catctggcga tcagactctg agcactgccc ctgctcgcct tggtcatgta 1900 cagcactgaa aggaatgaag caccagcagg aggtggacag agtctctcat 1950 ggatgccggc acaaaactgc cttaaaatat tcatagttaa tacaggtata 2000 tctattttta tttactttgt aagaaacaag ctcaaggagc ttccttttaa 2050 attttgtctg taggaaatgg ttgaaaactg aaggtagatg gtgttatagt 2100 taataataaa tgctgtaaat aagcatctca ctttgtaaaa ataaaatatt 2150 gtggttttgt tttaaacatt caacgtttct tttccttcta caataaacac 2200 tttcaaaatg tg 2212 <210> 55 <211> 346 <212> PRT <213> Homo Sapien <400> 55 Met Leu Lys Lys Pro Leu Ser Ala Val Thr Trp Leu Cys Ile Phe 1 5 10 15 Ile Val Ala Phe Val Ser His Pro Ala Trp Leu Gln Lys Leu Ser 20 25 30 Lys His Lys Thr Pro Ala Gln Pro Gln Leu Lys Ala Ala Asn Cys 35 40 45 Cys Glu Glu Val Lys Glu Leu Lys Ala Gln Val Ala Asn Leu Ser 50 55 60 Ser Leu Leu Ser Glu Leu Asn Lys Lys Gln Glu Arg Asp Trp Val 65 70 75 Ser Val Val Met Gln Val Met Glu Leu Glu Ser Asn Ser Lys Arg 80 85 90 Met Glu Ser Arg Leu Thr Asp Ala Glu Ser Lys Tyr Ser Glu Met 95 100 105 Asn Asn Gln Ile Asp Ile Met Gln Leu Gln Ala Ala Gln Thr Val 110 115 120 Thr Gln Thr Ser Ala Asp Ala Ile Tyr Asp Cys Ser Ser Leu Tyr 125 130 135 Gln Lys Asn Tyr Arg Ile Ser Gly Val Tyr Lys Leu Pro Pro Asp 140 145 150 Asp Phe Leu Gly Ser Pro Glu Leu Glu Val Phe Cys Asp Met Glu 155 160 165 Thr Ser Gly Gly Gly Trp Thr Ile Ile Gln Arg Arg Lys Ser Gly 170 175 180 Leu Val Ser Phe Tyr Arg Asp Trp Lys Gln Tyr Lys Gln Gly Phe 185 190 195 Gly Ser Ile Arg Gly Asp Phe Trp Leu Gly Asn Glu His Ile His 200 205 210 Arg Leu Ser Arg Gln Pro Thr Arg Leu Arg Val Glu Met Glu Asp 215 220 225 Trp Glu Gly Asn Leu Arg Tyr Ala Glu Tyr Ser His Phe Val Leu 230 235 240 Gly Asn Glu Leu Asn Ser Tyr Arg Leu Phe Leu Gly Asn Tyr Thr 245 250 255 Gly Asn Val Gly Asn Asp Ala Leu Gln Tyr His Asn Asn Thr Ala 260 265 270 Phe Ser Thr Lys Asp Lys Asp Asn Asp Asn Cys Leu Asp Lys Cys 275 280 285 Ala Gln Leu Arg Lys Gly Gly Tyr Trp Tyr Asn Cys Cys Thr Asp 290 295 300 Ser Asn Leu Asn Gly Val Tyr Tyr Arg Leu Gly Glu His Asn Lys 305 310 315 His Leu Asp Gly Ile Thr Trp Tyr Gly Trp His Gly Ser Thr Tyr 320 325 330 Ser Leu Lys Arg Val Glu Met Lys Ile Arg Pro Glu Asp Phe Lys 335 340 345 Pro 346 <210> 56 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 56 ttcagcacca aggacaagga caatgacaac t 31 <210> 57 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 57 tgtgcacact tgtccaagca gttgtcattg tc 32 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 58 gtagtacact ccattgaggt tgg 23 <210> 59 <211> 1049 <212> DNA <213> Homo Sapien <400> 59 gccgcagcaa tggcgctgag ttcctctgct ggagttcatc ctgctagctg 50 ggttcccgag ctgccggtct gagcctgagg catggagcct cctggagact 100 gggggcctcc tccctggaga tccaccccca gaaccgacgt cttgaggctg 150 gtgctgtatc tcaccttcct gggagccccc tgctacgccc cagctctgcc 200 gtcctgcaag gaggacgagt acccagtggg ctccgagtgc tgccccaagt 250 gcagtccagg ttatcgtgtg aaggaggcct gcggggagct gacgggcaca 300 gtgtgtgaac cctgccctcc aggcacctac attgcccacc tcaatggcct 350 aagcaagtgt ctgcagtgcc aaatgtgtga cccagccatg ggcctgcgcg 400 cgagccggaa ctgctccagg acagagaacg ccgtgtgtgg ctgcagccca 450 ggccacttct gcatcgtcca ggacggggac cactgcgccg cgtgccgcgc 500 ttacgccacc tccagcccgg gccagagggt gcagaaggga ggcaccgaga 550 gtcaggacac cctgtgtcag aactgccccc cggggacctt ctctcccaat 600 gggaccctgg aggaatgtca gcaccagacc aagtgcagct ggctggtgac 650 gaaggccgga gctgggacca gcagctccca ctgggtatgg tggtttctct 700 cagggagcct cgtcatcgtc attgtttgct ccacagttgg cctaatcata 750 tgtgtgaaaa gaagaaagcc aaggggtgat gtagtcaagg tgatcgtctc 800 cgtccagcgg aaaagacagg aggcagaagg tgaggccaca gtcattgagg 850 ccctgcaggc ccctccggac gtcaccacgg tggccgtgga ggagacaata 900 ccctcattca cggggaggag cccaaaccac tgacccacag actctgcacc 950 ccgacgccag agatacctgg agcgacggct gctgaaagag gctgtccacc 1000 tggcgaaacc accggagccc ggaggcttgg gggctccgcc ctgggctgg 1049 <210> 60 <211> 283 <212> PRT <213> Homo Sapien <400> 60 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr 1 5 10 15 Pro Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu 20 25 30 Gly Ala Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp 35 40 45 Glu Tyr Pro Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly 50 55 60 Tyr Arg Val Lys Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys 65 70 75 Glu Pro Cys Pro Pro Gly Thr Tyr Ile Ala His Leu Asn Gly Leu 80 85 90 Ser Lys Cys Leu Gln Cys Gln Met Cys Asp Pro Ala Met Gly Leu 95 100 105 Arg Ala Ser Arg Asn Cys Ser Arg Thr Glu Asn Ala Val Cys Gly 110 115 120 Cys Ser Pro Gly His Phe Cys Ile Val Gln Asp Gly Asp His Cys 125 130 135 Ala Ala Cys Arg Ala Tyr Ala Thr Ser Ser Pro Gly Gln Arg Val 140 145 150 Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr Leu Cys Gln Asn Cys 155 160 165 Pro Pro Gly Thr Phe Ser Pro Asn Gly Thr Leu Glu Glu Cys Gln 170 175 180 His Gln Thr Lys Cys Ser Trp Leu Val Thr Lys Ala Gly Ala Gly 185 190 195 Thr Ser Ser Ser His Trp Val Trp Trp Phe Leu Ser Gly Ser Leu 200 205 210 Val Ile Val Ile Val Cys Ser Thr Val Gly Leu Ile Ile Cys Val 215 220 225 Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val Ser 230 235 240 Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile 245 250 255 Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu 260 265 270 Glu Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His 275 280 283 <210> 61 <211> 1847 <212> DNA <213> Homo Sapien <400> 61 gccaggggaa gagggtgatc cgacccgggg aaggtcgctg ggcagggcga 50 gttgggaaag cggcagcccc cgccgccccc gcagcccctt ctcctccttt 100 ctcccacgtc ctatctgcct ctcgctggag gccaggccgt gcagcatcga 150 agacaggagg aactggagcc tcattggccg gcccggggcg ccggcctcgg 200 gcttaaatag gagctccggg ctctggctgg gacccgaccg ctgccggccg 250 cgctcccgct gctcctgccg ggtgatggaa aaccccagcc cggccgccgc 300 cctgggcaag gccctctgcg ctctcctcct ggccactctc ggcgccgccg 350 gccagcctct tgggggagag tccatctgtt ccgccagagc cccggccaaa 400 tacagcatca ccttcacggg caagtggagc cagacggcct tccccaagca 450 gtaccccctg ttccgccccc ctgcgcagtg gtcttcgctg ctgggggccg 500 cgcatagctc cgactacagc atgtggagga agaaccagta cgtcagtaac 550 gggctgcgcg actttgcgga gcgcggcgag gcctgggcgc tgatgaagga 600 gatcgaggcg gcgggggagg cgctgcagag cgtgcacgag gtgttttcgg 650 cgcccgccgt ccccagcggc accgggcaga cgtcggcgga gctggaggtg 700 cagcgcaggc actcgctggt ctcgtttgtg gtgcgcatcg tgcccagccc 750 cgactggttc gtgggcgtgg acagcctgga cctgtgcgac ggggaccgtt 800 ggcgggaaca ggcggcgctg gacctgtacc cctacgacgc cgggacggac 850 agcggcttca ccttctcctc ccccaacttc gccaccatcc cgcaggacac 900 ggtgaccgag ataacgtcct cctctcccag ccacccggcc aactccttct 950 actacccgcg gctgaaggcc ctgcctccca tcgccagggt gacactgctg 1000 cggctgcgac agagccccag ggccttcatc cctcccgccc cagtcctgcc 1050 cagcagggac aatgagattg tagacagcgc ctcagttcca gaaacgccgc 1100 tggactgcga ggtctccctg tggtcgtcct ggggactgtg cggaggccac 1150 tgtgggaggc tcgggaccaa gagcaggact cgctacgtcc gggtccagcc 1200 cgccaacaac gggagcccct gccccgagct cgaagaagag gctgagtgcg 1250 tccctgataa ctgcgtctaa gaccagagcc ccgcagcccc tggggccccc 1300 cggagccatg gggtgtcggg ggctcctgtg caggctcatg ctgcaggcgg 1350 ccgagggcac agggggtttc gcgctgctcc tgaccgcggt gaggccgcgc 1400 cgaccatctc tgcactgaag ggccctctgg tggccggcac gggcattggg 1450 aaacagcctc ctcctttccc aaccttgctt cttaggggcc cccgtgtccc 1500 gtctgctctc agcctcctcc tcctgcagga taaagtcatc cccaaggctc 1550 cagctactct aaattatgtc tccttataag ttattgctgc tccaggagat 1600 tgtccttcat cgtccagggg cctggctccc acgtggttgc agatacctca 1650 gacctggtgc tctaggctgt gctgagccca ctctcccgag ggcgcatcca 1700 agcgggggcc acttgagaag tgaataaatg gggcggtttc ggaagcgtca 1750 gtgtttccat gttatggatc tctctgcgtt tgaataaaga ctatctctgt 1800 tgctcacaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1847 <210> 62 <211> 331 <212> PRT <213> Homo Sapien <400> 62 Met Glu Asn Pro Ser Pro Ala Ala Ala Leu Gly Lys Ala Leu Cys 1 5 10 15 Ala Leu Leu Leu Ala Thr Leu Gly Ala Ala Gly Gln Pro Leu Gly 20 25 30 Gly Glu Ser Ile Cys Ser Ala Arg Ala Pro Ala Lys Tyr Ser Ile 35 40 45 Thr Phe Thr Gly Lys Trp Ser Gln Thr Ala Phe Pro Lys Gln Tyr 50 55 60 Pro Leu Phe Arg Pro Pro Ala Gln Trp Ser Ser Leu Leu Gly Ala 65 70 75 Ala His Ser Ser Asp Tyr Ser Met Trp Arg Lys Asn Gln Tyr Val 80 85 90 Ser Asn Gly Leu Arg Asp Phe Ala Glu Arg Gly Glu Ala Trp Ala 95 100 105 Leu Met Lys Glu Ile Glu Ala Ala Gly Glu Ala Leu Gln Ser Val 110 115 120 His Glu Val Phe Ser Ala Pro Ala Val Pro Ser Gly Thr Gly Gln 125 130 135 Thr Ser Ala Glu Leu Glu Val Gln Arg Arg His Ser Leu Val Ser 140 145 150 Phe Val Val Arg Ile Val Pro Ser Pro Asp Trp Phe Val Gly Val 155 160 165 Asp Ser Leu Asp Leu Cys Asp Gly Asp Arg Trp Arg Glu Gln Ala 170 175 180 Ala Leu Asp Leu Tyr Pro Tyr Asp Ala Gly Thr Asp Ser Gly Phe 185 190 195 Thr Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro Gln Asp Thr Val 200 205 210 Thr Glu Ile Thr Ser Ser Ser Pro Ser His Pro Ala Asn Ser Phe 215 220 225 Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro Ile Ala Arg Val Thr 230 235 240 Leu Leu Arg Leu Arg Gln Ser Pro Arg Ala Phe Ile Pro Pro Ala 245 250 255 Pro Val Leu Pro Ser Arg Asp Asn Glu Ile Val Asp Ser Ala Ser 260 265 270 Val Pro Glu Thr Pro Leu Asp Cys Glu Val Ser Leu Trp Ser Ser 275 280 285 Trp Gly Leu Cys Gly Gly His Cys Gly Arg Leu Gly Thr Lys Ser 290 295 300 Arg Thr Arg Tyr Val Arg Val Gln Pro Ala Asn Asn Gly Ser Pro 305 310 315 Cys Pro Glu Leu Glu Glu Glu Ala Glu Cys Val Pro Asp Asn Cys 320 325 330 Val 331 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 63 cagcactgcc aggggaagag gg 22 <210> 64 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 64 caggactcgc tacgtccg 18 <210> 65 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 65 cagccccttc tcctcctttc tccc 24 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 66 gcagttatca gggacgcact cagcc 25 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 67 ccagcgagag gcagatag 18 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 68 cggtcaccgt gtcctgcggg atg 23 <210> 69 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 69 cagccccttc tcctcctttc tcccacgtcc tatctgcctc tc 42 <210> 70 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 70 ggattctaat acgactcact atagggctcc tgcgcctttc ctgaacc 47 <210> 71 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 71 ctatgaaatt aaccctcact aaagggagac ccatccttgc ccacagag 48 <210> 72 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 72 ggattctaat acgactcact atagggctgt gctttcattc tgccagta 48 <210> 73 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 73 ctatgaaatt aaccctcact aaagggaggg tacaattaag gggtggat 48 <210> 74 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 74 ggattctaat acgactcact atagggcgca gcgatggcag cgatgagg 48 <210> 75 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 75 ctatgaaatt aaccctcact aaagggacag acggggcagc agggagtg 48 <210> 76 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 76 ggattctaat acgactcact atagggcgag tccttcggcg gctgtt 46 <210> 77 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 77 ctatgaaatt aaccctcact aaagggacgg gtgcttttgg gattcgta 48 <210> 78 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 78 ggattctaat acgactcact atagggcctc caagcccaca gtgacaa 47 <210> 79 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 79 ctatgaaatt aaccctcact aaagggacct ccacatttcc tgccagta 48 12

Claims (41)

Effective amounts of PRO179 (SEQ ID NO: 2), PRO207 (SEQ ID NO: 7), PRO320 (SEQ ID NO: 10), PRO219 (SEQ ID NO: 15), PRO221 (SEQ ID NO: 20), PRO224 (SEQ ID NO: 25), PRO328 (SEQ ID NO: 30), PRO301 (SEQ ID NO: 35), A composition useful for the inhibition of neoplastic cell growth comprising a PRO526 (SEQ ID NO: 43), PRO362 (SEQ ID NO: 48), PRO356 (SEQ ID NO: 55), PRO509 (SEQ ID NO: 60), or PRO866 (SEQ ID NO: 62) polypeptides with a pharmaceutically acceptable carrier. The method of claim 1, wherein the growth inhibition of PRO179 (SEQ ID NO: 2), PRO207 (SEQ ID NO: 7), PRO320 (SEQ ID NO: 10), PRO219 (SEQ ID NO: 15), PRO221 (SEQ ID NO: 20), PRO224 (SEQ ID NO: 25), PRO328 (SEQ ID NO: 30) ), PRO301 (SEQ ID NO: 35), PRO526 (SEQ ID NO: 43), PRO362 (SEQ ID NO: 48), PRO356 (SEQ ID NO: 55), PRO509 (SEQ ID NO: 60), or PRO866 (SEQ ID NO: 62) polypeptides. The cytotoxic amount of PRO179 (SEQ ID NO: 2), PRO207 (SEQ ID NO: 7), PRO320 (SEQ ID NO: 10), PRO219 (SEQ ID NO: 15), PRO221 (SEQ ID NO: 20), PRO224 (SEQ ID NO: 25), PRO328 (SEQ ID NO: 30). ), PRO301 (SEQ ID NO: 35), PRO526 (SEQ ID NO: 43), PRO362 (SEQ ID NO: 48), PRO356 (SEQ ID NO: 55), PRO509 (SEQ ID NO: 60), or PRO866 (SEQ ID NO: 62) polypeptides. The composition of claim 1, further comprising a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent. Therapeutic effective amount of PRO179 (SEQ ID NO: 2), PRO207 (SEQ ID NO: 7), PRO320 (SEQ ID NO: 10), PRO219 (SEQ ID NO: 15), PRO221 (SEQ ID NO: 20), PRO224 (SEQ ID NO: 25), PRO328 (SEQ ID NO: 30), PRO301 (SEQ ID NO: 35) A composition useful for treating tumors in a mammal, comprising a PRO526 (SEQ ID NO: 43), PRO362 (SEQ ID NO: 48), PRO356 (SEQ ID NO: 55), PRO509 (SEQ ID NO: 60), or PRO866 (SEQ ID NO: 62) polypeptides. The composition of claim 5, wherein the tumor is cancer. The composition of claim 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, kidney cancer, colorectal cancer, uterine cancer, prostate cancer, lung cancer, bladder cancer, central nervous system cancer, melanoma and leukemia. Effective amounts of PRO179 (SEQ ID NO: 2), PRO207 (SEQ ID NO: 7), PRO320 (SEQ ID NO: 10), PRO219 (SEQ ID NO: 15), PRO221 (SEQ ID NO: 20), PRO224 (SEQ ID NO: 25), PRO328 (SEQ ID NO: 30), PRO301 (SEQ ID NO: 35), Said tumor cells in a mammal, except humans, comprising exposing the PRO526 (SEQ ID NO: 43), PRO362 (SEQ ID NO: 48), PRO356 (SEQ ID NO: 55), PRO509 (SEQ ID NO: 60), or PRO866 (SEQ ID NO: 62) polypeptides to the tumor cells; To inhibit growth. delete delete The method of claim 8, wherein said exposing step is performed in vitro. The method of claim 8, wherein said exposing step is performed in vivo. (a) containers and (b) In the vessel, PRO179 (SEQ ID NO: 2), PRO207 (SEQ ID NO: 7), PRO320 (SEQ ID NO: 10), PRO219 (SEQ ID NO: 15), PRO221 (SEQ ID NO: 20), PRO224 (SEQ ID NO: 25), PRO328 (SEQ ID NO: 30), PRO301 ( A composition useful for inhibiting neoplastic cell growth, comprising as an active substance SEQ ID NO: 35), PRO526 (SEQ ID NO: 43), PRO362 (SEQ ID NO: 48), PRO356 (SEQ ID NO: 55), PRO509 (SEQ ID NO: 60), or PRO866 (SEQ ID NO: 62) polypeptides Product containing. The product of claim 13, further comprising a label attached to the container, or a package insert contained within the container, to refer to the use of the composition for inhibiting neoplastic growth. delete delete The product of claim 13, wherein the active agent is present in a therapeutically effective amount in a mammal. The article of claim 13, wherein said composition further comprises a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent. Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 20), Figure 12 (SEQ ID NO: 25), Figure 14 (SEQ ID NO: 30), and FIG. 16 (SEQ ID NO: 35), 18 (SEQ ID NO: 43), 20 (SEQ ID NO: 48), 22 (SEQ ID NO: 55), 24 (SEQ ID NO: 60), and 26 (SEQ ID NO: 62) selected from the group consisting of An isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence. Figure 1 (SEQ ID NO: 1), Figure 3 (SEQ ID NO: 6), Figure 5 (SEQ ID NO: 9), Figure 7 (SEQ ID NO: 14), Figure 9 (SEQ ID NO: 19), Figure 11 (SEQ ID NO: 24), Figure 13 (SEQ ID NO: 29), FIG. Nucleotide selected from the group consisting of the nucleotide sequence shown in 15 (SEQ ID NO: 34), Figure 17 (SEQ ID NO: 42), Figure 19 (SEQ ID NO: 47), Figure 21 (SEQ ID NO: 54), Figure 23 (SEQ ID NO: 59), and Figure 25 (SEQ ID NO: 61). Isolated nucleic acid comprising a sequence. Figure 1 (SEQ ID NO: 1), Figure 3 (SEQ ID NO: 6), Figure 5 (SEQ ID NO: 9), Figure 7 (SEQ ID NO: 14), Figure 9 (SEQ ID NO: 19), Figure 11 (SEQ ID NO: 24), Figure 13 (SEQ ID NO: 29), FIG. Consisting of the full-length coding sequence of the nucleotide sequence shown in 15 (SEQ ID NO: 34), FIG. 17 (SEQ ID NO: 42), FIG. 19 (SEQ ID NO: 47), FIG. 21 (SEQ ID NO: 54), FIG. 23 (SEQ ID NO: 59), and FIG. An isolated nucleic acid comprising a nucleotide sequence selected from the group. Isolated nucleic acid comprising the full length coding sequence of DNA deposited as ATCC 209281, ATCC 209358, ATCC 209670, ATCC 209384, ATCC 209262, ATCC 209263, ATCC 209438, ATCC 209432, ATCC 209704, ATCC 209620, ATCC 209422 or ATCC 209750. 23. A vector comprising the nucleic acid of any one of claims 19-22. The vector of claim 23, operably linked to regulatory sequences recognized by the host cell transformed with the vector. A host cell comprising the vector of claim 23. The host cell of claim 25, wherein said cell is a CHO cell. The method of claim 25, wherein said cell is E. E. coli. Host cell. The host cell of claim 25, wherein said cell is a yeast cell. 27. The host cell of claim 25, wherein said cell is a baculovirus-infected insect cell. delete Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 20), Figure 12 (SEQ ID NO: 25), Figure 14 (SEQ ID NO: 30), and FIG. Amino acids selected from the group consisting of the amino acid sequences shown in 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), and FIG. An isolated polypeptide comprising a sequence. delete Contains the amino acid sequence encoded by the full-length coding sequence of DNA deposited as ATCC 209281, ATCC 209358, ATCC 209670, ATCC 209384, ATCC 209262, ATCC 209263, ATCC 209438, ATCC 209432, ATCC 209704, ATCC 209620, ATCC 209422 or ATCC 209750 Isolated polypeptide. 34. A chimeric molecule comprising the polypeptide of any one of claims 31 and 33 fused to a heterologous amino acid sequence. The chimeric molecule of claim 34, wherein the heterologous amino acid sequence is an epitope tag sequence. The chimeric molecule of claim 34, wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin. 34. An antibody that specifically binds to a polypeptide according to any one of claims 31 and 33. The antibody of claim 37, wherein said antibody is a monoclonal antibody, humanized antibody, or a single chain antibody. (a) no accompanying signal peptide, 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). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). The nucleotide sequence encoding the polypeptide shown in the above, or (b) has the accompanying signal peptide and is shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 20), Figure 12 (SEQ ID NO: 25). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). The nucleotide sequence encoding the extracellular domain of the polypeptide shown in, or (c) no accompanying signal peptide, 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). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). Nucleotide sequence encoding the extracellular domain of the polypeptide shown in Isolated nucleic acid comprising a. (a) no accompanying signal peptide, 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). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). Polypeptide shown (b) has the accompanying signal peptide and is shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 24), Figure 14 (SEQ ID NO: 32), Figure 16 (SEQ ID NO: 37). ), FIG. 18 (SEQ ID NO: 42), FIG. 20 (SEQ ID NO: 50), FIG. 22 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 61), FIG. 26 (SEQ ID NO: 69), FIG. 28 (SEQ ID NO: 76), FIG. 30 (SEQ ID NO: 78) , The extracellular domain of the polypeptide shown in FIG. 32 (SEQ ID NO: 83) or 34 (SEQ ID NO: 91), or (c) no signal peptide accompanying, and FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 7), FIG. 6 (SEQ ID NO: 15), FIG. 10 (SEQ ID NO: 24), FIG. 14 (SEQ ID NO: 32), FIG. 16 (SEQ ID NO: 37). ), FIG. 18 (SEQ ID NO: 42), FIG. 20 (SEQ ID NO: 50), FIG. 22 (SEQ ID NO: 55), FIG. 24 (SEQ ID NO: 61), FIG. 26 (SEQ ID NO: 69), FIG. 28 (SEQ ID NO: 76), FIG. 30 (SEQ ID NO: 78) , Extracellular domain of the polypeptide shown in FIG. 32 (SEQ ID NO: 83) or 34 (SEQ ID NO: 91) Isolated polypeptide comprising a. 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt solution, sonicated salmon sperm DNA (50 μg / ml ), 0.1% SDS, and 10% dextran sulfate were used at 42 ° C., washed with 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C., and 50% formamide at 55 ° C., and EDTA at 55 ° C. Under the conditions of performing a strict wash with 0.1 x SSC contained, (a) no accompanying signal peptide, 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). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). The nucleic acid sequence encoding the polypeptide shown in (b) has the accompanying signal peptide and is shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 20), Figure 12 (SEQ ID NO: 25). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). The nucleic acid sequence encoding the polypeptide shown in (c) no accompanying signal peptide, 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). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). A nucleic acid sequence encoding the extracellular domain of a polypeptide shown in, or (d) has an accompanying signal peptide and is shown in Figure 2 (SEQ ID NO: 2), Figure 4 (SEQ ID NO: 7), Figure 6 (SEQ ID NO: 10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO: 20), Figure 12 (SEQ ID NO: 25). ), Figure 14 (SEQ ID NO: 30), Figure 16 (SEQ ID NO: 35), Figure 18 (SEQ ID NO: 43), Figure 20 (SEQ ID NO: 48), Figure 22 (SEQ ID NO: 55), Figure 24 (SEQ ID NO: 60), or Figure 26 (SEQ ID NO: 62). Nucleic acid sequence encoding the extracellular domain of the polypeptide shown in Isolated nucleic acid hybridizing with.