CA2632702A1

CA2632702A1 - Secreted and transmembrane polypeptides and nucleic acids encoding the same

Info

Publication number: CA2632702A1
Application number: CA002632702A
Authority: CA
Inventors: Kevin P. Baker; Dan L. Eaton; Ellen Filvaroff; Sherman Fong; Audrey Goddard; Christopher J. Grimaldi; Austin L. Gurney; Victoria Smith; Jean-Philippe Stephan; Colin K. Watanabe; William J. Wood; Zemin Zhang
Original assignee: Individual
Current assignee: Genentech Inc
Priority date: 2000-09-01
Filing date: 2001-08-29
Publication date: 2002-03-28
Also published as: AU1661002A; AU2002216610A1; WO2002024888A3; WO2002024888A2; EP1341814A2; JP4451059B2; CA2421056A1; JP2004526412A; JP2006223304A

Abstract

The present invention is directed to novel PRO 21434 polypeptides (SEQ ID
NO:112) and to nucleic acid molecules encoding those polypeptides which are overexpressed in liver tumor. 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

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SECRLTEI) AND T12ANS117EMBR.AN.E POLYPEPTIDES AND NUCLEIC ACIDS ENCODING Z'HE
S.A.ME

FIET D OF THE INVENTION
The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides.

BACKGROUND OF THE INVENTION
Extracellular proteins play iniportant roles in, among other things, the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction witli other cells, is typically governed by information received from other cells and/or the immediate environm.ent. This information is often transmitted by secreted polypeptides (for iustance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, rcceived aiid interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaiuig molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.
Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs available at present, such as thrombolytic agents, interferons, interleulcins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins.
Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts are being tindertaken by both industry and academia to identify new, native secreted pxoteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to ident,ify the coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proo. Natl. Acad. Sci. 93:7I08-7113 (1996); U.S. Patent No. 5,536,637)].
Membrane-bound prot.eins and receptors can play important roles in, among other things, the formation, differentiation and maintenance of tnulticellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins.
Such membrane-bound proteins and cell receptors include, but are not limited to, cytoltine receptors, receptor ldnases, receptor phosphatases, receptors irivolved in cell-cell interactions, and cellular adhesin molecules Iilae selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by pliosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and Z

nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor imtnunoadhesins, for instance, can be employed as therapeutic agents to block receptor-ligand interactions. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
Efforts are bein ; undertaken by both industry and academia to identify new, native receptor or membrane-bound proteins. Many efforts are focused on the screening of niammalian recombinant DNA libraries to identify the coding sequences for novet receptor or membrane-bound proteins.

SUMMARY OF THE INVENTION
In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide.
In one aspect, the isolated nuclelc acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82 % nucleic acid sequence identity, alternatively at least about 83 % nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86 % nucleic acid sequence identity, alternatively at least about 87 %
nucleic acid sequence identity, alternatively at least about 88 % nucleic acid sequence identity, alterna.tively at least about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternatively at least about 94 %
nucleic acidsequenee identity, alternatively at least about 95 % nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98 % nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a fiill-length amino acid sequettce as disclosed herein, an aznino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmenibrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82 % nucieic acid sequence identity, alternatively at least about 83 % nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86 % nucleic acid sequence identity, alternatively at least about 87 %
nucleic acid sequence identity, alternatively at least about 88 % nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternatively at least about 94%

WO 02/24888 PCT/USOA(27099 nucleic acid sequence identity, alteinatively at least about 95 % nucleic acid sequence identity, alternatively atleast about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least aboat 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full-length PRO polypeptide cDNA
as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length atn.ino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
In a, fiirther aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82 % nucleic acid sequence identity, alternatively at least about 83%
nucleic acid sequence identity, alternatively at least about 84 % nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86%
nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89 % nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nueleic acid sequence identity, alternatively at least about 93%
nucleic acid sequence identity, alternatively at. least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96 % nucleic acid sequence identity, altematively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequemce encoding a PRO polypeptide wbich is either transnlembrane domain-deleted or transmembrane domain-inactivated, or is compleinentary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide are disclosed herein. Therefore, soluble extracellular domains of the herein described PRO
polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that niay fiund use as, for example, hybridization probes, for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes. Such nucleic acid fragments are usually at least about 10 nucleotides in length, alternatively at least about 15 nucleotides in length, alternatively at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in lengtlr, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, altemativeiy at least about 170 nucleotides in length, altematively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, altematively at least about 500 nucleotides in length, altematively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced lengtli. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide-encoding nucleotide sequence with other known nuoleotide sequences using any of a number of well known sequence alignment programs and determining which PRO
polypeptide-encoding nucleotide sequence fraginent(s) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody.
In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerus an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, altematively at least about 81% amino acid sequence identity, altematively at least about 82% amino acid sequence identity, altematively at least about 83 %
amino acid sequence identity, alternatively at least about 84% aTnino acid sequence identity, alternatively at least about 85% aniino acid sequence identity, alternatively at least about 86%
amino acid sequence identity, altematively at least about 87% amino acid sequence identity, altematively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90%
amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alterXlatively at least about 92% an3ino acid sequence identity, alternatively at least about 93%
amino acid sequence idemtity, alternatively at least about 94% amino acid sequence identity, altematively at least about 95% amino acid sequence identity, altematively at least about 96% anuno acid sequence identity, altematively at least about 97%
amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99 % amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacicing the signal peptide as disclosed herein, an extracellu?ar domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein.
In a further aspect, t'te invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, altematively at least about 82% amino acid sequence identity, altematively at least about 83%

amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about. 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90%
amino acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97%
amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% aniino acid sequence identity to an amino acid sequence encoded by any of the human protein eDNAs deposited with the ATCC as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence andlor the initiating methionine and is encoded by a nucleotide sequence that encodes such an amlao acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturiuig a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO
polypeptide from the cell culture.
Anotlier aspect the invention provides an isolated PRO polypeptide which is either transmernbrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are'also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises tb.e appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein. In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule.
;(n, a iurther embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO
polypeptide which comprise contacting the PRO polypepdde with a candidate moleeule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
In a stiti further embodiment, the invention concerns a composition of matter comprising a PRO
potypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier. Optionall,y, the carrier is a pharmaceuticaU.y acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicanaent useful in the treatment of a condition which is responsive to the PRO
polypeptide, an agonist or antagonist thereof or an anti-PRO antibody.
In other embodi.nlents of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cell coniprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing any of the herein described WO 02/24888 PCT/iJS01/27099 polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering tba desired polypeptide from the cell culture.
In other embodiments, the inventianprovides chimeric molecules comprising any of the herein described polypeptides fased to a beterologous polypeptide or amino acid sequence.
Example of sueh chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an irmntmoglobulin.
In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes which may be useful for isolating genomic and eDNA nucleotide sequences, measuring or detecting expression of an associated gene or as antisense probes, wherein those probes may be derived from any of the above or below described nucleotide sequences. Preferred probe lengths are described above.
In yet other embodiments, the present invention is directed to methods of using the PRO polypeptides of the present invention for a variety of uses based upon the functional biological assay data presented in the Examples below.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID N0:1) of a native sequence PR0281 cDNA, wherein SEQ ID NO:1 is a clone designated herein as "DNA16422-1209".
Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID
NO:1 shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequence PRO1560 eDNA, wherein SEQ ID NO:3 is a cloue designated herein as "DNA19902-1669".
Figure 4 shows the amino acid sequence (SEQ ID NO:4) derived from the coding sequence of SEQ ID
NO:3 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequence PRO
189 cDNA, wherein SEQ ID NO:5 is a clone designated herein as "DNA21624-1391".
Figure 6 shows the amino acid sequence (SEQ 1D NO:6) derived fTom the coding sequence of SEQ ID
NO:5 shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequence PR0240 eDNA, wherein SEQ ID NO:7 is a clone designated herein as "DNA34387-1138".
Figure 8 shows the amino acid sequence (SEQ ID NO:8) derived from the coding sequence of SEQ ID
NO:7 siiown in Figure 7.
Figure 9 shows a nucleotide sequence (SEQ I) NO:9) of a native sequence PR0256 cDNA, wherein SEQ ID NO:9 is a clone designated herein as "DNA35880-1160".
Figure 10 shows the amino acid sequence (SEQ ID NO: 10) derived from the coding sequence of SEQ
XD NO:9 shown in Figure 9.

Figure t I shows a nucleotide sequence (SEQ ID NO:11) of a native sequence PRO306 cDNA, wherein SRQ ID NO:I1 is a clone designated herein as "DNA39984-1221".
Figure 12 shows the aniino acid sequence (SEQ ID NO:12) derived from the coding sequence of SEQ
ID NO:11 shown in Figure 11.
Figure 13 shows a nucleotide sequence (SEQ ID NO: 13) of a native sequence PR0540 oDNA, wherein SEQ ID NO:13 is a clone designated herein as "DNA44189-1322".
Figure 14 shows tne amino acid sequence (SEQ ID NO:14) derived from the coding sequence of SEQ
ID NO: 13 shown in Figure 13.
Figure 15 shows a nucleotide sequence (SEQ ID NO: 15) of a native sequence PRO773 cDNA, wherein SEQ ID NO: 15 is a clone designated herein as "DNA48303-2829".
Figure 16 shows the amino acid sequence (SEQ ID N0:16) derived from the coding sequence of SEQ
ID NO:15 shown in Figure 15.
Figure 17 sliows a nucleotide sequence (SEQ ID NO: 17) of a native sequence PR0698 cDNA, wherein SEQ ID NO: 17 is a clone designated herein as "DNA48320-1433".
Figure 18 shows the amino acid sequence (SEQ ID N0:18) derived from the coding sequence of SEQ
ID NO:17 shown in Figure 17.
Figure 19 shows a nucleotide sequence (SEQ ID NO:19) of a native sequence PR03567 cDNA, wherein SEQ ID NO:19 is a clone designated herein as "DNA56049-2543 Figure 20 sliows the amino acid sequence (SEQ ID NO:20) derived from the coding sequence of SEQ
ID NO:19 sbown in Figure i9.
Figure 21 shows a nucleotide sequence (SEQ ID NO:21) of a native sequence PR0826 cDNA, wherein SEQ ID NO:21 is a clone designated herein as "DNA57694-1341 Figure 22 shows the amino acid sequence (SEQ ID NO:22) derived froni the coding sequence of SEQ
ID NO:21 shown in Figure 21.
Figure 23 sliows a nucleotide sequence (SEQ ID NO:23) of a native sequence PRO1002 cDNA, wherein SEQ ID NO:23 is a clone designated herein as "DNA59208-1373".
Figure 24 shows the amino acid sequence (SEQ ID NO:24) derived from the coding sequence of SEQ
ID NO:23 shown in Figure 23.
Figure 25 shows a nucleotlde sequence (SEQ ID NO:25) of a native sequence PRO1068 eDNA, wherein SEQ ID NO:25 is a clone designated herein as "DNA59214-1449'.
Figure 26 shows the amino acid sequence (SEQ ID NO:26) derived from the coding sequence of SEQ
ID NO:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID NO:27) of a native sequence PRO1030 eDNA, wherein SEQ ID NO:27 is a clone designated herein as "DNA59485-1336".
Figure 28 shows the amiuo acid sequence (SEQ ID NO:28) derived from the coding sequence of SEQ
ID NO:27 shown in Figure 27.
Figure 29 slaows a nucleotide sequence (SEQ ID NO:29) of a native sequence PRO1313 cDNA, wherein SEQ ID NO:29 is a clone designated herein as "DNA64966-1575".

Figure 30 shows the amino acid sequence (SEQ ID NO:30) derived from the coding sequence of SEQ
ID NO:29 shown irt Figure 29.
Figure 31 shows a nucleotide sequence (SEQ ID N0:31) of a native sequence PR06071 cDNA, wherein SEQ ID NO:37. is a c3one designated herein as "DNA82403-2959".
Figure 32 shows the amino acid sequence (SEQ ID NO:32) derived from the coding sequence of SEQ
ID NO:31 shown in Figure 31.
Figure 33 shows a nucleotide sequence (SEQ ID NO:33) of a native sequence PR04397 eDNA, wherein SEQ ID NO:33 is a clone designated herein as "DNA83505-2606".
Figure 34 shows the amino acid sequence (SEQ ID NO:34) derived from the coding sequence of SEQ
ID NO:33 shown in Figure 33.
Figure 35 shows a nucleotide sequence (SEQ ID NO:35) of a native sequence PR04344 cDNA, wherein SEQ ID NO:35 is a clone designated herein as "DNA84927-2585".
Figure 36 shows tLe amino acid sequence (SEQ ID NO:36) derived from the coding sequence of SEQ
ID NO:35 shown in F'igure 35.
Figure 37 shows a nucleotide sequence (SEQ ID NO:37) of a native sequence PR04407 cDNA, wherein SEQ ID NO:37 is a clone designated herein as "DNA92264-2616".
Figure 38 shows the am.ino acid sequence (SEQ ID NO;38) derived from the coding sequence of SEQ
ID NO:37 shown in Figure 37.
Figure 39 shows a nucleotide sequence (SEQ ID NO:39) of a native sequence PRO4316 cDNA, wherein SEQ ID NO:39 is a clone designated herein as "DNA94713-2561".
Figure 40 shows the amino acid sequence (SEQ ID NO:40) derived from the coding sequence of SEQ
ID NO:39 shown in. kigirxe 39.
Figure 41 shows a nucleotide sequence (SEQ ID NO:41) of anative sequence PR05775 eDNA, wherein SEQ ID NO:41 is a clone designated herein as "DNA96869-2673".
Figure 42 shows the amino acid sequence (SEQ ID NO:42) derived from the coding sequence of SEQ
ID NO:41 shown in Figure 41.
Figure 43 shows a nucleotide sequence (SEQ ID NO:43) of a native sequence PR06016 cDNA, wherein SEQ ID NO:43 is a clotxe designated herein as "DNA96881-2699".
Figure 44 shows the amino acid sequence (SEQ ID NO:44) derived from the coding sequence of SEQ
ID NO:43 shown in Figure 43, Figure 45 shows a nucleotide sequence (SEQ ID NO:45) of a native sequence PR04499 cDNA, wherein SEQ ID NO:45 is a clone designated herein as "DNA96889-2641".
Figure 46 shows the amino acid sequence (SEQ ID NO:46) derived from the coding sequence of SEQ
ID NO:45 shown in Figure 45.
Figure 47 shows a nucleotide sequence (SEQ ID NO:47) of a native sequence PR04487 cDNA, wherein SEQ ID NO:47 is a clone designated herein as "DNA96898-2640".
Figure 48 shows the amino acid sequence (SEQ ID NOr48) derived from the coding sequence of SEQ
ID NO:47 shown in Figure 47.

WO 02124888 PCT/US(11/27099 Figure 49 shows a nucleotide sequence (SEQ ID NO:49) o~ a native sequence PRO4980 cDNA, wherein SEQ ID NO:49 is a clone designated herein as "DNA97003-2649".
Figure 50 shows the amino acid sequence (SEQ ID NO:50) derived from the coding sequence of SEQ
ID NO:49 shown in Figure 49, Figure 51 shows a nucleotide sequence (SEQ ID NO:51) of a native sequence PR06018 cDNA, wherein SEQ ID NO:51 is a clone designated herein as "DNA98565-2701".
Figure 52 shows the amino acid sequence (SEQ ID NO:52) derived from the coding sequence of SEQ
ID NO:51 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ ID NO:53) of a native sequence PRO7168 eDNA, wherein SEQ ID NO:53 is a clone designated herein as "DNA102846-2742".
Figure 54 shows the amino acid sequence (SEQ ID NO:54) derived from the coding sequence of SEQ
ID NO:53 shown in Figure 53.
Figure 55 shows a nucleotide sequence (SEQ ID NO:55) of a native sequence PRO6308 eDNA, wherein SEQ ID NO:55 is a clone designated herein as "DNA102847-2726".
Figure 56 shows the amino acid sequence (SEQ ID NO:56) derived from the coding sequence of SEQ
ID NO:55 shown in Figure 55.
Figure 57 shows a nucleotide sequence (SEQ ID NO:57) of a native sequence PRO6000 eDNA, wherein SEQ ID NO:57 is a clone designated herein as "DNA102880-2689".
Figure 58 shows the amino acid sequence (SEQ ID NO:58) derived from the coding sequen.ce of SEQ
ID NO:57 shown in Figure 57.
Figure 59 shows a nucleotide sequence (SEQ ID NO:59) of a native sequence PR06006 cDNA, wherein SEQ ID NO:59 is a clone designated herein as "DNA105782-2693".
Figure 60 shows the amino acid sequence (SEQ ID NO:60) derived from the coding sequence of SEQ
ID NO:59 shown in Figure 59.
Figure 61 shows a nucleotide sequence (SEQ ID NO:61) of a native sequence PRO5800 cDNA, wherein SEQ ID NO:61 is a clone designated herein as "DNA108912-2680".
Figure 62 shaws the amino acid sequence (SEQ ID NO:62) derived from the coding sequence of SEQ
ID NO:61 shown in Figure 61.
Figure 63 shows a nucleotide sequence (SEQ ID NO:63) of a native sequence PR07476 cDNA, wherein SEQ ID NO:63 is a clone designated herein as "DNA1152.53-2757".
Figure 64 shows ti~e aFnino acid sequence (SEQ ID NO:64) derived from the coding sequence of SEQ
ID NO:63 shown in Figure 63.
Figure 65 shows a nucleotide sequence (SEQ ID NO:65) of a native sequence PR06496 eDNA, wherein SEQ ID N0:65 is a clone designated herein as "DNA119302-2737".
Figure 66 shows the amino acid sequence (SEQ ID NO:66) derived from the coding sequence of SEQ
ID NO:65 shown in Figure 65.
Figure 67 shows a nucleotide sequence (SEQ ID NO:67) of a native sequence PR07422 cDNA, wherein SEQ ID NO:67 is a clone designated herein as "DNA119536-2752".

WO 02/24888 PCT/'QS01127099 Figure 68 shows the amino acid sequence (SEQ ID NO:68) derived from the coding sequence of SEQ
ID NO:67 shown in Figure 67.
Figure 69 shows a nucleotide sequence (SEQ ID NO:69) of a native sequence PR07431cDNA, wherein SEQ ID NO:69 is a clone designated herein as "DNA119542-2754".
Figure 70 shows the amino acid sequence (SEQ ID NO:70) derived from the coding sequence of SEQ
ID NO:69 shown in Figure 69.
Figure 71 shows a nucleotide sequence (SEQ ID NO:71) of a native sequence PR010275 cDNA, wherein SEQ ID N0:71 is a clone designated herein as "DNA143498-2824".
Fib ire 72 shows the amino acid sequence (SEQ ID NO:72) derived from the coding sequence of SEQ
ID NO:71 shown in Figure 71.
Figure 73 shows a nucleotide serluence (SEQ ID NO:73) of a native sequence PR010268 eDNA, wherein SEQ ID NO:73 is a clone designated herein as "DNA145583-2820".
Figure 74 shows the amino acid sequence (SEQ ID NO:74) derived from the coding sequence of SEQ
ID NO:73 shown in Figure 73.
Figure 75 sltows a nucleotide sequence (SEQ ID NO:75) of a native sequence PR020080 cDNA, wherein SEQ ID NO:75 is a clone designated herein as "DNA161000-2896".
Figure 76 shows the amino acid sequence (SEQ ID NO:76) derived from the coding sequence of SEQ
ID NO:75 shown in Figure 75.
Figure 77 shows anucleotide sequence (SEQ ID NO:77) of a native sequence PR021207 eDNA, wherein SEQ ID NO:77 is a clone designated herein as "DNA161005-2943".
Figure 78 shows the amisio acid sequence (SEQ ID NO:78) derived from the coding sequence of SEQ
ID NO:77 shown in Figt,re 77.
Figure 79 shows a nucleotide sequence (SEQ ID NO:79) of a native sequence PR028633 eDNA, wherein SEQ ID NO:79 is a clone designated hereiu as "DNA170245-3053 Figure 80 shows the amino acid sequence (SEQ ID NO:80) derived from the coding sequence of SEQ
ID NO:79 shown in F'igure 79.
Figure 81 shows a nucleotide sequence (SEQ ID NO:81) of anative sequence PR020933 cDNA, wherein SEQ ID NO:81 is a clone designated herein as "Di"IA171771-2919".
Figure 82 shows the asnino acid sequejice (SEQ ID NO:82) derived from the coding sequence of SEQ
ID NO:81 shown in Figure 81.
Figure 83 shows a nucleotide sequence (SEQID NO:83) of a native sequence PR021383 eDNA, wherein SEQ ID NO:83 is a clone designated herein as "DNA173157-2981".
Figure 84 shows 97e amino acid sequence (SEQ ID NO:84) derived from the coding sequence of SEQ
ID NO:83 shown in Figure 83.
Figure 85 shows a nucleotide sequence (SEQ ID NO:85) of a native sequence PR021485 cDNA, wherein SEQ 1D NO:85 is a clone designated lierein as "DNA175734-2985".
Figure 86 shows the amino acid sequence (SEQ ID NO:86) derived from the coding sequence of SEQ
ID NO:85 shown in Figure 85.

WO 02/24888 PCTI[ISOI/27499 Figtire 87 shows a nucleotide sequence (S)3Q XD NO:87) of a niat:ive sequence PR028700 eDNA, wherein SEQ ID NO:87 is a clone clesignated herein as "DIl~A176108-3040".
Figure 88 shows the ainino acid sequence (SEQ ID NO:88) derived from the coding sequence of SEQ
ID NO:87 shown in Figure 87.
Figure 89 shows a nucleotide sequence (SBQ ID NO:89) of a native sequence PRO34012 cDNA, wherein SEQ ID NO:89 is a clone designated herein as "DNA190710-3028".
Figure 90 shows the amino acid sequence (SEQ ID NO:90) derived from the coding sequence of SEQ
ID NO:89 shown in Figure 89.
Figure 91 shows a nucleotide sequence (SEQ ID NO:91) of a native sequence PR034003 cDNA, wherein.
SEQ ID NO:91 is a clotie designated herein as "DNA190803-3019".
Figure 92 shows the arnino acid sequence (SEQ ID NO:92) derived from the coding sequence of SEQ
ID NO:91 shown in Figure 91.
Figtire 93 shows a nucleotide sequence (SEQ ID NO:93) of a native sequence PR034274 cDNA, wherein SEQ ID NO:93 is a clone designated herein as "DNA191064-3069".
Figure 94 shows the amino acid sequence (SEQ ID NO:94) derived from the coding sequence of SEQ
ID NO:93 shown in Figure 93.
Figures 95A-95B shows a nucleotide sequence (SEQ ID NO:95) of a native sequence PR034001 cDNA, wherein SEQ ID NO:95 is a clone designated herein as "DNA194909-3013".
Figure 96 slzows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ
ID NO:95 shown in Figures 95A-95B.
Figure 97 shows a nacleotide sequence (SEQ ID NO :97) of a native sequence PR034009 cDNA, wherein SEQ ID NO:97 is a clone designated herein as "DN.A.203532-3029".
Figure 98 shows the amino acid sequence (SEQ ID NO:98) derived from the coding sequence of SEQ
ID NO:97 shown in Figure 97.
Figure 99 shows a nucleotid.e sequence (SEQ ID NO:99) of a nat.ive sequence PR034192 cDNA, wherein SEQ ID NO:99 is a clone designated herein as "DNA213858-3060".
Figure 100 shows the amino acid seguence (SEQ ID NO: 100) derived from the coding sequence of SEQ
ID NO:99 shown in Figure 99.
Figure 101 shows a ttucleotide sequence (SEQ 11) NO:101) of a native sequence PR034564 cDNA, wherein SEQ ID NO:101 is a clone designated herein as "DNA216676-3083".
Figure 102 shows the amino acid sequence (SEQ ID NO: 102) derived from the coding sequence of SEQ
ID NO:101 shown in Figtu=e 101.
Figure 103 shows a nucleotide sequence (SEQ ID NO:103) of a native sequence PR035444 eDNA, wherein SEQ ID NO: 103 is a clone designated herein as "DNA222653-3104".
Figure 104 shows the amino acid sequence (SEQ ID NO:I04) derived from the coding sequence of SEQ
ID NO: 103 shown in Figure 103.
Figure 105 shows a nucleotide sequence (SEQ ID NO: 105) of a native sequence PR05998 eDNA, wherein SEQ ID NO: 105 is a clone designated herein as DNA96897-2688 .

7.~

Figure 106 shows the amino acid sequence (SEQ ID NO:106) derived from the coding sequence of SEQ
ID NO:105 shown in Figure 105.
Figure 107 shows a nucleotide sequence (SEQ ID NO: 107) of a native sequence PR019651 cDNA, wberein SEQ ID NO: 107 is a clone designated herein as "DNA142917-3081".
Figure 108 shows the auino acid sequence (SEQ ID NO: 108) derived from the coding sequence of SEQ
ID NO:107 shown in Figure 107.
Figure 109 shows a nucleotide sequence (SEQ ID NO: 109) of a native sequence PR020221 cDNA, wherein SEQ ID NO: 109 is a clone designated herein as "DNA 142930-2914".
Figure 110 shows the amino acid sequence (SEQ ID NO: 110) derived from the coding sequence of SEQ
ID NO:109 shown in Figure 109.
Figure 1 I1 shows a nucleotide sequence (SEQ ID NO:111) of a native sequence PR021434 eDNA, wherein SEQ ID NO:111 is a clone designated herein as "DNA147253-2983".
Figure 112 shows the amino acid sequence (SEQ ID NO: 112) derived from the coding sequence of SEQ
ID N0:111 shown izz Figure 111.
Figure 113 shows a nucleotide sequence (SEQ ID NO: 113) of a native sequence PR019822 eDNA, wherein SEQ ID NO: 113 is a clone designated lzerein as "DNA149927-2887".
Figure 114 shows the amino acid sequence (SEQ ID NO: 114) derived from the coding sequence of SEQ
ID N0:113 shown in Figut'e 113.

DETAILED DiJSCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions The terms "PRO polypeptide" and "PRO" as used herein and when immediately followeed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein. The ternis "PRO/nuniber polypeptide" and "PRO/number" wlterein the term "niunber" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are flirther defined herein). The PRO polypeptides described herein may be isolated from a variety of soiuces, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The ternm "PRO polypeptide" refers to each individual PRO/number polypeptide disclosed herein. All disclosures in this specification which refer to the "PRO polypeptide" refer to each of the polypeptides in.dividually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, focmation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The terrn "PRO
polypeptide" also inclucies variants of the PRO/number polypeptides disclosed herein.
A "native sequence PRO polylzeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be prodaced by recombinant or synthetic means. The term "native sequence IIRO polypeptide"
specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g., an extracellular domain sequence), naturaliy-occurring variant fornis (e.g., altecnatively spliced forms) and WO 02124888 PCT1i7S01/27099 naturally-accurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length am.ino acids sequences shown in the accompanying figures.
Start and stop codons are shown in bold font and underlined in the figures. However, wbile the PRO polypeptide disclosed in the accompanying figures are shown to begin with nzet.hionine residues designated. herein as amino acid position 1 in the figures, it is conceivable and possible ttiat other methionine residues located either upstream or dowast.ream from the amino acid position I in the figures may be employed as the starting amino acid residue for the PRO polypeptides.
The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmeznbrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1% of such tr=ansmembrane and/or cytoplasniic domains and preferably, will have less than 0.5 9'0 of such domains. It wi12 be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at eitner end of the domain as initially identified lierein. Optionally, therefore, an extracelZular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domainlextracellulax domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are comtemplated by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are shown in the present specification andlor the accompanying figures. It is noted, however, that the C-terminai boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von.
Heinje et al., Nucl. Acids. Res, 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no inore than about 5 amSno acids on either side of the C-terminal bo"andary of the signal peptide as identified herein, and the polynucleotides encoding them, are conterztplal:ed by the present invention.
"PRO polypeptide variant" nieans an active PRO polypeptide as defin.ed above or below having at least about 80 % amino acid sequence identity witb a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, witi or without the signal peptice, as disclosed herein or any other fragment of a full-length ?'ItO polypeptide sequence as disclosed herein. Such PRO polypeptide variants include, for instance, PRO
polypeptides wlierein one or more am.ino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively CA UZ4G1Vbb GVV3-V6-Gti WO Uzi24888 PC'[7qS41129099 at least about 841%, aniino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88 % amino acid sequence identity alternatively at least about 89%
amino acid sequence identity, alternatively at ieast about 90% amino acid sequence identity, alternatively at least about 91% arnino acid sequence identity, alternatively at least about 92%
amino acid sequence identity, alternativeiy at least about 93% amino acid seqnence identity, alternatively at least about 94% ami3ao acid sequence identity, alternatively at least about 95% antitto acid seqneuce identity, atternatively at least about 96%
amino acid sequence identity, alternatively at least abcut 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99%
arnino acid sequence identity to a full-length native sequence PRO pvlypeptide sequence as disclosed herein, a PRO polypeptide sequence lacldag the signal peptide as disclosed herein, ati extracellular domain of a PRO
polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full=length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20 amino acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino aoids in length, alternatively at least about 50 a.mino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in leugth, alteniatively at least abotrt 80 amino acids in length, alternaatively at least about 90 amino acids i.n length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or rnore, "Percent (%) aniino acid sequence identity" with respect to the PRO
polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introdueing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutians as part of the sequence identity, Alignment for purposes of determiniug percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software sucli as BLAST, BLAST-2, ALIGN or Megaligzt (DNASTAR) software, Those slcilled in the ast can determine appropriate parameters for measuring alignrnent, including any algorithms needed to achieve maximal alignment over the fvll length of the sequetices being compared. For purposes herein, however, % amin.o acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, vvherein tlte complete source code for the ALIGN-2 program is provided in Table 1 below. The AT.TCEN-2 sec(aeme comparison computer program was authored by Genentech, I'nc, and the source code shown in Table I below has been filed with user documentation in the U, S. Copyright Offfce, Washington D,Cõ 20559, where it is registerad under U.S. Copyright Registration No. TXCJ510087. Tne ALTt3N 2 program is publicly available through Genentech, Inc., South San Frssu:iseo, California or may be compiled from the source code provided in Tabie 1 below. The ALIGN-2 program should be compiled for use oa a UNIX operating system, preferably di.gital UNIX V4.OD. All scquence comparison pzsameters are set by the ALIGN-2 program and do not vary.
In situations where AI.,IGN'-2 is employed for amino acid sequence comparisons, tbe % anaino acid WO 02i24888 PCTIUS01/27099 sequence identity of a givea arui.uo acid sequence. A to, with, or against a given, amino acid sequance B(whfch ean alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y
S
where X is the uuniber of amino acid residues scored as identical matdhes by the sequence alignment program ALION-2 in that program's alignment of A and B, and where Y is the total n7unber of amino acid residues in B, It will be appreciated that where the length of amino aeid sequence A is not equal to the length of atnino acid sequence B, the % arnino acid sequence identity of A to B vrill not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity eaicnlations using this method, Tables 2 and 3 demonstrate how to calculate the % anLiato acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PItO", wherein "PItO" represents the amino acid sequence of a hypothetical PRO polypeptidee of interest, "Coraparison Protein"
represents the amino acid sequence of a polypeptide against whicb tbe "PRO" polypeptide of interest is being compared, and "X, "Y" and "Z" eaeta represent different hypotbetical auiino acid residues.
Unless specifically stated otherwise, atl iqo amino acid secliience identity values used herein are obtained as described in the immediatel.y preceding paragraph using tlie ALIGN-2 computer program. However, % amino acid sequence identity values may also be obtained as described below by using the 'QVU-I3LAST-2 computer program (AltschW et al., ethods in Bnzymn.Logy 266:460-480 (1996)). Most of the WU-BLAST-2 searoh parameters are set to the default values. Those uot set to default values, i.e., the adjustable parameters, are set with the following valites: overlap span = 1, overlap fraction = 0.125, word threshoid (T) = 11, and scoring matrix = BLOSUM62. Wb.erz I'V"U-BI..AST-2 is employed, a%'o amino acid sequence identity value Is determined by dividing (a) the number of matching identicai amino acid residues between the amino acid sequenee of the PRO
polypeptide of interest having a sequence derivad from the native PRO
polypeptide and the conlparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being ooxnpared wbioh may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest, For example, in the statement "a polypeptide comprising an the amino acid sequence A which bas or having at Ãeast 807o amino acid sequence identity to the autino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
Paroent arnino aeid sequence identity may also be deternined using the sequence comparison program NCBI-I3LAST2 (Altschul et al., tquc ei cids Res. 25:3389-3402 (Ã997)). The bÃCBI-BLAB'P2 sequence comparison program may be downloaded or otherwise obtained frotn the National Tnstitute of Iiealth, Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask =
yes, strand = all, expected occurrences = 10, minimum low cornpiexity length 1515, multi-pass e-value =
0.01, eonstant for muki-pass = 25, dropoff for final gapped alignment = 25 aud scoring matrix = BLOSUM62.

.

In situations where NCBI-BLAST2 is employed for ancino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be pin-ased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction XIY

where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity ofBtoA.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence la.cking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragnent of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, a PRO
variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 %
nucleic acid sequence identity, alternatively at least abcut 8296 nucleic acid sequence identity, altematively at least about 83% nucleic acid sequence identity, alternatively at least about 84%
nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, altematively at least about 88 %
nucleic acid sequence identity, alternatively at least about 89 % nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, altet-natively at least about 91%
nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94 % nucleic acid sequence identity, alternatively at least about 95 %
nucleic acid sequence identity, aiternatively at least about 96 % nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98%
nucleic acid sequence identity and alternatively at least about 99 % nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacldng the signal peptide as disclosed herein, an extraeellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragnient of a full-length PRO polypeptide sequence as disc]osed herein. Variants do not encompass the native nucleotide sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, altematively at least about 60 nticleotides in length, alternatively at least about 90 nucleotides in length, altexnatively at least about 120 nucleotides in lengta, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in lengtb, alternatively at least about 210 nucleotides in length, alternatively at least about 240 CA 02421056 2oo;3-vVe-Za WO 02124888 PCTlUS01/27099 nut:leotides in length, aiternatively at least about 270 nucleotides in length, alternatively at least about 300 nuc2eotides in length, alternatively at least about 450 nucleotides In length, altBrnatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides In length, or rnore.
"Percent (%) nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequences identified herein is def%ned as the percentage of nucleotides in a oandidate sequence that are identical witb ft nucleot'ides in the PRO nucleic acid sequence of interest, after aligning the sequeum and 'antroducing gaps, if necessary, to achieve the znaximum percent sequence idei:tity. Alignrrtent for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are withict the sk7l in the art, for instanae, using publicly availab:e computer software such as BT,AST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nueleic acid sequence identity valaes are genetated using the sequence eosnparison computer program A'LIC3N-?., wherein ttte cotplete sowrt:e code for the AUGN-2 program is provided in Table I below. The 'A1.IGlq-2 sequence comparison cotnputar program was authorW by Genentech, Inc, and the source code shown in'iable 1 below has been filed with user documentation in the U.S.
Copyrigttt Office, Vdashington. D.C., 20559, wbere It is registered under U.S.
Copytight Registration No.
'= TXUS10087. The ALIGN-2 program is publicly ava9lable through Genentech, Inc., Souih San Framaisao, California or may be compiled from the source code provided in Table I below.
TTte AY,IGN-2 prcVam. ahonid be compiled for use on a UN1X operating system, preferably digital UNIX V4.OD, All sequenca comparfaoa parameters am set by the ALIGN-2 program and do not vary.
Ia situations vOhere AL1CiN-2 Is employed for nucleie acid sequence cornparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D(whiah can alternatively be phrased as a given mucleic acid sequence C that has or comprises a certain ~ nuclaic acid sequence identity to, with, or against a$iveu nucleic acid sequence D) is calculated as followsc Io0 idmes the fraction WiZ

2.5 where W is the number of nucleotides scored as identical matches by the sequence alignment program AY.ICN-2 in that program's alignment of C and D, artd whea Z is the total number of nucleotides in D. It w111 be appreciated that where the length of nncleic acid sequence C is not equal to the length of nucleic acid seqttenae D, the % nuoleic acid sequence identity of C to D will not equal the % nucleic acid sequerice identity of D to C.
As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid seqaence identity of the nucleic acid sngucnce desigaatsd "Comparison DNA" to the nucleic acid seqnence designated "PRO-DNA", wherein "PRO-DNA" represents a hypothetioal PRO-encoding nucleic acid sequernoe of interest, "Coznparison DNA" represents the nucleotide sequer.ce of a nocleic aeid molecule againet which the "I'RO-DNA" nueleie acid molecule of interest is being compared, and "N", l." at-d "V" each reprmnt different hypothetical nucleoticles.
Unless specifically stated otherwise, a:l % nueleio acid sequence identity values used herein are obtained as described in the rmmediately preceding paragraph using the ALIGN 2 eoinputer program. However, % nuoleic acid sequeuce identity values may also be obtained as desoribed below by using the WU-BI.,AST'-2 computer tl-1 vG~iL.L.uJV .!.vu:a-vc-c.v WO 02/24888 PC'TJI1S01127099 progratn (Altschui et al., Mexiods in E,nzy~iglogX 266:460-480 (1996)). Most of the Wl~-BI,AST-2 seareh parameters are set to the default values. Those not set to default values, i.e., the adjustable paraineters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix - BLOSUMG2. When WCJ-BI.AST-2 is employed, a % nucleic acid sequence identity value is detetnRified by dividing (a) the number of matching identical nucleotides betwe@n the nucleic acid sequence of the PRO
polypeptide-encod:ng nucleic acid molecule of interest having a sequence derived from the native sequence PRO
polypeptide-encoding nucleio acid and the comparison nucleic acid molecule of interest (i.e., the sequence against wlaich the PRO polypeptide-eneod'u,g nucleic acid znolecute of interest is being compared which may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the PRO
polypeptide-encodin.g nucleic acid molecuie of interest. For example, in the statement "an isolated nucieic acid moleeule comprising a nucleic acid sequence A which has or having at least 80%
nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleie acid molecule of interest and the nucleic acid sequence B is the nuclelc acid sequence of the PRO
polypeptide-encoding aucleio acid molectile of interest_ Percent nucleic acid sequence idantity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., ~1 ie o Aeids Res. 25:3389-3402 (1997)), The NCBI-BLASTZ sequerice comparison pron a.~n may be dowtiloaded or otherwise obtained from the iVational Jnstitute of I-Iealth, Bethesda, MD, NCBI-BI.AS';1'2 uses several search parameters, wherein all of titose search parameters are set to default values including, for example, unmask =
yes, strand = all, expected occurrences = 10, minimum low complexity length = 1515, multi-pass e-value =
0.01, constant for multi-pass = 25, dropoff for f'inal gapped alignznent = 25 and scoring matrix = BLOSUM62.
In situations vtihore NCBI-BI,AS'1'2 is employed for sequence comparisons, the % nucleic acid sequen..ce identity ox a given nucleic acid seqaence C to, with, or agalnst a given nucleic acid seguence D(which een altematively be phrased as a given nucleic acid sec}uenae C that has or comprises a certain % nucieic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraetion wlz where W is tYte number of nucleotides scored as identical matahes by tho sequence alignment program NCBI-Bl=_.AS'1'2 in that program's alignmeat of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid seqnence C is not equal to the length of nucleic acid seqpenoe D, the o nueleic acid sequence identity of C to D wili not equat the % nucleic acid sequeuce identity of D to C.
In offier embodirnents, PRO variantpolynucteotides are nucleic acid molecules that eztcode an active PRO
polypeptide and wliich are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequ.ences encod'uig a full-length PRO polypeptide as disclosed herein. PRO variant polypeptides may be those that are encoded by a PRO variant polynueleotide.
"Tsolated," when used to describethe varioua polypeptides disclosed herein, means polypeptide thatltas been identified and separated and/or recovered from a component of its natural environment. Contaminant 1&

WO 02/24888 PC'i'1US01/27099 components of its natural enviro.rment are materials that would typically intsrfere with diagnostic or therapeutic uses for the polypeptide, and may i.nclnde enzymes, hot7nones, and other proteinaceous or non-proteinaceous solutes. In preferred emtiodiments, the polypeptide will be p;.uified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions usnig Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide rn situ within recombinant cells, since at least one component of the PRO polypeptide natural cnvironnient will not be present.
Ordinarily, hovcrever, isolated polypeptide will be prepared by at least one purification step.
An "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ord'uiaril,y associated in the natural source of the polypeptide-encodittg nucleic acid. An isolated polypeptide-encodino nucleic acid molecule is otlier than in the form or setting in which it is found in nature.
Isolated polypeptide==encoding nucleic acid molecules therefore are distinguished fzom the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic aeid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucieic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an aperator sequence, and a zibosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably lin.ked" when it is placed into a fun.ctional relationship wittt another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preproteib that participates in the secretion of the polypeptide; a promoter or enbaneer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linlced"
means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in. reading phase. Ho-a+ever, enhancers do not have to be contiguous.
I,inki.ng is accomplished by ligation at conveuient restriction sites. If suc;i sites cio not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with corventionall practice.
The terna "antibody" is used in the broadest sense and specifically covers, for example, single anti-PRO
monoclonal antibodies (incl.udi.ng agonist, antagonist, andneutralizing antibodies), anti-PRO antibody cornpositions withpolyepitopic specificitv, single chain anti-PRO antibodies, and fragments of auti-PRO antibodies (see below).
The terni "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.o., the individua: antibodies comprising the population are identical except for possible naturally-occirzriilg mutations that may be present in minor amounts.
"Striugency" of hybridization reactions is readily determinable by one of ordinary sldli in the art, and generaIly is an empirical calculation dependent upon probe length, washing temperature, and salt concentration.

WO 02124888 f'C7'RIS01/27099 In general, longer probes require higlier temperatures for proper annealing, while shorter probes need lower temperattyres, Hybridization generally depends on the ability of denatured DNA
to reanneal when complerruentary strands are present in an environnent below their melting temperature, The higher the degree of desired homology between the probe and hybridizable seqtience, the highef the relative temperature which can be used, As a result, it follows that higher relative temperatures would tend to make the reaction c,ond'itions more stringent, whilh e lower temperatures less so. 1~or additional details and explauation of stringency of hybrid'tzation reaotions, see Ausubel et al., Campt Protocols in Molecular Biolosay, Wiley Xnterscience Publishets, (1945).
"Stringent conditions" or "high stringency oonditions", as defined herein, may be identitiai by those that:
(t) employ low ionic sirength and high temperature for washing, for example 0.015 M sodium chloride10.0015 M sodium citrate10.1 % sodintu dodecyl sulfate at 50 C; (2) employ during hybridization a denaturing agent, such as forraauude, for example, 50% (v/v) formacnide with 0.1% bovine serum albumin/0. I%, Ricolll0.1 %
polyvinylpyrrolidone150mM sodium p'hosphate buffer at pH 6.5 with 750 mM
sodium chloride, 75 mM sodium citrate at 42 C; or (3) employ 50% formamide, 5 x SSC (0.751Vi NaC1, 0.075 M
sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 glml), 0.1 % SDS, and 10% dextran sulfate at 42 C, with washes at 4Z C in 0.2 x SSC (sodium chioridelsodituu citrate) and 50% formarnide at 55 C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 C.
"Moderately stringent conditions" may be identified as described by Sannbrook et al,,141olecular Clonias:
La oratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solut9on and hybridization conditions (e, gõ temperature, ionic strength and %SDS) less stringent that those described above.
An example of moderately stringent conditions is overnight incubation at 37 C
in a solution comprising: 20%
formarnide, 5 x SSC (150 mM NaCi, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denbardt's solution, 10% dextran sulfate, and 20 mglnxl denatured sheared salrnon sperm DNA, followed by washing the falters In i x SSC at about 37-50 C. The skilled ardsan will recognize how to adjust the tamperature, ionic strength, etc, as necessary to acconunodate factors such as probe length and the like, The term "epitope tagged" when used herein refers to a cltimeric poiypeptide coraprisnng a PRO
polypeptide fused to a"tag polypeptide'. The tag polypeptide has enough residues to provide an epitope against wtuch an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fased. The tag polypeptide preferabiy also is fairly unique so that the antibody does not substantially cross-react wifn other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 auxirto acid residues).
Asusedherein, the term "imxnunoadhesin" designates antibody-like moleesiles whichcor[tbine thebinding specificity of a hete.rologous protein (an "adhesin") witlt the effector functions of immtmogiobuii.a constant domains. Stracturaliy, the izntnunoadhesins comprise afusion of an atnino acid sequence with the desired binding specificity which is ok,her than the antigen recognition and bulding site of an antibody (i.e., is "heterologous"), and an unmunogiobulin constant domain sequence. The adhosin part of an immzmoadhesin molecule typioally is a contiguous amivo acid sequence comprising at least the binding site of a receptor or a Iigand. Tho imrr,unoglcbal ~ eocastant domain se<Iuence in the immunoadhesin may be obtained from any immurtogiobulin, *-trademaric WO 02/24888 PCT/LIS(11/27099 such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
"Active" or "activity" for the purposes herein refers to fortn(s) of a PRO
polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring PRO, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO
other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring PRO.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native PRO
polypeptide disclosed herein. In a sizta.il.ar manner, the term "agonist" is used in the broadest sense and 'n7cludes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist ntolecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, sniall organic molecules, etc. Methods for identifying agonists or antagonists of a PRO polypeptide mav comprise contacting a PRO polypeptide with a candidate agonist or antagonist nxolecule and zzzeasur:uig a detectable change in one or more biological activities normally associated with the PRO polypeptide.
"Treatment" refers to both therapeutic treatment and propbylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
"Chronic" administration refers to adnainistxation of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain tlie inetial therapeutic effect (activity) for an extended period of time. "Intermittent"
administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, sucb as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the fuammal is human.
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutivc administration in any order.
"Carriers" as used lherein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable caxrier is an aqueous pH buffered solution.
Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and otlier organic acids;
antioxidants including ascorbic acid;
low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymezs such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and ot.her carbohydrates including glucose, mannose, or dextrius; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; mdlor nonionic surfactants such as TWLPNTm, polyethylene glycol (PEG), and PLLTRONICS''T~.

2,1 WO 02/24888 PCT/LISOI./27099 "Antibody fragments" cotnprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')Z, and Fv fragments;
diabodies; linear antiLrodies (Zapata et al., Protein Bag: 8(10): 1057-1062 119951); single-chain antibody molecules; and multispecific antibodies fortned from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designat3on reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab'), fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
"Fv" is the minimwn antibody fragment which contains a complete antigen-recognition and -binding site.
This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interaet to define an antigen-bindiug site on the surface of the Vf,-V, dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an .Fv comprising only three CDRs specific for an antigen) lias the ability to recognize and bind antigen, although at a lower affmity than the entire binding site.
The Fab fragm.ent also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for. Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')z antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between thern. Other ehemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
Depending on the arnano acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, Igo, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2.
"Single-chain Fv" or "sFv" antibody fragments coinprise the V. and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide liraker between the Vx ancl Vi, domains which enables the sFv to form the desired structure for antigea binding. For a review of sFv, see I'luckthun in The Pharmacology of Monoclorial Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-nbain variable domain (VH) connected to a light-chain variable doinain (VJ in the same polypeptide chain (VH-VL), By using a linlcer that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another clzain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP
404,097; WO 93111161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

WO 02/248$8 PCT/LfS41127099 An "isolate3" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminaitt oomponents of its natural environment are materials which would 'uzter.fere with diagnostic or t:aerapeutic uses Por the antibody, and n-ay include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. hi preferred embodiments, the antibody wi.11 be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99%
by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal atnino acid sequence by use of a spinning cup secluenator:. or (3) to honiogeneity by SDS-PAGE
under reducing or nonxedueing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present.
Ordinarily, however, isolated antibody will be prepared by at least one purification step.
An antibody that "specit3cally binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to iliat particular polypeptide or epitope on a particular polypeptide without substantially binding to any otlier polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound or composition wbich is conjugated directly or indirectly to the antibody so as to generate a"labeled" antibody.
The label may be detectable by itself (e.g. radioisotope labels or iluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
By "solid pl.ase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on, tlje cont:ext., the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., au affinity cflromatography column). This term also includes a discontiiiuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.
A "liposome" is a sniall vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal. The components of the liposozne are commonly arranged in a bilayer formation, simiiar to the lipid arrangement of biological membranes.
A"snzall nrolecule" is defined lierein to laave a molecular weiglit below about 5001)altons.
An "effective amount" of a polypeptide disclosed herein or an agonist or antagonist thereof is an amouukt sufficient to carry out a specif.ically sta'ed plirpose. An "effective atnount" may be determined empirically and in a routine mantier, in relation to the stated pupose.

Table 1 ~
* C-C increased from 12 to 15 * Z is avexage of EQ
B is average of ND
match with stop is M; stop-stop 0; S(joke.r) match = 0 *1 #define _M -8 1''~ valne of a matclr with a stop tu1 8ay[261(261 - {
!* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/
!* A*/ { 2, 0,-2, 0, 0,-4, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 01, /* B*1 { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1), /* C */ 15 /* D*/ { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2), I* E*/ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_,M,-1, 2r1, 0, 0, 0,-2,-7, 0,-4, 3), /* F (-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-1, 0, 0, 7,-5}, /* G*/ { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,,M1, 0, 0,-1,=7, 0,-5, 0), /* 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,._141, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2), /* 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-1, 0, 0, 4,-5, 0,-1,-2}, /*J*! {0,O,0,0,0,0,0,0,0,0,0,0,0,0,_M,0,0,0,0,0,0,0,0,0,0,0}, I* K*1 {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1, 3, 0, 0, 0,-2,-3, 0,-4, 01, I* L *1 { 2>-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_hI, 0, 2,-2, /* M*! 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-1, 0,-2,-1, 0, 2,-4, 0,-2,-1}, /* 1 d -1 { 0, 2,-4, 2, 1,-4, U, 2,-2, 0, 1,-3, 2, 2,-_M,-1, 1 , 0, 1, 0, 0,-2,-4, 0,-2,1}, /* O*/ {_M,_M,_M,_M, M_M,_M,M,_M,_M, M>,,.M,_M, M, 0,_M,_M _M,M,_M _TvT _M,_M, M, M,, M}, 1~' P*1 0,2, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0}7, /* Q*/ { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1, 2,-1, 1,_M, 0, 4, 0,-2,-5, 0,-4, 31, 1* R*1 {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 01, S*/ { 1, 0, 0, 0, 0,-3, 0, 0,-3, 2, 1,-_M, 1,-1, U, 2, 1, 0r1r2, 0,-3, 0), /* T*/ { 1, 0,-2, 0, 0,-3, 0r1, 0, 0, 0, M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-3, 01, f*U*/ {0,0,0,0,0,0,0,0,0,0,o,0,0,0,_M,0,0,0,0,0,0,0,0,0,0,0}, /* V x! { 0,-2,-2,-2r2r4, 0,-2, 2, 2, 2, IvI,-1,-2,-2,-1, 0, 0, 4,-6, 0,-2,-2), /* W {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3, 2,-4,-4 _M,-6,-5, 2,-2,-5, 0,-6;17, 0, 0,-6}, /* x { o, o, o, o, o, o, o, o, a, o, o, o, o, o,M, o, o, o, o, o, o, o, o, o, o, a}, /* Y fi! {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-2, 0, 0,10,-4}, !* Z*1 { 0, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4}

'1'able 1 coi t' _ 1*
#include <stdio.h>
#include <ctype.h>

#define M.AXJMP 16 1* max jumps in a diag *1 #dePLxe MAXGAI' 24 11' don't continue to penalize gaps larger than this *!
#define JMPS 1024 /* max jmps in an path #defwe MX 4 (* save if there's at least MX-1 bases since last jmp */
#define DMAT 3 /* vahie of matching bases */
#define DMIS 0 1* penalty fnr mismatched bases */
#define DINSO 8 1* penalty for a gap #define DTNS1 1 /* penalty per basc *1 #define PiNSO 8 !* penalty for a gap *1 Jfdefxne PINS1 4 /* penalty per residue structjmp {
--T
short n[AL4%JMP]; /* size of jmp (neg for dely) *1 uns-gned short x(MAXJMP7; /* base no. of jmp in seq x }; J*Iimitsseqto2'16-1 struct diag {
int score; 1* score at last jmp *I
Iong offset; /* offset of prev block *1 short ijmp; /''~ current jmp index struct jmp jp; /* list of jmps struct path {
int spc; i* nuttaber of leading spaces short n[JMPS); /* siz.e of jmp (gap) *;
int x[JMI'5];1* Ioc of jmp (last elem before gap) */
cbar *ofile; /* output file name char *namex[2]; I* seq names: getseqsQ
cbar *prog; prog name for err msgs *1 char *seqx[2]; !* secls: getseqs() */
int dmax; 1* best diag: nw() */
int dmaxO; /* ffnat diag int dna; f* set if dna: main() *1 int endgaps; /* set if penalizing end gaps int gapx, gapy; /* total gaps in seqs int IenO, lenl; /* seq lens */
int ngapx, ngapy; /* total size of gaps *1 int smax; /* max score: nw() */
int *xbm; /* bitmap for matching */
long affset; 1* current offset in jmp file */
struct diag *dx; /* holds diagonals */
struct path pp[2]; /* holds padi for seqs i char *calÃocO, *maÃloc(), *indexQ, *strcpy();
char '~gelseq{, *g calloc();

'Table.l cont' !* Needleman-Wimsch alignment program * usage: progs filel ftle2 where tiÃel and file2 are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity * Any lines beginning with ';', '>' or '<' are ignored * Max file length is 65535 (limited by urisigned short x in the jmp struct) A sequence with 113 or more of its elements ACGTU is assumed to be DNA
Ontput is in the file "align.out"
* The progrant may create a tmp file in /trnp to hold nifo about traceback.
* Original version developed under 13SD 4.3 on a vax 8650 *1 #lticlude "nw.h"
#include "day.h"

static _dbval[261 = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,Q0,5,6,8,8,7,9,0,10,0 }
static -pbval[26) 1, 21 (1 <<('D'-'A')) I (1 <<('N' 4, 8, 16, 32, 64, 128, 256, oxFFk+PFFF, 1 10, 1 11, 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 1 20, 1 21, 1 22, 1<<23, 1<<24, 1<<25~(1<<('E'-'A'))(1<<('Q'-'A')) rnain(ac, av) Inain char *avp;

prog = avfOJ;
if (ac 1= 3) {
fprintf(stderr,"usage: %s filel frle2ln", prog);
fprintf(stderr, "where filel and file2 are two dna or two protein sequences.'n");
fprintf(srderr, "The sequences can be in upper- or lower-caseln");
fprintf(stderr, "Any lines beginning with ';' or '<' are ignored\n");
fprintf(stderr, "Output is in the file 1"align.outl"1n");
exit(i);
}
namex[0] = av[1.1;
ttamex[1] = avj2];
seqx[0) = getseq(namex[0], &len0;;
seqx[i] = getseq(namex[1], &1en1);
xb:n = (dna)7 dbval : _pbvai;

endgaps = 0; 1* 1 to penalize endgaps ofile = "align.out"; I* output file *I

nw{); /* fill in the matrix, get the possible jmps readjmps(); get the actual jmps printp; print stats, alignment */
cleanup(0); 1* unlink any trnp files *1 }

WO 02/24888 PCTlUS01127099 Tab1e fconf' /* do the aligrement, return best score: inainQ
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983 * pro: PAN1250 values When scores are equal, we prefer mismatches to any gap, prefer * a new gap to extending an ongoing gap, and prefer a gap hi seqx ~ to a gap in seq y.

nw() !1W
{ char *px, *py; /* seqs and ptrs int *ndely, *dely; /* keep track of dely *!
int ndelx, delx; /* keep track of delx */
int *unp; /* for swapping rowO, rowl int mis; /* score for each type int ins0,insl; I*insertionpenalties register id; /* diagonal index register ij; 1" jmp index ''/
register *col0, *coll; !* score for curr, last row *I
register xx, yy; h" index into seqs *1 dx = (struct diag *)g_calloc("to get diags", len0-l-len1+1, sizeof(struct diag));
ndely = (int*)g_calloc("to getndely", lenl+1, sizeof(int));
dely =(tnt *)gõ calloc("to get dely",lenl + 1, simof(int));
col0 =(int *)g_calloc( to get col0", 1enI+1, sizeof(int));
coll =(int *)g._calloc("to get coll lenl+1, sizeof(int));
ins0 = (dna)? DINSO PINSO;
insl = (dria)? DINSI 1'IN'S1;
smax = -10000;
if (endgaps) {
for (co10[0; = dely[0] =-ins0, yy = 1; yy <= lenl; yy++) {
co10[yy] = dely[yy] = co10[y,y-1] - insl;
+.idely[yyI = yy;
}
0010[0] =~J; /* Waterman Bull N/cath Biol 84 }
else for (yy = 1; yy <= ienl; yy -i--I-) dely[yyl = -inso;

(* fill in rnatclk matrix for (px = seqx[Oj, xx = 1; xx <= lenO; px-}-+, xx-F,-) {
I* initialize first entry in col if (endgaps) {
iÃ(xx==1) coll[01 = delx -(ins0+insi);
else coil[J] == delx col0[0] - ins1;
ndelx = xx;
~
else {
coli [0] = 0;
delx = ==ins0;
ndelx = 0;

WO 02/24888 PCT/USO1l27099 Table 1 eon ...nw for (py = seqx[1], yy = 1; yy <= lenl; py'}'+, yy++) {
mis = co10[yy-1];
iF(dna) mis += (xbm[*Px-'A']8cxbnt[*py-'A'])7 DMAT ; DMIS;
else mis += _day[4'px-'A'1['"py 'A'l;
!P' update penalty for del in x seq;
x favor new del over ongong del * ignore MAXGAP if weighting endgaps if (endgaps I I ndely[yyj < MAXGAP) {
if (co10[yy] - ins0 > = dely[yy]) {
dely[yyl = co10[yy] - (ins0+insl);
ndely[yy] = 1;
}eise{
delyfyy] -= insl;
ndely[yy]++;
}
} else {
if (colo[yyl - (ins0+yns1) > = dely[yy]) {
dely[yy] = colO[yy] - (ins0+ins1);
ndely[yy7 = 1;
} eise ndely[yy]++;
}

!* update penafty for del in y seq;
* favor new del over ongong del if (endgaps ndelx < MAXGAP) {
if (col l [Yy-1] - ins0 > = delx) {
delx = coll[yy-11 - (ins0+ins1);
ndetx = 1;
} else {
de)x -= insl;
ndelx++;
~
} else {
if (coll[;ry-11 - (ins0+insl) > = delx) {
delx = coll[yy-1] - (ins0+ins1);
ndelx = 1;
} else ndelx+ +;
}

!* pick the maximuin: score; we're favoring mis over any de1 and delx ove: dely ~i WO 02/24888 PCT/USOi/27099 Table 1 cont' ...nw id = xx - yy + tenl - 1;
if (mis > = delx && mis > = deiy[yy]) coli[yyj - mis;
else if (deix > = dely[yy]) {
co11 [yy] - deix;
ij - dx[idl.ijmp;
iÃ(dx[id3 jp.n[01 && (Idna I I (udeix > = MAXJMP
&& xx > dx[idl.jp.x[ij]+MX) j I mis > dx[id].score+DIN'SO)) {
dx[idl.ijrnp++;
if (+ +ij > = MARJMP) {
writejmps(id);
ij = dx[idj.ijmp 0;
dx[id].offset = offset;
offset + = sizeof(struet jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = ndeix;
dx[idI.jp=x[ijj = xx;
dx[id].score = deix;
}
else {
coll[yy] = dely[yy];
ij = dx[idj.ijmp;
if (dx[id].jp.n[0] && (Idna I I (ndely[yy] > = MA~f' && xx > dx[id3.jp.x[ij]+Zv1X) I I mis > dx[idJ.score+DINSO)) {
dx[id].ijmp+ +;
if (++ij > = MAXJMP) {
wzitejmps(id);
ij = dx[idl.ijmp 0;
dx[id].offset = offset;
offset + = sizeof(strnct jmp) + slzeof(offset);
}
}
dx[id].jp.n[ij] = -ndely[yy];
dx[id].jp.x[ijI = xx;
ds[id].score = dety[yy];
t , if (xx = = 1en0 && yy < lenl) {
/* last col *j if (endgaps) coll[yy] -= ias0+iuzsl*(1enl-yy);
if (coil[yy] > smax) {
smax = coll jyy];
dmax - id;
}
~
if (endgaps && xx < len0) coll[yy-I] - ius0-i-insl*(IenO-xx);
if (colljyy-1] > smax) Ã
smax = coll[yy-1J;
dmax = id;
~
tmp col0; coIO = coil; coil - tmp;
}
(void) free((cbar *)ndely);
(void) free((char *)dely);
(void) free((ahar *)colO);
(void) free((char *)coll); }

Table 1 (cont') *
* print() -- only routine visible outside this module *
* static:
* getmat() - trace back best path, count matebes: print() * pr align() -- print alignment of described in array p(]: print() s dtunpblock() -- dump a block of lines with numbers, stars: pr_align() * numsQ -- put out a number l'uie: dtunpblock() * put[ine() -- put out a line (name, [num], seq, [num]): dunipblock() * starsQ - -put a line of stars: dtunpblock() " stripname() -- strip any path and prefix from a seqnanme */

#include "nw.h"
#def5ne SPC 3 #dek"iue P L1NE 256 1* maximuna output line #def'nie PSPC 3 /* space between name or num and seq'"J
extern day[26][267;
int olen; /* set output line length *Y
k7LE *fx; output file */

printp print {
9nt Ix, ly, firstgap, lastgap; /* overlap */
if ((~ ' fopen(ofrle, "w")) = = 0) {
fprintf(stderr,"%s: can't write %s1n", p:og, ofle);
oleanup(i);
}
fprintf(fx, "<first sequence: %s (length %ad)1n", namex[0], lenO);
fprintf(fx, "<second sequence: %s (length = %d)1n", namex[I], lenl);
olen = 60;
lx = len0;
ly = lenl;
firstgap = lastgap = 0;
,lY (dmax < lenl - 1) { /* leading gap in x pp[0].spc = firstgap = lenl - dmax - 1;
ly - PP[0].spc;
}
else if (dmax > lenl - 1) {/* leading gap in y*!
ppji].spc = firstgap = dmax - (lent - 1);
Ix -- pp[l].spc;
}
if (dmax0 < len0 - 1) { /" trailing gap in x lastgap = len0 - dmax0 -1;
} lx -= lastgap;
else if (dmax0 > len0 - 1) {/* trailing gap in y"I
lastgap = dmaxO - (len0 - 1);
ly -== lastgap;

getmat(lx, ly, firstgap, lastgap);
pr_alignn;

'I"abie x (cont') r*
* trace back the best path, count matches static J getmat(!x, Iv, firstgap, Iastgap) getIIlat int Ix, ly; /"' "core" (minus endgaps) *1 int firstgap, lastgap; /* leading trailing overlap {
iut nm, i0, il, siz0, sizi;
char outx[32];
double pet;
register nO, ni;
register char *1)0, "'pI;
get total matches, score *1 i0={1=si20=sizl=0;
p0 = seqx[0] + pp[i].spc;
pl = selxt1] + ppE0l.spc;
nO = pp[l].spc -}- 1;
nl = pp[01,spc + ?;
nm=0;
iFhile ( *p0 &8c *pl if (siz0) {

n1++;
else if (sizl) {
p0~+;
n0+ -t-;

else {
if (xbm[*p0-'A']&xbm[*pl-'A']) nm++;
if (n0+-+- pp[01=x.[iO]) siz0 = pp[OJ.n[i0++1;
it(nl -I- a- ==pp[l].x[il)) sizl = pp[ll.xi[il-+-+];
()0,-+;

}
}

/* pct homology:
* if penalizing endgaps, base is the shorter seq * else, 1mocY, off overhangs and take shorter core if (endgaps) ix = (lenO < lenl)? len0 : lenl;
else lx = (lx < ly)? ix : ly;
pet = 100.*(c3onbCe)mn1(douh1e)lx;
fprintf(fx, "\n );
fprintf(fx, "<%d match%s in an overlap of %d: %u.2fpercent sim'slarityln", nm (.nrn, es ', lx, pct);

WO 02/24888 PC'17[)S01/27099 Table 1 (cont') fprintf(fx, "<gaps in fsrst sequence: %d", gapx); ...getmat If (gapx) {
(void) sprintf(outx, " (%d %s%s)", ngapx, (dna)? "base":"residtie", (ngapx == 1)? " :"s");
fprintf(fx,s", outx);

fprintÃ(fx, ", gaps in second sequence: %d", gapy);
if (gapy) {
(void) sprintf(outx, " (%d %s%s)", ngapy, (dna)? "base":"residue", (ngapy 1)? " :"s");
fprintf(fx," %s", outx);
~
if (dna) fprir.tf(fx, "\n<score: %d (matcli = %d, misniatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT, DMIS, DINSO, DINSI);
else fprintf(fx, "1n < score; %d (Dayhoff PAM 250 matrix, gap penalty = % d + % d per residue)\n", smax, PLNSO, YINS1);
i~(endgaps) fprintf(fx, <endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n", firstgap, (dua)? "basa" "residue", (firstgap 1)? "" : "s lastgap, (dna)? "base" "residue", (lastgap == 1)? s");
else fprintf(fx, " < er,dgaps not penalized\n");
}

static nm; /* matches in for checking 'k/
static Imax; /* lengths of stripped file names static ij[2]; /"' jmp index for a path *1 static nc[2]; /* number at start of current line */
static ni[2]; /* current elem nuniber -- for gapping *1 static siz[2];
static char *ps[2]; /* ptr to current element *1 static char *po[2]; /* ptr to next output char slot *!
static char out[2][P_I.LhBI; !r' output line *1 static char star[p L1Nr]; !* set by stars() */

print alignment of described in stnict path pp[]
static pr alignO pr align {
int nn; /"' char count */
int more;
re.gister for (i = 0, lmax = 0; i< 2; i+-E=) {
nn = stripnaMe(namex[il);
if fn a > Imax) Imax=nn;

ni[i] 1;
siz[t] = 8[il = 0;
ps[il = seqxii];
po['a) = out[i]; }

WO 02/24888 )?CT/iIS01/27099 Table I (cont') for (nn = nm = 0, more = 1; more; ) { ...pY' align for(i = more = 0; i < 2; i-}-+) { ' !*
do we have more of this sequence7 if C'"Ps[il) Contitriie;
more++;

if (PPG)=sPc) { I* leading space *l *po[i]++
Pp[i]=spc"';
}
else it (siz[i]) { /* in a gap */
*PD[il++
siz[i]--;

edse { I* we're putting a seq element *Po[i] = *Ps[il;
i~f tislower(*Ps[il)) *Ps[i) = touppcr("ps[i));
Po[ij++;
ps[il -+- +;

~ ate we at next gap for this seq?
X/
if (ni[il == PP[il=xli.l[ill) {
l*
* we need to merge all gaps at tlus location y siz[il = PPIi]=n[i3[i1++];
wwte (ni[i7 == PP[il=x[iJ[lll) siz[il -E-= pP[il=n[i,j[il+
uiil+i;
}
}
if'(+ +nn =- olen !more && nn) {
dumpblockQ;
:or (i = 0; i< 2; i-1-+) po[ia - out[il:
nn = 0;
}
* dump a block of lines, including numbers, stars: pr_ aiign0 static dumpblockp dumpblock {
register i;

for (i = 0; i~ 2; i++) *Po[i7--WO 02/248$8 PCTIiJSQ1l27099 Tabie 1 cont' ...dumpblock (void) putc('In', fx);
for(i0;i<2.;i++){
if (*Out[i] && (*OIIt[i] I -- , , I I *(po[i]) I= ' ')) {
if(i--0) nums(i);
if (i 0 && *out[i]) starsO;
putline(i);
fif (i == 0 && *out[1)) fprintf(&, star);
if (i == 1) nums(i);
}
}
}
r*
put out a number line: dumpblock() */
static nums(ix) nums int ix; /* index iu out[I holding seq line {
cbar nline[P_L1NE];
register i, j;
register char *pn, *px, *py;

for (pa = niine, i 0; i < iznax+P SPC; i+ a-, pn++) *pn for (i = nc[ix{, py = aut(ix7; *PY; pY-}-+, pn+=i=) {
if (*PY = = ' ' 11 *py == ' ') *un else {
if(i%100II (i==1&&nc(ix]i= 1)) }=(i<0)?-i:i;
for (px = pn; j; j I= 10, px--) *px=j4610+'';
if (i < 0) *p,c }
else *pn _ i+-+, }
}
*pn nc[ix] = i;
for (pn = nline; *pn; pn+ +) (void) putc("pn, fx);
(void) putc('\n', fx);

I*
* put out a line (name, [nuttz], seq, [num]): dumpblock() *1 static puttine(ix) putume i.nt ix; {

WO 02/24888 PCT/[JS01/27099 Tab1e 1 cont' ...pntline int i;
$ register char *px;

for (px = namex[ix], i = 0; *px && 'kpx ! px++, i+ +) (void) putc(*px, fx);
for (; i < Ienax+P_SPC; i+ +) (void) putc(' ', fx);

1* these count from 1:
* nifl is cuzrent element (from 1) * nco is number at staet of current line for (px = out[ix]; *px; px+ +) (void) putc(*px&ux7F, fx);
(void) putc('1n', Ãx);
}

* put a line of stars (seqs alvvays in out[0], out[1]): dumpblock() static starsO stars ~
9nt i;
register char *p0, *pl, cx, *px;
if (1*out[oa (*out[O] && ~(PaEo]} __ ' ') I i 1*ont[l] (*out[i] && s'(Po[1]) == ' ')}
returzx;
px = star;
for (i = linax+PSPC; i; i-) *px++

for (p0 = aut[0], pi = out[1]; *p0 && *pl; p0++, p1++) {
if (isalpha('ep0) 8c&, isalpha(*pl)) {

if (xbm[*p0-'A'I&xbni[*p1-'A']) {
Cx nm++;
}
else if (tdna && _dayE*p0-'A']E*Pl-'A'] > 0) cx eise cx =
}
else cx =
*px++ = cx;

",px-'r-+ = '1ti ;
*Px }

WO 02/24888 PCT(USO1/27099 Tabie 1 (cont') ~ strip path or prefix from pn, return len: pr_alignO
static stripname(pn) stripname c.har *pn; r* file name (may be path) */
{
register char *px, *py;
py=0;
for (px = pn; *px; px++) if ("px = _ '/') py = px + 1;
~ (py) (void) strcpy(pn, py);
rehirn(strlen(pn));
~
2s Table 1 (cont') * eleanup() -- cleanup any taip file * getseq() -- read in seq, set dna, len, maxden g calioc() -- calloc() rlvitti error checkin readjmpsO -- get the good jmps, from tmp file if necessary * writejmpsO -- write a filled array of jmps to a tmp file: nwO
*1 #include "nw,h"
!tÃnclude <sys/file.h>
char *jname ="/tmp/homgXXX)C]CV; /* tmli file for jmps *1 FILE *fj;

int cleanup(); /* cleanup tmp file *1 long Ãseek();

" remove any tmp file if we blow cieanup(i) cleanup int i;
~
if (f1) (void) unlink(jname);
exit(i);
}
1*
* read, return ptr to seq, set dna, len, maxÃen * skip lines starE~.ng with ';', '<', or * seq in upper or lower case char *
getseq(file, len) getseq char *file; 1* file nazne int *len; Pr seq len */
{
char Ãine[1024], *pseq;
register char *px, *py;
int natgc, tien;
PILF "!p;
iP((fp = fopen(fi:e,"r")) 0) {
fpr6atf(stderr,"%s: can't read %sln", prog, file);
exit(l);
}
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line *line == '<' 11 'rline contÃnue;
for (px = line; *px i = 'tn'; px++) if (isupper(*px) islower(*px)) tlen++;
if ((pseq - malloc((nnsigned)(tlen+6))) 0) {
fprintf(stderr,";bs: malloco failed to get %d bytes for %s1n", prog, tlen+6, fiZe);
exit(l);
}
pseqlP) = pseq[z) pseq[21 = pseq[3] _ "S01;

WO 02/24RRR p'C'IYIIS01/27099 Table 1 cont' . , getseq py = pseq + 4;
*Ien = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line = = ';' I I *line = _ ' <' ~ I *line = _ ' >') coutinue;
for (px = line; *px (= '1n'; px++) {
if (isupper("'px)) *py.}. + = *px;
else if (islower(*px)) *py+ + = toupper(*px);
if (index("ATGCI,J",*(Py-1))) natgc++;
}
}
*py+ -?-*py - \0';
(void) fclose(fp);
dna = natgc > (tlen/3);
return(p'seq4-4);
}
cbar *
g_calloc(msg, nx, sz) g ca]jpc char *msg; / * program, calling routine int mc, sz; /* number and size of elements *!
{ char *px, *calloc();

if ((px = catloc((uasigned)ax, (unsigned)sz)) 0) {
if (*msg) {
fprintf(stderr, %s: g_callocO failed ys (n= %d, sz=%d)1n", prog, msg, nx, sz);
exit(1);
}
}
return(px);
}

~ get final jmps from dx(] or tmp file, set ppiJ, reset dmax: main( readjmpsQ readjmps {
int fd = -1;
int siz, i0, il;
register i, j, xx;
i~ (tj) {
(void) fclose(fj);
if ((fd open(jname, 0 RDONi.Y, 0)) < 0) {
fprintf(stderr, %s: can't openO % s1n", prog, jpame);
cleanup(1);
}
}
for(i=i0=ii =0,dmax0-dmax,xx=len0;;i+-E-){
wtule (1) {
for (j = dx[dmaxj.ijmp; j > = 0 && dx[dmaxl.jp.x(jl >= xx; j--) ~

Table 1 (cont') ...readJmps if (j < 0 && dx[daoax],offset && fj) {
(void)]seek(fd, dx[dtnax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(strnct jmp));
(void) read(fd, (char *)&dx[dinax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
}
else breal~;
}
if (i > = IMPS) {
fprintf(stderr, %s: too many gaps in alignmentln", prog);
cleanup(1);
}
if (1 > =- o) {
siz = dx[dmax].jP=n[j];
xx = dx[drnax].3P=X[j];
dmax + = sizr if ;siz < 0) { /* gap in second seq s'1 pp[1].n[ii] = -siz;
xx += siz;
'sd=xx-yy-1- lenl-1 *!
PP[i].x[il] = xx - dmax + lenl - 1;
gapy+4=;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps */
siz =(.-siz < MAXGAI' endgaps)? -siz MAXGAP;
il++;
}
else If (siz > 0) Ã/'" gap in first seq *1 PP[0].n[i01 = siz;
PP[0].x[i0] = xx;
gapx+ +;
ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz (siz < MAXGAP 1 endgaps)? siz : MAXGAP;
eLse l~reaic;
}

/* reverse the order of jnips *1 for (J = 0, iO-; j < iO; j}- -I-, i'[)--) {
i = Pp[o]=n[i]; PP[e]=n[iJ = PPTo],n[i0]; Pp[0].n[i0] = i;
i= PP10741; PP[0]4] = PP[0].x[i0]; PPIO].x[i0] = i;
}
for (j - 0, il.- ; j < ii; j-h-1-, i1--) {
i= PP[i]=na]; PP[i].nCl] = PP[11.n[il]; PP[1].n[i1) = I;
i= PP[i]=xCll; PP[i]=xCJ] = PP[i].x[il]; PP[1].x[ii] = i;
if(fd >= 0) (void) close(fd);
ie (fj} {
(void) iualink(jname);
t) = 0;
offset = 0;
} }

WO 02/24888 PCTfEJS(fI/27099 Table 1_ (c~n.t'Z

* write a filled jmp struct oÃfset of the prev one (if any): nwO
*1 writejmps(ix) ppriwmm int ix;
{
char *mktemp();
if(!f) ( if (mktemp(jnarxte) < 0) ( fprintf(stderr, "%s: can't m.ktempO %s1n", prog, jname);
olcanup(1);
}
if ((f = fopen(jname, "w )) 0) ~
fprintf(stderr, "%s: can't write %s1n", prog, jname);
exit(1);
}
(void) fwrite((char *)&dx['rx].jp, sia.eoÃ(struct jmp), 1, f);
(void) fwrite((char *)&dx[ixj,offset, sizeof(dx[ix].offset), 1, fj);
}

WO 02/24888 PCT/[JS01/27099 Table 2 PRO 7~xxxxXXxXXXXxXX (Length = 15 amino acids) Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids) % amino acid sequence identity =

(the number of identically ntatching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total nutnber of amino acid residues of the PRO
polypeptide) 5 divided by 15 = 33.3 %

T-abip, PRO X.XX.XXXXXXX (Length = 10 amino acids) Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids) % amino acid sequence identity =

(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) =

5 divided by 10 = 50%

iable 4 PIZO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNI3NLLLLLLI,LLL (Length = 16 nucleotides) % nucleic acid sequence identity =
(the number of identicaIIy matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =

6 divided by 14 = 42.9 %

Table 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =

(the number of identically matching nucleoddes between the two nucleic acid sequences as determined by AI,IGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) _ 4 divided by 12 = 33.3 %

R. Co ositio s and ethods of the I venti n A. 1 ull-Lenath PRO Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides. In particular, cDNAs encoding various PRO
po3ypeptides have been identified and isolated, as disclosed in further detail in the Exacnples below, It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed.
However, for sake of simplicity, in the present specification the protein encoded by the full length native nucleic acid molecules disclosed herein as well as aff furtber native homologues and variants included in the foregoing definition of PRO, will be referred to as "PRO/number", regardless of their origin or mode of preparation.
As disclosed in the Examples below, various eDNA clones have been deposited with the ATCC. The actual nucleotide sequences of those clones can readily be determined by the slcilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routi.u:e skill. For the PRO polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the readiug frame best identifiable with the sequence information available at the time.

B. PRO Polypeptide Variants In addition to the fuÃl-length native sequence PRO polypeptides described herein, it is contemplated that PRO variants can be prepared. PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, andlor by synthesis of the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variatiom in the native full-length sequence PRO or in various domains of the PRO described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instanee, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO that results in a change in the amino acid sequence of the PRO
as compared with the native sequence PRO. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or tnore of the domains of the PRO. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by coinparing the sequence of the PRO with that of homologous lrnownproteinmolecules and minimizing the number of amino acid sequence ch<~nges znade in regians of high homology. Amino acid substitutions can be the result of replacing one amino acid with anorlrer amisno acid having similar strnetural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the f.ull-Iength or mature native sequence.
PRO polypeptide fragments are provided herein. Such fragments may be tnmcated at the 1V-terminus or C-terminus, or may lacl: internal residues, for example, when compared with a fuli length native protein.
Certai.n fragments lack amino acid residues that are not essential for a desired biological activity of the PRO
polypeptide.
PRO fragments may be prepared by any of a number of conventional techniques.
Desired peptide fragments may be chemically synthesized. An alternative approach involves generating PRO fragments by enzymatic digestion, e.g., by treating the protein with aii enzyme k7-own to cleave proteins at sites defined by particular atztino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonueleotides that defzne the desired termini of the DNA fragment are enlployed at the S' and 3' primers in the PCR.
Preferably, PRO polypeptide fragments share at least one biological v.zd/or immtmological activity with the native PRO polypeptide disclosed heiein.
In particular embodinients, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exempiary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.

WO uZ/24888 PCT/IIS01/27099 Table 6 OrigiZZal Exemplary Preferred Residue Substitutions b'ubstitutions Ala (A.) val; leu; ile vaI
Arg (R) lys; gln; asn lys Asn (N) gla; his;'lys; arg gin.
Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Giu (E) asp asp Gly (G) pro; ala ala His (B) asn; gln; lys; arg arg lle (I) leu; val; met; ala; phe;
norleucine leu Lett (L) norleucine; ile; val;
met; ala; phe ile Lys (K) arg; gln; asn arg Iviet (M) leu; phe; ile leu Phe (P) leu; val; ile; ata; tyr Ien Pro (P) ala aIa Ser (S) tlzr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (X) trp; phe; thr; ser phe Val (V) ile; leu; met; phe;
ala; norleucine Ien Substantial modifications in fitnction or immunological identity of the PRO
polypeptide are accomplished by selecting substitutions that differ significantly in their eifect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (o) the bulk of the side chain. Naturally occurring residues are divided into groups based on cominon side-chain properties:
(1) hydropltobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that infiuence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
Non conservative substitutions will entail exchanging a member of one of these classes for another class.
Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonucleotide-tnediated (site-directed) mutagenes3s, alanine scannin.g, and PCR mutagenesis. Site-directed rnutagenesis [Carter et al., kjugj:
Acids Res., 1õ3:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette m.utagenesis [We]Is et al., WO 02/24888 PCT[US01/27099 Gene, 34:315 (1985)], restriction selection nautagenesis [Wells et al., Philos. Trans R Soc. London SerA
317:415 (1986)] or other known techiiiclues can be performed on the cloned DNA
to produce the PRO variant nNA.
Scanning amino acid analysis can also be employed to iden.tify one or more amino acids along a contiguous sequence. Among the preferred scanning atnino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and eysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the naaim-cliain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.fl. Freeman & Co., N.Y.);
Chotbia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
C. Modifications of PRO
Covalent modifications of PRO are included within the scope of this invention.
One type of covalent modification includes reacting targeted amino acid residues of a PRO
polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the PRO.
Derivatization with biftuzetional agents is useful, for instance, for crosslinlci.ng PRO to a water-insoluble support matrix or surface for use i. the nlethod for purifying anti-PRO antibodies, and vice-versa. Commonly used crosslinldng agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuceinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(suecinimidylpropionate), biiiznctionai maleimides such as bis-N-maleimido-1,8-octane and agents such as metnyl-S-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residaes, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, PriDteins: Strncture and Molecular Properties, W.H.
Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal annizte, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the PRO polypeptide included within the scope of this invention comprises altering tbe naaave glycosylation pattern of the polypeptide.
"Altering tize native glycosylation pattern"
is intended for purposes hereii~ to mean deleting one or more carbohydrate moieties found in native sequence PRO
(eitlier by removing t13e underlying glycosylation site or by deleting the glycosylation by chemical and/or enzyjnatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO.
In addition, the phrase includes quaiitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate nioieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The aIteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native secluence PRO (for 0-linked glycosylation sites). The PRO anuno acid sec)uence may optionally be altered tln=oitgh changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that cocions are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO
polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO
87105330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemieally or enzymatically or by mutational substitution of codons encodding for amino acid residues that serve as targets for glycosylation. Chen-tical deglycosylation techniques are Iazown in the art and described, for instance, by Haldmuddin, et al., Arch. Biocher_z. Bionhys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate tnoieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakeua et al., Meth. Enzymol., 13$:350 (1987).
Another type of covalent modificatiou of' PRO comprises linldng the PRO
polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
The PRO of the present invention may also be modified in a way to form a chimeric molecule comprising PRO fused to another, beterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
The epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO. The presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag, polypeptide. Also, provision of the epitope tag enables the PRO to be readily purified by affinity purit'ication using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are we11 known in the axt. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., MoI. Cell. Biol., 8:2159-2165 (1988)); tlte c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the I-Ierpes Simplex virus glycoprotein D(gD) tag and its antibody [Paborsky et al., Protein EnjZineerin~, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnologv, 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Scze~g, 255:192-194 (1992)]; an a-tubulin epitope peptide jSkinner et at., J. Biol. Chem., 266:15I63-15166 (1991)]; and the T7 gene 10 protein peptide tag [I.utz-Freyermuth et al., Proc. Nati. Acad. Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a partiouiar region of an immunoalobulin. For a bivalent form of the chimeric molecule (also referred to as an "inmunoadhesin"), such a fitsion could be to the Fc region of an IgG molecule. The lg fusions preferably include the substitution of a soluble (trausmembrane domain deleted or inactivated) form of a PRO
polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGl moIecule. For the production of imniunoglobulin fusions see also US
Patent: No. 5,428,130 issued June 27, 1995.

D. Pre axation of PRO
The description below relates primarily to production of PRO by culturing cells transformed or transfected with a vector containing PRO nucleic acid. It is, of course, contemplated that alternative methods, which are well knovm in the art, may be employed to prepare PRO. For iuistance, the PRO sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e. g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969);
Merrifield, J. Am. Chem, Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation.
Automated synthesis maybe accomplished, for instance, using anApplied Biosystems Peptide Synthesizer (Poster City, CA) using manufacturer's instractions. Various portions of the PRO may be chemically synthesized separately and combined using cheniical or enzymatic methods to produce the full-length PRO.

1. Isolation of DNA Encoding PRO
DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level. Accordingly, human PRO DNA
can be conveniently obtained from a eDNA library prepared from human tissue, such as described i.n the Examples. The PRO-encoding gene may also be obtained from a genomic library or by kuown synthetic procedures (e.g., automated nucleic acid synthesis).
Libraries can be screened with probes (such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratorv Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding PRO is to use PCR
methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer:. A Laboratory IvZanual (Cold Spring Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened, Methods of labeling are well known in the art, and include the use of radiolabels like "P-labeled ATP, biotinylation or enzynie labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., su ra.
Sequences identifieci iui such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within defined regions of the nioleeule or across the full-length sequence can be determined using methods known in the art and as described herein.
Nucleic acid having protein coding sequence may be obtained by screening selected eDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al.,- su ra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into eDNA.

2. Selection and Transformation of Host Cells Host cells are transfected or transfonned with expression or cloning vectors described herein for PRO
production and cultured in conventional rnitrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skiiled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in MamnJplian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL
Press, 1991) and Sambrook et al., sunr.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily sidlled artisan, for example, CaC1z, CaPO4, liposonze-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calciuni chloride, as described in Sambrook et al., suvra, or electroporation is generally used for prokaryotes. Infection with Agrobacteriurn turnefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virolo ,L2:456-457 (1978) can be employed. General aspects of mammalian cell host systent transfections bave been described 9n U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl.
Acad. Sci. (USA), 76:3829 (1979).
However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyoraitb.i.ne, may also be used. For various tecl2niques for transforming mamnnalian cells, see Keown et al., Methods in Enz=olo185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eikaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC
31,537); E. coll strain W3110 (ATCC 27,325) and Jf.5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia,e.g., E. coli, Enterobacter, Erwinia, Kiebsiella, Proteus, Saltnonella, e.g., Salnaonella typhi3nuriu7i, Ser=ratia, e.g., Serratia rnarcescans, and Sfiigella, as well as Bacilli such as B.
subtilis and B. licheniforrnis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 April 1989), Pseudornonas sucli as P. aeruginosa; and Streptornyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentat.ions. Preferably, the host cell secretes minizsial amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain IA2, which has the complete genotype tonA ; E. coli W31 10 strain 9E4, which has the complete genotype tonff ptr3;
E. coli W3110 strain 27C7 (ATCC

CA. UE4I'LJ,VD0 GVv.~-v4-=4o WO 02124888 PC'i'/US01/27099 55,244), which has tne complete genotype totrrl ptr3 phoA Gr15 (argP=lac)169 clegl' otnpT kart'; E. coli W3110 strain 37D6, which has the complete genotype toM ptr3 phaA E75 (argF-1ac)169 degP onrpT rbs711vGkan';
E. colt W3110 strain 40B4, which is strain 37176 with a non-icanaatycirc resistant degP dalotion mutatlon; and an E. coli strain having mutant periplastnie protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.
Alternatively, lri vitro methods of cloning, e,gõ PCR or oJier nacleic acid polymerase rcjactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as fjIaraentous fwtgi or yeast are suitable clontttg or expression hosts for PTtO-eneoding vectors. 5'accharomyces cerevisiae is a commonly used lower eukaryotie bost microerganisnt. Others inelude 5'Mosaccharom.yces pombe (Beach and Nurse, a e, 290: 140 [19813;
BP 139,383 pubiished 2 May 1985); lUuyvermrFyces hosts (U.S. Patent No.
4,943,529; Pleer et al., 13iotTecl,no!M, 9:968-975 (1991)) such as, e.g., E. G:cctis (3V1W98-8C, CBS683, CI3S4574; Louvettcourt et al,, J. Bac 1eriol,, 154(2):737:?42 (1983]), X.fragilis (ATCC 12,424), K.
buigartcus (ATCC 16,045), K, wickerumli (ATCC 24,178), K. wullii (ATCC 56,500), K. rCrosophilarutn (ATCC 36,906; Van den Berg et al., >3io/Technaloav, 8:133 (1990)), K. th.ermototertuis, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (BP 183,070; Sreekrislma et al,, J. Basic~Microbiol , 28:265-278 (1988j);
Candidrr, Trlchoderrna reesia (EP
244,234); N'eurospora crassa (Case et al., -nc; Nat). Acad. Sci. TJSA, 76:5259-526311979J); SchvYannBonryees such as SchFVarriiomyces occideretaifs (EP 394,538 published 31 October 1990);
and fllamentous fungi such as, e.g., ATeuro,spara, !'erciciflium, 7blypocladium (WO 91/00357 published 10 January 1991), amtAspergillus iroats bem. Biopliys, Res. CommjR,, 112:284-289 [1983); Tilburu et al., sach as A. rtidularts (Ballance et al., Bim e e, 26:205-221 (19831; Xelton et al., kMq. Natt. AcaIL &j. USA, 81: 1470-1474 11984]) and A. niger (Kelly and I3ynas, _ v1BQL, 4:475-479 [19851). Methylotropic yeasts are suitable herein and include, but are not Iimited to, yeast capable of grovrth on methanol selected from the gonera consisting oP Flansenula. Candtda, lfloeckera, Pichia, SaccFttzramyces, Ybrulopsis, and Rlzodotorula. A list of specific speeies that are exemplary of this class of yeasts may be found in C, Anthony, The Biochernisirv of Methylotronhs, 269 (1982).
Suitable host cei?s for the expresslozt of glycosylated PRO are derived from multicellular organisuts.
Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera St9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells, More specific e7tauiples include tno:ilcey kidney CVI line transformed by SV40 (COS-7, ATCC Cltl.. 1651); hutpan embryonic lddney Iine (293 or 293 cells subolon.ed for growth In suspension culture, Graham et al., 7 Gen V'uoi., 36.59 (1977)); Cliiu?ese haFn.ster ovary celis/-pllptt (CIIQ, Urlaub and Chasin, P ar. NBtI, Aead. Sei. US,F1, 7 r:4216 (1.980)); mouse sertoli cells (TIv14, Math.er, niol.ReArod., 23;243-251(1980)); humanlrmg cells (W138, ATCC CCL 75); luunan liver cells (fiep C2., FIB 8065); and mouse mammary tumor (MMT 060562, ATCC
CC1.51). The selection of tlze appropriate l:ost cell is deemed to be within the 00 in the art.

3, $election and'fbe of a Replicable Veecor 7he nnaloic acid (e.g., cDNA. or p,enomic DNA) eneoding PRO may be inserted into a replieable veetbr for cloning (amp=lifi.caEion of the DNA) or for expression. Various vectors are publicly available. The vector may, for exampte, be uz the forrn ox' a ptasuiid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may he iuserted into t7ie vector by a variety of procedures. In generat, DNA is inserted into an WO 02124888 PCT1tJS01/27099 appropriate restriction endonuclease sit:e(s) asi,ig techniques known in the art. Vector components generally include, but are not limited to, oue or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termirtation sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
The PRO may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a eignal seqtienee or other polypeptide having a specific cleavage site at the N-teminus of the mature protein or poIypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the PRO-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccliarornyces andKluyverornyces a-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C.
albicans glucoamylase leader (FP
362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, man2malian sigual sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
The origin of replication from the plasmid pB.R322 is suitable for most Gram-negative bacteria, the 2 plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex znedia, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is t.he CHO cell line deticient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trpl gaae present in the yeast plasmid YRp7 [Stinchcomb et al., ature, 282:39 (1979);
Kingsman et al., Gene, 7,141 (1979); Tscheinper et al., Gene, 10:157 (1980)].
The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptopktan, for example, ATCC No.
44076 or PEP4-1 [7ones, Geneties, 85:12 (1977)].
Expression and clwaing vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are welt known. Promoters snitabie for use widt prokaryotic hosts include the j3-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,7761, and hybrid promoters such as the tac promoter [deBaer et al., Proc. Nati. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will cor.tain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding PRO.
Examples of suitable promoting secluences for use with yeast hosts include the promoters for 3-phosphoglycerate kfnase [I-Sitzeman et al., J. BioE. Cheni., 255:2073 (1980)]
or other glycolytic enzymes [Hess et a1., J. Adv.Enzvme 12-&, 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructoldnase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, wnich are inducible pronioters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metailothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and gaIactose utilization. Suitable vectors and promoters for use in veast expression are ftuther described in EP 73,657.
PRO transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 7uly 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-13 virus aud Simian Vin3s 41 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an imniwioglobufin promoter, and from heat-shock promoters, provided such promoters are compatfible with the host cell systems.
Transcription of a DNA encoding the PRO by higher eulcaryotes may be increased by inserting an enhancer sequence into the vector. l:nlzancers are cis-acting elements of DNA, usualIy about from 10 to 300 bp, that act on a promoter to inc.rease its transcription. Many enhancer sequences are now knovtm from mammalian genes (globin, elastase, albumin, a-fetoprotein, and 'uisulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Exarnples include the SV4t) enhancer on the late side of the replication origin (bp 100-270), the cytoznegalovirtis early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the PRO
coding sequence, but is preferably located at a site 5' fror.i the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from otlzer multiceilular organisms) will also contain sequences necessary for the terinination of transcription and for stabiliv-ing the znRNA. Such sequences are comznonly available from the 5' and, occasionally 3', untranslated regioiis of eukaryotic or viral DNAs or cDNAs. T'nese regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO.
Sdil other methods, vectors, and host cells suitable for adaptationto the 3ynthesis of PRO in recombinant vertebrate cell culture are described iv. Cething et al,, Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); BP 117,060; and EP 117,058.

l.J'). VEY44V:7V GVV.J-V.a--VVO 02/24888 PCT/[)S01/27099 4. Detectina Gene AmAlinoationlExoression Gene arxzpliCieation andlor expression may be moasured in a sample directly, for example, by conventional Southern blotting, Narthern blotting to qizantitate the tmucription of mRNA [Thomas, Proc NalL
Acad. 5ci. USA, 77:5201-52fl5 (19$0)1, dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
Alternatively, antibodies may be eistployed that can recognize specific duplexes, i.naluding DNA duplexes, RNA duplexes, and 17NA-RNA hybrid duplexes or DNA-protein dupiexes. The antibodies in Curn may be labeled and the assay may be carried out where the duplex is bound to a suraace, so that upon the foraration of duplex on the surface, the presence of antibody bound to the duplex can be dotected.
Gene expression, alternatively, may be measured by immunoiogicat methods, such as innrnunohistochenlic.al stzircing of cells or tissue sections and assay of cell cutture or body fluids, to quantitate directly the expression of gerte product. Antibodies usefut for immunohistochemical stain'tug and/or assay of sample fluids may be either monoclonal or polyalonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepated against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided hereirr or against exogenous sequence fused to PRO
DNA and encoding a specifio antibody epitope.

5. ;i'ur)rication of Polyneptide Forms of PRO may be recovered from culture mediam or from host ce11 iysates.
If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g.
Triton X 100) or by enzymatic cleavage, Cells employed in expression of PRO can be disrupted by various physical or chemteal means, suoh as treeze-thavr cycling, sonication, mechanicaml disruption, or cell lysing agents.
It may be desired to purify PRO from recombinant cell proteins or potypeptides. The following procedures are exetnpiary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase TIt'iC; chrotnatography on silica or on a cation-exchange resin such as D'BAB;
etn:omatofocnsing; 5DS-I'AGPs; ammonium sulfate precipitation; gel ftltrationusing, forexample, Sephadex?Q-'15;
protein A Sepharose colurnris to remove contacninants such as IgC3; and metal chelati.ng columns to bind epitope-tagged forms of tlie PRO. Various methods of protein purification may be employed and such mellrods are loaown in the art and clescribed for example in Deutsoher, Methods in Enzymolaav, 182 (1990); Scopes, r i Parifica ion; Prinoinlas an JPrac 'ce, Springer-Verlag, New York (1982). The pnrificatioa step(s) se).ected wiil depend, for example, on the natnre of the production process used and the particular PRO produoed.

i?. C1se~_ for P ) 14ucleotide= sequences (or their complement) encoding PRO have various applioations in the art of moleculu= biology, iaicluding uses as hybridization probes, in cbromosozne and genemapping an.d in the generattott { 35 of anti-sense RNA and DNA. PRO niicleic acid will also be useful for the preparation of PRO polypEpt,idea by the reeombinant tecbniques described herenl.
The full-length native sequence PRO gene, or portions thereof, may be used as bybridization probes for tr ade:r:~172 52 WO 02/24888 PCT/tIS01/27099 a cDNA library to isolate the fiill-lengtli PRO eDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of PRO or PRO from otlier species) which have a desired sequence identity to the native PRO sequence disclosed herein. Optionally, the lengtlt of the probes will be about 20 to about 50 bases.
The hybridization probes rnay be derived from at least partially novel regions of the full length native nucleotide sequence wherein, those regions tnay be determined without undue experimentation or from genomic sequences including promoters, enaancer eleinents and introns of native sequence PRO. By way of example, a screening metYiod will comprise isolating the coding region of the P.RO gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the PRO gene of the present invention can be used to screen libraries of human cDNA, genomic DNA
or inRNA to determine which members of sucli libraries the probe liybridizes to. Hybridization techniques are described in fu:ther detail in the Examples below, Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
Otlter usefut fragments of the PRO nacleic acids include antisense or sense oligonucleotides comprising a singe-strandecl nucleic acid sequence (either :RNA or DNA) capable of binding to target PRO mRNA (sense) or PRO DNA (antisense) sequerices. Antisense or sense ol'zgoniicleotides, according to the present invention, comprise a fragment of f,he coding region of Pl2O DNA. Sucli a fragment generally comprises at least about 14 nucleotides, preferably froin about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA. sequence encoding a given protein is described in, for example, Stein and Cohen (_Qane,er. Res. 48:26-59, 1988) and van der Krol et al. (Bio'Z'eehniques 6:958, 1988).
Binding of antisease or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, inoluding enhanced degradation of tt e dupiex.es, premature termination of transcription or translation, or by other means.
The antisense oligonucIeotides thus may be used to block expression of PRO
proteins. Antisense or sense oligonuclecitides furtlaer coxnprise oligozrucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as tbose described in WO 91/06629) and viherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides viith resistant sugar linlcages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucieotide sequences.
Otlier exaniples oi sense or antisense oligonucleotides include those oligonucleotides which are eovalently linked to organic moieti:s, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide f'or a target nncleic acid sequence, sucli as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alirylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nueleotide sequence.
Antiseztse or sense oligonucleot9des may be introduced into a cell containing the target nucleic acid sequence by any gene transfctr method, includ.ing, for example, CaPOcmediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
In a preferred procedure, an antisense or sense oligonucleodde is inserted i,ttto a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or nx vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCTSA, DCT5B and DCT5C (see WO
90/13641).
Sense or antisense o3igonncleatides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligaud binding molecule, as described. in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other eytoltines, or other ligands that biud to cell surface receptors. Preferably, conjugation of the ligand bind'zng m.olecule does not substantially ir terfere witii the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
Alternatively, a sense or an antisenae oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid contplex, as described in WO 90/10448. The sense or antisense oligonucleotide-lip;.d complex is preferably dissociated within the cell by an endogenous lipase.
Antisense or sense R1~IA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in lengtli, about 20 bases in Iengiii, about 25 bases in length, about 30 bases in length, about 35 bases in IengtL, about 40 bases in length, about 45 bases in length, about 50 bases in length, about 55 bases in length, about 60 bases in length, about 65 bases in length, about 70 bases in length, about 75 bases in length, about 80 bases in length, about 85 bases in length, about 90 bases in length, about 95 bases in length, about 100 bases iu length, or more.
The probes niay also be employed in PCR techniques to generate a pool of sequences for identification of closely related PRO coding sequences.
Nucleotide sequences encoding a PRO can also be used to construct hybridization probes for nZapping the gene w'czich encodes that PRO and for the genetic analysis of individuals witb genetic disorders. The nueleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, suc1T, as in situ hybridi-zation, linkage analysis against lmown chromosomal markers, and hybridization screening with libraries.
When the coding sequences for PRO encode a protein which binds to another protein (example, where the PRO is a receptor), the PRO can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified.
Proteins involved in such bizzding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor PRO can be Used to isolate correlative ligand(s).
Screening assays can be designed to find :ead compounds that mimic the biological activity of a native PRO or a receptor for PRO. Such screening assays will include assays amenable to high-througbput screening of chemical lzbraries, making them particuiarly stritable for identifyinb small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic co.aipounds. Tke assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, imrnunoassays and cell based WO 02/24888 PCTI[1501/27099 assays, wltich are well c3 :acictericed in the art.
Nucleic acids which encode PRO or its modified forms can also be used to generate either transgenic animals or "knock out" atuntals which, in n;.txt, are useful in the development and screening of therapeutically Useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the aniznal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DN A which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding PRO can be used to clone genomic DNA encoding PRO in accordance with establ ished techniques and the genomic sequences used to generate transgenic aniunals that contain cells which express DNA encoding PRO. Methods for generating transgenic animals, particularly animals such as niice or rats, have beconie conventional itr, tt:e art and are described, for example, in U.S. Patent Nos.
4,736,866 and 4,870,009. Typically, particular cells would be targeted for PRO
transgene incorporation with tissue-specific enhancers. Transgenic ani.Fnals that incltide a copy of a transgene encoding PRO introduced into the gernx line of the ar:imal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding PRO. Such ani_mals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing dte transp,ene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non=hutnan.homologues of PRO caztbe used to constntct a PRO
"kn.ockout" animal which has a defective or altered gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced into an embryonic stem cell of the animal. For exazuple, eDNA encoding PRO can be used to clone genoniic DNA
encoding PRO in accordance with established techniques. A portion of the genomic DNA encoding PRO can be deleted or replaced with another gene, such as a gene encoding a selectable marker whicb can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchl, Celi, 5+_:503 (1.987) for a descriptioii of homoiogous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA
has homologousty recombined with the e.ndogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)].
The selected cells are tlien injected into a blastocyst of an animal (e,g., a mouse or rat) to form aggregatiort chimeras [see e.g., 13radley, iz. 2er-atocarcinornas artd .8rnbryortic Stent Cells: A Practical Approach, E. J.
Robertson, ed. (IRL, Oxford, 1987), pp. 113-1521. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster anuual aud the embryo brought to term to create a"knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed ani.mals in wb.ioh all cells of the animal contain tlLe homologously recombined DNA. Knockout animals can be characteriLed for instance, for their ability to defend against certain pathological conditions and for their development of pathological coMitions due to absence of the PRO
polypeptide.
Nucleic acici encoding th, PRO polypeptides may also be used in gene therapy.
In gene therapy applications, genes are i,ntroduced into cells in order to achieve in vivo synthesis of a therapeutically effective ct .iJ

wu uZ/24tt88 PCTf[IS01/27099 genetic product, for examp;e for replacenient of a defective gene. "Czene therapy" includes both conventional ger-e therapy where a lasting effect is achieved 6y a single treatment, and the administration of gene therapeutic agents, whi;.h involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo.
Xt has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intraeellula, concentrations caused by their restricted uptake by the cell membrane.
(Zamecaik et al., Proc. Natl. Acad. Sc. I;"A 83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g, by substituting their negatively charged phosphodiester groups by uncharged groups.
There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, micro.in.jection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer teclwiques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Bioteclanologv 11, 205--2t0 [1993]). In sotaie situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a ceil surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis niay be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tro ~c for a articular cell type, antibodies for r oteuu which under o internalization in cycling, A~ P' P' $ proteins that target intracelauiar localization atid enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for exainple, by NVu et al., J. Biol.
Chern. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410 3414 (1990). For review of gene marlang ancl gene therapy protocols see Anderson et al., Science 256, 808-813 (1992).
The PRO polypeptides described herein may also be employed as molecular weight markers for protein electrophoresis purposes and the isolated nucleic acid seruences may be used for recombinantly expressing those markers.
The nucleic acid molecules encodixtg the PRO polypeptides or fragrxten.ts thereof described herein are useful for chromosoine identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chrornosome rr?ariang reagents, based upon actual sequence data are presently available. Each PRO nucleic acid molecule of the present invention can be used as a chromosome marker.
The PRO polypeptides and nucleic acid molecttles of the present invention may also be used diagnostically for tissue typing, wherein the PRO polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type. PRO nucleic acid molecules will fznd use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
TIxe PRO polypep?ides described herein may also be employed as therapeutic agents. The PRO
polypeptides of the present invention can be formulated accord'utg to known methods to prepare pharmaceutically useful coinpositions, wlietc:by the PRO p.roduct hereof is combined in admixture with a ph.armaceutieally :zEy acceptable carrier vcliicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabiti2ers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formuiations or aqueous solutions. Acceptable carriers, exeipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other orgaiiic acids; antioxidants including ascorbic acid; low molecular weight (less tlian, about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or humunoglobultas; hyd.rcphilic polymers such as polyvinylpyrrolidone, amino acids sueh as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; cne;ating agents such as L?DTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; andlor nonionic surfactants such as TWEENTM, PLURONICST"d or PEG.
The formulations to be used for in vivo administration niust be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
Therapeutic compositions herein gen.erally are placed into a container having a sterile access port, for exazuple, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The route of adnciuistration is in accord witlz known methods, e.g, injection or infusion by intravenous, intraperitoneal, intracerebral, intraniuscular, intraocular, intraarterial or intralesional routes, topical adnninistration, or by sustained release systems.
Dosages and desired clrug concentrations of pl;armaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is weli within the sldil of an ordinary physician. Animal experiments provide reliable guidance for. the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Cbappell, W.
"The use of interspecies scaling in toxicolcinetics" In Toxicok.~netics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.
When in vivo adnrird.stra*ion of a PRO polypeptide or agonist or antagonist thereof is employed, normal dosage amounts may vary from abont 10 Lg/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 ug/kg/clby to 10 mg/kg/day, depending upon tlze route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos.
4,657,760; 5,206,344; or 5,225,212. lt is anticipated that different forniuiations will be effective for different treatment compounds and diffei-ent disorders, tiiat adTninistration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
Where sustained-release adnkinistration of a PRO polypeptide is desired in a formulation with release characteristics suitable for the treatment of any disease or disorder requiring administration of the PRO
polypeptide, microencapsuiation of the PPO polypeptide is contemplated.
Microencapsulation of recombinant proteins for sustained release lias been successfitlly performed with hiunan growth hormone (rhGH), interferon-(rhIFN- ), uiterleuldn-2, and MN rgp12U. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 2 7:122 i-1223 (1993): Hora ct ai., Rzo/ feclnzo g~L
8:755-758 (1990); Cleland, "Design and Production of Single 5~
, WO 02/24885 PC'F/IISOI./27099 immunization Vacc'vnes Using Polylactide Polyglycolide Microsphere Systems,"
in Vaccine Design; The Subunit and Adjuvant Approaeh, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO
97/03692, WO 96/40072, WO 96107399; and U.S. Pat, hro. 5,654,010.
The sustaiuted-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradationproducts of PLGA, lactic and glycolic acids, ca.n be cleared quickly within the hutnan body. Moreover, the degradability of this polymer can be adj'ust.ed from months to years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer," in:
M. Chasin and R. Langer (Eds.), iode radable P_ olyrners as Drug Deliver =y Systems (Marcel Dekker; New York, 1990), pp. 1-41.
This invention encompasses methods of screening compounds to identify those that mimic the PRO
polypeptide (agonists) or prevent the effect of the PRO polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify conipounds that bind or coniplex with the PRO polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, i=.nnutmoassays, and cell-based assays, which are well chazacter3zed in the art.
All assays for antagonists are cotnznnn iuk that they call for contacting the drug candidate with a PRO
polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to L'1tP.r'dCt, In binding assays, the intei~action is binding and the complex formed ean be isolated or detected in the reaction mixture. In a pzrticular embodhn:.nt, the PRO polypeptide encoded by the gene identified herein or the drng candidate is iu unobilized on a solid phase, e.g., on a niicrotiter plate, by covalent or nou-coval.ent attachments, Non-covalent attachment generally is accoznplished by coating the solid surface with a solution of the PRO polypeptide and drying. Alternatively, an z:nntobiiized antibody, e.g., a monoclonal antibody, specific for the PRO polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobiiized component, wiiich may be labeled by a detectable label, to the immobilized component, e, g., the coated surface contahting the anchored component. When the reaction is complete, the non-reacted components are rernoved, e.g., by washing, and complexes anchored on the solid surface are detected.
When the originally non-irr.mobilizc:d coniponent carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the original'ly nan-immobilized component does not carry a label, complexing canbe detected, for example, by using a labeled antibody specifically binding the immobilized complex.
If the candidate compour,d interacts vaitwh but does not bind to a particular PRO polypeptide encoded by a gene identiiied lierein, its interaction witla that polypeptide can be assayed by methods well known for detecting protein-protein interactions, Such assays include traditional approaches, such as, e.g., cross-linldug, oo-irnmuuoprecipit.a*ion, and co-purification thrauglz gradients or chromatographic columns. In addition, protein-protea.t interactions can be monitored by using a yeast-based genetic systetn described by Fields and co-workers ~ 58 WO 02124888 PCTliJS01l27099 (Fields and Song, Nature fi.oridonI, ;40:245-246 (1989); Chien et al., Proc.
Nati. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. l'atl. Aoad. Soi. USA, 89:
5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the othef one functioning as the transcription-activation domain. The yeast expression systet-o described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of t]his property, a:id employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and anollier, ~a vrhich caredid.ate activating proteins are fused to the activation domain.
The exuression of a GAL1-In:cZ reporter getze under c,ontrol of a GAl.4-acti.vated promoter depends on reconstitution of GAL4 activity via protein-protein. interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for 0-galactosidase. A complete kit (MATCHMAKERTM) for identifying protein-protein interactions between two specific prateins us:ng the two-ltybrid technique is commerciaily available from Clonteeb. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint <anino acicl residues that are crucial for these interactions.
Compounds that interfere with the interaction of a gene encoding a PRO
polypeptide identified herein and other intra- or extracelltaar caniponents can b. tested as follows:
usually a reaction mixture is prepared containiutg the product of the gene aatd the intra- or extracellular component under conditions and for a time allowing for the interaction :m.d binding of the two products. To test th,e ability of a candidate compound to inlubit binding, the reaction is rtrn in the absence ar,d in tb.e presence of the test compouud. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular compouent present in the mixture is monitored as described hereinabove, The formation of a complex in the control reaction(s) but not in the reaction znixtut=e containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
To assay for antagonists, *1te PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of ttie compound. to inhibit the activity of interest in the presence of the PRO polypeptide iizdicates that the coinpound is an antagonist to the PRO polypeptide. Alternatively, antagonists niay be detected by combining the PRO polypeptide and a potential antagonist with membrane-bound PRO polypeptide .receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The PRO polypeptide can be labeied, such as by radioactivity, such that the number of PRO polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panz;ing and FACS sorting. Coligan et al., Ctnrent Protocols in, lmznun., 1(2): Chapteir 5 (1991).
Preferably, e:cpression ciox:irig is emplcyad whereii polyadenylat:ed RNA is prepared froin a cell responsive to the PRO polynep'_ide and a cDNA library created from this RNA is divided into pools and used to transfect COS
cells or other cells that are not responsive to tlse PRO porypeptide.
Transfeoted cells that are groum on glass slides are exposed to iabeled PP.O polypeptide. '1'l.ie PRO polypeptide can be labeled by a variety of ineans including iodination or incl.usion of a recognition site for a site-specific protein Ic.'vaase. Following fixation and incubation, the slides are sutyjectecl to autoradiographic analysis. Positive pools are identified and sub-pools are WO 02/24888 PC'TfUS01127099 prepared and re-transfected using an interactive sub-pooling anci re-screening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, Iabeled PRO
polypeptide can be photoaffinity-lin-ked with cell membrane cr extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray filtn. The labeied complex containi.ng the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing, The amino acid sequence obtained from ukicro- sequencing would be used to .ivsign a set of degenerate oligonucleotide probes to screen a cDNA
Iibrary to ident:fy the gene encoding the putativP receptor.
In another assay for antagor.ists, mani:natian cells or a membrane preparation expressing the receptor wotild be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enbance or block this interaction could then be meastued, iViore specific examples of potential antagonists include an oligonucleotide that binds to t.tie fusions of immunoglobulin with PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragntertts, single-chain aiitibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
Alternatively, a potenrn;ial antagonist may be a closely related protein, for example, a mutated form of the PRO
polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the PRO polypeptide.
Another potential PRO polypeptide antagonist is an antisense RNA or DNA
construct prepared using antisense technology, wliere, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to tarFcted niRNA and prever~ting protein translation.
Antisense teclmology can be used to control gene expression d},roagh triple-lzelix forxnation or antisense DNA
or RNA, botla of wl3ich methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, whicli encodes the matnre PRO polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 4(I base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -- see Lee et al., Nuol.
Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 2S1! 1360 (1991)), thereby preventing transeription and tlte production of the PRO pol}lr:;ptide, Tiae antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the niRNA inolecule into the PRO polypeptide (antisense -Okano, Neurochem., 56:560 (1991); Oliu_odeoxyni:cleotides as Antisense Iniiibitors of Gene Expression (CRC Press; Boca Raton, FL, 1988).
'Phe oligonucleotides described above cau also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the PRO polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the ranslation-initiation site, e, g., between about -t0 and + 10 positions of the target gene nucleotide secfience, are pr.ef'erred.
Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the PRO polypeptide, thereby blocldng the normal biological activity of the PRO palypepcide. b;x.azn,3les of snzall molecules include, but are not limited to, small peptides or peptide-like moleciiles, preferabty soluble peptides, and synthetic ilon-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing t'ne specific cleavagd of RNA, Ribozymes act by sequence-specific hybridizatioci to the cornplementary target R11A, followed by endonucleolytia cleavage. Specific ribozyme cleavage ,ites within a potential RNA target can be identified by knoNvn techniques.
For furtlaer details see, c.g., Rcssi, CurrLnt Biol6gy; 4:469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997).
Nucleic acid molecules ia triple-helix formation used to arMbit transcription should be single-stranded and composed of deoxynuvleotides. The base composition of these oligonueleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duf,.ex. For further details see, e.g., PCT publication No. W0 97/33551, supra.
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by aLiy oiiter sc.eening techniques well known for tl:ose skilled in the art.
Diagnostic and therapeutic uses of the herein disclosed molecules may also be based upon the positive functional assay hits disclosed and described below.

F. Anti-PRO Ant~boflics The present invention fttrt:her provides anti-PRO antibodies. I3xemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconiugate antibodies.

1. F'ocon A4tibo4es The anti-PRO antibodies rnay comprise polyclonal antioodies. Methods of preparing polyclonal antibodies are knowri to the skilled artisan, Polyclonal antibodies can be raised in a mammal, for example, by one or rnore injections of an irntuw-iizing agent and, if desired, an adjuvant. Typically, the immunizing agent aud/or adjuvant will be injecteri in. the mammal by znultiple subcutaneous or intraperitoneal iajections. The immunizing agent rnay incAaae the PRO polypeptide or a fusion protein thereof.
It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the inanunai being itnmunized. Examples of such immu_nogenic proteiuls include but are not limited to keyhole litnpet hemocyanin, sernm aibumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete acTjuvant and MPJ.MMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The imtuuzzization protocol may be selected 'uy one slalIed in the art without undue experimentation.
2. Lvtonoclonal Antibodies The anti-PRO antibodies may, alternatively, be monoclonai antibodies.
Monoclonal antibodies may be prepared using hybridoma nxethods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
ln; a hybridonia method, a mouse, hatnster, or other appropriate host animal, is typically immunized with an iunmunizing agent to elicit lymphocytes ttiat produce or are capable of producing antibodies that will specifically bind to the i.ron.tuniziug ageni. Alternatively, ti3e lymphocytes may be immunized in vitro.
The immunizing agezit wili typioalty include Ltze PRO polypeptide or a fusion protein t$ereof. Generally, either peripheral blood l}rnp,bocytes ("Pl3Ls") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-hurnar: tnainmalian sources are desired. The lymphocytes are then fused with an inxxraortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridorna cell [Goding, Monoclonal Antibodies: Princivles and Praatiee, Academic Press, (1986) pp. 59-1031. Immortalized cell lines are usually trausforrned mapatnalian cells, particularly myeloma celis of rodent, bovine and human origin, Usually, rat or mouse myeloma cell lines are eniployed. The hybridoma cells may be cultttred in a suitable cultrue medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For exainple, if the parental cells lack the enzyme hypoxanthine guaaine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thynlid'ute ("HAT mediuni"), which substances prevent the growth of HGPRT-deficient cells.
Preferred imcnortalized cell lines are those that fuse eff.iciently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a mediutn such as HAT medium. More preferred inunort.alized cell lines are mnrin.e myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, (:alifornia azad the American Type Culture Collection, Manassas, Virginia. Human niyeloma and naouse-human heteromyeloma cell lines also have been described for the production of hunian m.onoclonal antibodies [Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., MonocIonal Antibody Production Techr,iques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture wedium iij which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioinununoassay (RIA) or enzyme-lin:ked imznunoabsorbent assay (ELISA). Such teclmiques and assays are imown in the art. The binWing affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and 13ollard, Anal. I3iochem.., 107:220 (1980).
After the desired hybridorna cells are identified, the clones may be subcloned by limiting dilution procedures and grown by s-mdard methods [Goding, su ra . Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 rnediuni.
Alternatively, the hybridoma cells may be grown in vivo as asci[es in a mainmal.
The monoc?onal wltibodies secreted by the subclones may be isolated or purified from. the culture medium or ascites f(uid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite clzromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibcdies may also be made by recombiuiant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encodiog tt-ie monoclonal antibodies of the invention can be readily isolated and sequenced using eonventior<al procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred soarce of such DNA. Once isolated, the DNA may be placed into expression vectars, which are tkken tra.usfeued into host cells such as simian COS cells, Chinese hamster ovary (CHO) eells, or myelonta cells that do not otherwise produce imnkunoglobulin protein, to obtain the s}ntthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for htutian lteavy and light chWn constant domains in place of the homologous murine sequences (U.S.
Patent No. 4,816,567; Morrison et aI. su ra or by covalently joining to the inimunoglobulin coding sequence all or part of the coding sequence for a non-imrnunoglobulin polypeptide. Such a non-immunoglobul'uipolypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigezt-combix-ting site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be mc7ovaiert antYbodias. Metlaods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobuiin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crossiitilciag. Alternatively, the relevant eysteine residues are substituted with another amino acid residue or are deleted so as to pr vent crossl=utlang.
Irc vitra methods are also suitnble for preparing monovalent antibodies.
Digestion of antibodies to produce fragments ihereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

3. Human and 1-Tunzanized Antibodies The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies. ITumaizized forms of non-liuman (e.g., inurine) antibodies are chizneric immunoglobulins, immunoglobulin chains or fragments thexeof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain tninimal sequence derived from non-human immunoglobulin.
liiunanized antibodies include human i:nmunoglobuli.-ts (recipient antibody) in which residues from a complementary detetminirig region (CDR) of the recipient are replaced by residues from a CDR of a tion-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affuuty and capacity. In some instances, Pv framework residues of the hunzan 'r.nmmunoglobulin are replaced by corresponding non-hutxtan residues. Humanize.d antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR, or framework sequences. h-t general, tlre humanlzed antibody will comprise substantially all of at least one, and typically tv/o, variable donlains, in which all or substantially all of the CDR regions correspond to those of a non-iituuatF inununoglobbul'ui and all or substantially all of the FR regions are those of a ltuman immunoglobulin consensus sequence. 7'he hunianized antibody optimally also will comprise at least a portion of aiz imntunoglobulin constant region (Fc), typically that of a human immunoglobttlin [Jones et al., ature, 321:522-525 (1986); Riechznann et al., Nature, 332,323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing tion-hunxan antibodies are well known in the art.
Generally, a humanized antibody has one or zriore arnino acid residaies hrtroduced into it frorn a source which is non-human. These non human amino acid residues are often referred to as "impor.t" residues, which are typically taken from an "import"
variable doniain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (198(i); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Soience, 239:1534-1535 (1988)J, by substituting rodettt CDRs or CDR sequences for the corresponding sequences WO 02/29888 PCT/IiSOIl27099 of a human antibody. Accordingly, such "kzunianized" antibodies are chilneric antibodies (U.S. Patent No.
4,816,567), wherein substantially less than an intact Iitmian variable domain has been substituted by the corresponding sequence fro.m a non=hutnan specles. In practice, humanized antibodies are typicaily human autibodies in which soaze CI'jR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques Icaown in the art, including phage display libraries [Hoogenboom and Winter, l. Mol. Biol., 227.381(1991); Maxks et al., J. Moi. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner ot aI. are also available for the preparation of human monoclonal antibodies (Cole et aL, Monoclonal Antibodies_and Cancer Theranv, Alan R. Liss, p. 77 (1985) and Boerner et aL, 3. Imiirmno1_ l_4711:86=95 (1991)]. Sttuilarly, human aatibodies can be made by intxoducing of human ittummoglobulin loci into transgenic animals, e.g., xnice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, Iranian antibody production is observed, which closely resentbles that seen in humans in ail respects, including gene rearrangement, assembly, and antibody repertoire. This approach is clescribed, for example, in U.S. Patent Nos.
5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific publications:
Marks et al., Bio/Teehnolou- 10, 779-783 (1992); Lonberg et al,, Nature 328 856-859 (1994); ;'diorrison, Nature 3 ,8, 812-13 (1994); Fishwild et al., Nature Bioteclutoloav 14, 845-51 (1996); Neuberger, Natuse Biotccbnology 14, 826 (1996); Lonberg and 1luszar, Internõltev. lnunuuoi. 17 65-93 (1995).
The antibodies may also be affinity maturecl using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies liave an affinity which is five times, more preferably 10 times, even more preferabiy 20 or 30 tnnes greaier than the starting antibody (generally murine, humanized or human) from which the rnatmed antibody is prepared.

4. Bispecific Antibodies I3ispecific autiboaies are monoclcnal, preferably hunian or liuntanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the PRO, the other one is for any of:ief ar;tigen, and preferably for a cell-surface protein ar receptor or receptor subunit.
Methods for niakingbispecific antibodies are knov,n iu the art.
'1'radit'ronai3.y, the recombinant production of bispecific antiboclies is based on :lte co-expression of two immunoglobulin heavy-chaiuz/light-chaia pairs, where the two lteavy chains have different specificities IMilstein and Cuello, Nature, 305:537-539 (1983)]. Beeause of tlte random assortment of imrnunoglobulin heavy and light chains, these hybridornas (quadromas) produce a potential niixture of ten diffbrent antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is iisaally accomplished by affinity ehromatograpliy steps. Similar procedures are disclosed in WO 93108829, publisne.d 13 May 1993, and, in Traunecker et al., B,MBO Jv, 30:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen conzbining sites) eaCc be fused to immunoglobulir' c.onstat.t domain seqttences. 'the fusionpreferably is with an irnmttnoglobttiinheavy-chairx constant domain, comprising at least part of the hinge, CH2, and. CH3 regions. It is preferred to have the WO 02/24888 PC'TIUS01127099 first heavy-cbain con.stant iLpioa (Cl-l:1) containing ilie site necessary for light-chain binding present in at least one of tho fusions. DNAs erec,d;.ne the i ztr.unoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inszrted in,o separatz exl)ression vectors, and are co-transfected into a suitable host organism.
For further details of generating bispecific antibodies see, for example, Suresh et al., 17ethod5 in Bnzymolo 121:210 (1986).
According to anot?:er approach described ir; WO 96/27011, the interface between a pair of antibody molecules can be engiricered to ,r,iaximize i:he percentage of heterodinzers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of tlie CH3 region of an antibody constant domain.
In this method, one or more small amino aeicl side ehains from the interface of the first antibody molecule are replaced witb. l.a.rgar side claains (e,g, tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g- alanine or tbreonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as hoinodiiners.
Bispecific antibodies can be prepared as full lengtla an.tibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispeesfic antibodies can be prepared can be prepared using chemical linlcage. Brennan et al., Science 229:81 (3.985) descr ibe a procedure wherein intact antibodies are proteolyt.ically cleaved to generate F(ab'), fragtnents. These fragments are reduoed in the presence of the dithiol complexing agent sodium arsenite to staiDitize vicinal ditl~.iols and prevent intermolecular disulfide formation. The Fab' fragments generated are then coa.vertecl. to th.i.onitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylanuae and is mixed wztla an equimolar amount of the other Fab'-TN23 derivative to forrix the bispecific antibody.
The bispecific antibodies produced can be used as agents for the setective immobilization of en7ymes.
Fab' fragntents nray be directly recovered froin E. coli and chemically coupled to form bispeeific antibodies. Shalaby et al., IjUxp. sUled, 175,217-225 (1992) describe the production of a fully lntmanized bispecific antibody F(ab')z molecule. Eaclt Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling i: a vi!ro to form the bispecific antibody. The bispecific antibody tlius formed was able to bind to cells overexpressing the ErM receptor an;i .no.rnral human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various teclinique for malcing and isolating bispecific antibody fragtnents directly from recombinant cell culture have aiso been described. For example, bispecifxc antibodies have been produced using leuciae zippers.
Kostelny et rr.l., J. lrnmiu~oL 148(5):1547-1553 (1992). The leucine zipper peptldes froni the Fos and Jun prot.eins were linked to the Fab' portions of two different antibodies by gene fusion.
The antibody homodimers were reduced at the hinge region to forin monomers azzd ttien re-oxidized to form the antibody fieterodimers. This method can also be utilired ~or the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc _ Natl. Acad. Sei. USA 90:6444-644$ (1993) has provided au alternative niechauism for making bispecific antibody fragÃnents. The fra.gznents comprise a lzeavy-chain variable domain (VH) connected to a light-chai.*t variable domain (V; ) by a linl:er which is too short to allow pa:ring between the two domains on WO 02/24888 PCT/iJS01/27099 the same chain. Accordingly, tlre Vx and V,, domains of one fragment are forced to pair with the complementary Vt, and Vu domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol_, 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared.
Tutt et al., J. Immunol_ 147:60 (1991).
Exemplary bispecific antibodies ;nay bind to two different epitopes on a given PRO polypeptide herein.
Alternatively, att auti-1'ItO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leulcocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(FcyR), sucii as FcyRl (CD64), FeyRI1(CD32) and FcyRIH (CD 16) so as to focus ceilular defense mechanisms to the cell expressing the particular PRO polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells wh.ich express a particular PRO polypeptide. These atitibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Anoflier bispecific antibody of irFterest binds the PRO polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies Heteroconjugate antibodies are also withinth.e scope of the present invention.
Heteroconjugate antibodies are composed of two covalentiy joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [tJ',S. Patent No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089:]. It is contemplated that the antibodies may be prepared in vitro using known methods in s-;rnthetic protein clremistry, including those involving crosslinldng agents. For example, immunotoxins may be consttucted using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate artd methyl-4-mercaptobutyrinnidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

6. Effector Funetion Engincerin_g It may be ciesirable to tnodiiy the antibody of the invention with respect to effector function, so as to enl.tance, e.g., the effeciiveness of the ant.ibody hn.treating cancer. T'or exanlple, cysteine residue(s) may be introduced into t'.he I'c region, thereby allowing inte;.chain disulfide bond formation in this region. The homodimeric aiitibody thus ge;ierated may have i.*nproved internalization capabi]ity and/or increased complement-mediated cell lalling and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., 3.Exa Med., ,17~:
1191-1.195 (1992) and Shopes, 1. mmunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and may tltereby have enhanced complement lysis anc; ADCC capabilities. See Stevenson et al., Anti-Cancer Dntg Desim 3: 219-230 (1989).

WO 02/24888 PCTlUS01127099 7. ~I1lIPUROC~l3 Y-.~,.at~S
'The invention also pertains to unm.anoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e. g. , an enzynaatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thcreofl, or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useiul in the generation of such immunoconjugates have been described above.
Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (fronz ,t'sea3:dosnoturs aerugiiiosa), ricin A chain, abrin A chain, modecein A chain, alpha-sarcin, .4leuritesJw-dii pcoteins, dianthin proteins, Plhytolaca amerfcanaproteins (PAPI, PAPII, and PAP-S), moniordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictoci.n, plxenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for the production of radioc=jugated antibodies. 13:amples include 212Bi 131I
1341n 90Y, and !ssRe.
Conjugates of the antibody and cytotoxic agent: are made nsing a variety of bifnnctional protein-coupling agents such as N-succiniinidyl-3-(2-pyridyldithioi) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as nin2ethyi adipimidate .HCT ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-aliclo compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-etk'tylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (suc:h as 1,5-difhioro-2,4-diaitrobenzene). For exaznple, a ricin immunotoxin cm be prepared as described in ViL-tta et al., Scieiace, 239:. 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylcne triami:sepentaacetic aaid (M.{-I)TPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/1;t02r.
In another embodiment, tlte antibody may be conjugated ta a"receptor" (such streptavidin) for utilization in tumor pretargeting wherein Zie antibody-receptor conjugate is administered to the patient, followed by removal of unbotmd conjugate from the circulation. using a clearing agent and then adaninistration of a"l'zgand" (e.g., avidin) that is conjugated to a cy--totoxic ag;;nt (e.g., a radionucleotide).

8. Ifr.muTtoiipos_asnes Tne antibodies d.is: xoseci ~ererst may also be formulated as inuvunoliposocnes. I.,iposomes containing the antibody are preparec'; by rr,etdhc3ds In3ovni :n ti.e art, sur,h as described in. Epstein et al., Proc. Nati. Acad. Sci.
USA, $2; 3688 (1985j; Iiw<t,ag et al., I'roc. !Vaii cad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.
4,485,045 and 4,544,545. 1.iposo1nes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useiiil !iposomes can be generated by the reverse-phase evaporation niethod with a lipid composition comprising p.osl U.atidylchol'ztte, claolesterot, and I'EG
=derivatized phosphatidylethanolamine (PEG-PE). Liposomes arc: extrucied throngh filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragnients of tbe antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 25'7: :>,&6-288 (1982) vza a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionall; cLnitained within the liposome. See Gabizon et al., J. National Cancer Inst., 019):
1484 (1989).

WO 02!24888 PCTf(JSf-]/27099 9. Pharmaceutical Compositions of Antibodies Antibodies specifically binding a PRO polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
If the PRO po2ypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells, 'Where atit;.body fragments are used, the smattest inhibitory fragment that specifically binds to the binding domain of the tazget protein is preferrecl.
For example, based upon the variable-region seqtiences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequenee. Such peptides car: be synthesized chemieaity andlor produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formutation herein may also contain more than one active compound as necessazy for Aze particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytoldne, chemotherapeutic agent, or growth-inhibitory agent. Such niolectxles are suitably present in combiutation in amourats that are effective for the purpose i,ntended.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interf icia! poiyrnerization, for exarnple, hydroxymethytcellulose or gelatin-vaicrocapsules and poly-(methylmethacylate) microcapsuies, respectively, iaacolloidal drug delivery systems (for example, liposomes, albumin microspheres, raicroemaisions, nP.no-pa.c ticles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remii7gton's Phannaceutical Sciences, supra.
The forzn.nlations to be used for in vivo administration znust be sterile.
This is readily accomplished by filtration througli sterile filtration membranes.
Su~tained-reJease preparat;ons may be prepared. Suitable examples of sustained-release preparations include seniipermealrle matrices of solicf hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., tilrrrs, or microcapsuies. Examples of sustained-release matrices include polyesters, hydrogels (forexa,nple, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S.
Pat. No. 3,773,919), capolVxners of i.,-giutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lacf:ic acid-glycolic acid copclyaners such as the LUPRON
DEPOT rM (injectable microspheres composed of lactic acid-glyco:tiu acid copolyrner and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
While polymers sucii as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins f.or shorter time periods. When encapsulated antibodies remain in the body for a long time, tliey may denature or aggregate as a result of exposure to nioisture at 37 C, resulting in a loss of biological activity aud possible changes in immanogenicity.
Rational strategies can be devised for stabilization depending on the mecha.tism involved. For example, if the aggregation meehanism is discovered to be interm.olecular S-8 S bond fo-nnation through thio-disulfide interchange, stabiiization may be achieved by modifying sulfhydryl residues, lvophilizing from acidic solutions, controliing tnoisture content, using appropriate additives, and developing spvcafic potymer maLTix compositions.

CA 02421056 GOU:i--V't-Za WO 02/24888 I'CTIC7S01/27099 G. ~ses f.~r anti-PI20 Antibor'ias The anti-PRO zrstiCodies of the inve?.tion have various utilities. Nor example, anti-PRO antibodies may be used in diagnostic assays for PRO, eõg., detecting its expressiop. (and in some cases, differential expression) In speci:Clc cells, t.isstles, or serun, Various diagizost.ic assay tecbniques known in the art may be used, such as cornprtitive biatding assays, direct or indirect sar.dwioh assays and immtmoprecipitation assays conducted in either S h,eterogeneous or nomc,geneous pliases [Zola, YI r.oc rrai AntiUadies: A
Mannal of Techniaues, CRC Press, Ine, (1987)) pp. 147-1581. The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable tttoiety sliouid be capable ofpzodt5cing, either directly or indireotly, a detectable signal. For example, tlae detectab;e aloie.ty rs:ay be a radioisotope, such as 3H,'SC 'zP,'sS, or 1211, a fluorescent or ohemilurninesceat com.pouzid, such as fiuoresoe"vi isothiooyanate, rhodamine, or luciferin, or an enayure, such as alknline phosplaatase, beta-g,alactosidas9 or hoi.seradish peroxidase. Any method knewn in the art for conjugating the antibody to the detectr?bTe moiety may be employed, including those methods descri.bed by Hunter et al., Nat~
1-,L4:945 (1962); Lavi.d et al., Rioflernistry, 3:?014 (1974); Painet al., 7, Inununol. Meth., 40:219 (1981); and Nygren, 3. Histnclratrt._and C~ocl~eur~, 3Qr4~07 (1982).
Ao.ti-PRCI aait,ibodies also are vse,iul for the afiinity purification of PRO
from recombinant cell cttltare or natural souroes. Ir. tlzis procPss, the antSbodies against PRO are immobilized on a suitable support, such a SephadYx resin or fi?ter paper, usutg methods we]l Inzown in the art, The imniobilized antibody then is contacted with a sample contai iag the PRO to be puxified, and thereafter the support is washed with a suitable solvent that will remove substaritially all the material in the sample except the PRO, which is bound to the iunnobilized antibody. Fizially, L:ie support is washed with anot.'ier suitable solvent diat will release the PRO from the antibody.
Thv followiatg exampies are offered for illustrative purposes only, and are not intended to fimit the soope of the present irrvention in any way, f,~C_AMPC ~S
Coriamercitt:;y avai?abte reager:ts referred to in the examples were used according to manufacturer's instxuctions unless ot11erwise indicate-d. Tl-se source of those cesls identifred In the following examples, and throUghout ttie specii;caiion; by ATCC acdession ntirnbers is tlie Ameriean'['y;?e Culture Collection, Manassas, VA.

LXAViPL:2 I; ~,~ t acell:a foinain Ilomola~v 3creenin~to Ide ti -Nova Po1YPPAtides and cDNA Bncod' ~
Lhere or Tlze extraceliular doma;n (BCD) seqaences (Including the se.cretion signal sequence, if any) from about 950 kxu,wn secreted proteir:s frox;r 1re Sv: iss-ProE: publi.c database wore used to search EST databases. The R5'I' databases inc]uded ptiblic databases (e,g., Dayhoff, Cen)~ank), and proprietary databases (e.g. LIF'IBNQTM, Lncyte Phaicn;tcetirticals, Palo Alt(,,, t'A). The .search was performed using the computer program 13LAST or CA 02421Ub5 'I,VU::5-UZ--eu BI.AST-2 (AltsclJUl er ai., Me hods ig PnzYmolopy, M:460-480 (1996)) as a comparison of the BCD protein sequerxces to a 6 frame trnns7atzon of the EST sequences. Those comparisons with a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA
sequences wiC}t the program "phxap" (Phil Green, University of Washington, Seattle, WA).
Using tais extracellular domain homology screen, consensus DNA sequences were assembled relative to the other identified EST sequences using pluap. In addition, the covsensus DNA sequences obtair,ed wete often (but not always) extendsd using repeated cycles of BLAST or BLAST-2 and pbrap to extend the consensus sequence as far as possible using ttie sources of ~:ST sequences disoussed above.
Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to idantify by PCR a eDNA library ttiat contained the sequence of interest and for use as probes to isolate a clone of tlte PAl-length coditig sequence for a PRO polypeptide.
Porward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR
product of abottt 100-1000 bp in length. The probe sequene,es are typically 40-55 bp in length. In some cases, additional oligonucleotides are syatbesired when tae consensus setluence is greater than about I-1.5kbp. I.n order ta screen several libraries for a fiill-length cloi;e, DN.:a. from ttae libraries was screened by PCR
arnplification, as per Ausubel et al., Camt rotocols irjj~Iolecular Bio qgy, with the PCR primer Dair, A positive libzary was then used to isolate clonas encoding the gene of interest using the probe ollgonucleotide and one of the primer pairs.
The eDNA libraries used to isolate tlx cDNA clones were constructed by standard methods using commercially available reagents suclt as those from Invitrogen, San Diego, GA, The cDNA was primed with oligo dT contaiawig a NotI site, linked with blunt to SalI hemi.kinased adaptors, cleaved with Notl, sixscl appropriately by gel electrophoresis, and cloned in a tiefSned orientation into a suitable cloning veator (sach as pRXB or pltl;.D; pIZY:5T3 is a precursor of pRK5D that tloes not contain the Sfil site; see, Holmes er a1. , ss:im, 253,:1278-1280 (1991)) in the unique XJzol and Notl sites.

UA~PL)soi tsou oUPNA clones v P m las S ree in 1, preparation of oliao d'1' yrinre~pNA fibraz mitNA was isolated r"rom a human tissue of iniezest using reagents and protocols fxom Invitrogen, San Diego, CA (.Fast 'i'racfc 2). Tlzis RNA was used to generate an oliga dT
primed cDNA library in the vector pRK51) using reagents and protocols from l.,ife Technolories, Gaithersburg, MD
(Super ScriptT'lasmid Systern).
In t'us procedure, +:he double str anded cDNA was si7ed to greater than 1000 bp and the SalllNotl tiuikered eDNA
was cloned into Xb.of/Notl cleaved vector. pltK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriciion enzynae site preceding the XhoIiNotl eDNA
cloning sites.

2. Pr?paratton u ar;dom primed ci7NA libr A seoonda;y :;D\'A library was generated in or.der to preferentially represent the 5' ends of the primary cDiNA cloncs. Sp6 PNA was generated fr.oin the primary library (described above), and this RNA was used to generate a randotn primed cI3NA library in the vector pSST-AMY.0 using reagents and protocols from Life Tealuiologies (Sup r Script Plasmid System, referenced above). In this procedure the double stranded eDNA was WO 02124888 PC'I'/CJSOl/27099 sized to 500-1000 bp, linkered wicL blunt to Noti adaptors, cleaved with SfiI, and cloned into SfiI/Notl cleaved vector. pSST-AMY.0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding ttie cDNA
cloning sites and the mouse aznylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase term;nator, after the cloning sites. Thus, cDNAs cloned into this vector that are fused in frame with anrylase sequence will lead to the secretion of a.mylase from appropriately transfected yeast colonies.

3. Trar:sformation and 37etectiotl DNA from the library described in. paragraph 2 above was chilled on ice to which was added electrocompetert DTI?OT3 bacteria (Life Teclinologies, 20 ml). The bacteria and vector mixture was then electroporated as reconunenfled by the manufacture.r. Subsequently, SOC media (Life'I'echnologies, 1 mi) was added and the mixture vms i.tctibated at ?7 C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampiciilin and incubatecl for 16 hours (37 C). Positive colonies were scraped of, the plates and tlie DNA was isolated from tlte bacterial pellet using standard protocols, e.g. CsC1-gradient. The purified 37N<~, was then carried oit to the yeast protocols below.
The yeast n,e~aods were divided into trrree categories: (1) Transformation of yeast with the plasmidlcDNA combined vector=, (2) Detecti.on and isolation of yeast clones secreting amylase; and (3) PCR
amplification of the iwe. t directly frorn the yeast c.olony and purilkcation of the DNA. for sequencing and further analysis.
The yeast strain used was H:1156-5A (.r1.TCC-90785). 'I'his strain has the following genotype: MAT
alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL"t, SUC', GAL'''.
Preferably, yeast mutants can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient alleles in sec71, sec72, sec62, with truncated sec 11 being most preferred.
Alternatively, antagonists (including antisense nucleoti(les and/or ligands) which interfere witki the nornial operation of these genes, other proteins implicated in this post translation Pat:hwaY e. ~ SEC61p, SEC72p, SEC62P, SEC63p, TDJ1P
or SSAIp-4p) or the complex ( b=>
formation of these proteius ~nay also be preferably employed in combination with the amylase-expressing yeast.
T'ransform~itior.- wa~ 1a~,-rformcd based on the protocol outlined by Gietz et ad., Nucl. Acid. Res., 20:1425 (1992). Transformed cells arc+re titer inociilated from agar into YEPD
complex media broth (100 tnl) and grown overnight at 30'C. 7'he Y-T-?.F'D bro-c:h was prepared as described W Z{aiser et a1,, Mathods in Yeast Genetics, Cold Spring Harbor Press, Cold :Ipring Harbor, NY, p. 207 (1994). The overnight culture was ttten diluted to about 2 x 106 cells/nxl (app*cx. C 1) intv f'reslr'YEl'I3 brotlk (500 ml) and regrown to 1 x 107 cells/ml (approx.
vDeao = 0.4-0.5).
The cells were then harvested and pz-epared for trarsformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,003 rpm for 5 miriutes, the stfuer.aatant discarded, and then resuspended into st.erile water, and centrifuged again in 50 ml falcon td.~es at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ntl, 10 m-M
'1'ris-THCI, 1 mM EDTA pH 7.5, 1.00 mM I.iZOOCCli3), and resuspended into L:iA.c/TE (2.5 ml).
Transfortnation took place by niixing the prepared cells ;100 tzl) with fteslily denatured single stranded WO 02124888 PCTlfJS01127099 salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transfotming DNA (1 wg, vol. < 10 l) in nzicrofuge tubes. Tl<v mixtere was mixed briefly by vortexing, then 40% PFG/TE
(600141, 40% polyethylene glycol-400D, 10 mM Tris-HCI, 1 mM EDTA, 100 mM Li.O0CCH3, pH 7.5) was added.
Tlus mixtura was gently mixed and incubated at 30 C while agitating for 30 naizzutes. The cells were then heat shocked at 42 C
for 15 nninutes, and the reaction vessel centrifuged in a inicrofuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TF (500 pl, 10 miM Tris-HCI. 1 rn1Vl EDTA pH 7.5) followed by recentrifngation. The cells were then diluted into TE (1 xri) and alicfarots (200 l) were spread onto the selective media previously prepared in 150 mm growth ptatee (VW'IZ).
Alternatively, instead of ttiultiple small reactions, the txansformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
The selective inedia used was a syntlictic complete dextrose agar lacking uraeil (SCD-Ura) prepared as described in Kaiser et a1., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p.
208-210 (1994). Transformants were grown at 30 C for 2-3 days.
Tl:e detection of colonies secreting amylase was performed by including red starch in the selective growth media. Starch was c.oupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely el al., Anal. Bioehent., 172:176-179 (1988). The coupled starch was incorporated into the SCD-Ura agar plates at a final concentration of 0.15 % (w/v), and was buffered with potassium phosphate to a pH of 7.0 (50-100 anM
final concentration).
Tiie positive colones were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies. Well isolated siugle colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD-Ura agar. Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly.

4. Isolation of DVA lsv PCR Amplif'ication When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 y.l) in a 96 well plate. At this tiine, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified. An aliquot of cells (5 l) was used as a template for the PCR reaction in a 25 l volume contaioing: 0.5 ,ul Ki.entaq (Clonteclr, Palo Alto, CA); 4.0 ~41 10 mM dNTP's (Perldn Elmer-Cetus);
2.5 l Kewmq buffer (Clontech); 0.25 l forward oli.go 1; 0.25 l reverse oligo 2; 12.5 l distilled water. The sequence of the forward oligonucleotide 1 was:
5' TGTAAAAC:GACGGCCAGTTAAA'1'~AGACCTGCAATTATTAA l CT-3' (SEQ ID N0:115) The sequence of reverse oligonucleotide 2 was:
5'-C.AGGAAALAC3CI'ATGACCACTGCACACCTGCAAA.TCCATT-3' (SEQ ID N0:116) PCR was then performed as follows:
a. Denabare 92 C, 5 minutes b. 3 cycles c4: L:enatazre 92 C, 30 seconds Anneal 59 C, 30 seconds Extend 72 C, 60 seeonds c. 3 cycles of: Denature 92 C, 30 seconds Anneal 57 C, 30 seconds Extend 72 C, 60 seconds d. 25 vycles of: Denatu.rc 92 C, 30 seconds Anneal 55 C, 30 seconds Extend 72 C, 60 seconds e. Hold 4 i.

The underliued regions of tla.e oli~onucleotides annealed to the ADH promoter region and the an-ylase region, respectively, and amplified a 30Ti bp tegion from vector pSST-AMY.0 when no insert was present.
Typieall.y, the first 18 nucleoticles of the 5' end ef these oligonucleotides contained annealing sites for the sequencing priiners. Thus, the total p:oduct of the PCR reaction from an empty vector was 343 bp. However, signal sequence-fused cD;vA resulted in considerably longer nucleotide sequenoes.
Following the PCR, an aliquot of the reaction (5 pl) was examined by agarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA (TI3E) buffering system as described by Sambrook et at., supra.
Clones resulting in a single strong PCR product larger than 400 bp were ftuther analyzed by DNA sequencing after purifi.cation with a 96 Qiaquick PCR c:lean-up column (Qiagen Inc., Chatsworth, CA).

E'<Y.AMPLE 3: Isolation ot cllNA Clones t:sing Si ual Al or~ ittun Analysis Various polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence findizig algorithni. develope(I by Genenteeli, Inc. (South San Francisco, CA) upon ESTs as well as clustered and asseml)ied P',5'l' fragments f'ront public (e.g., CenBank) andlor private (I.,IFESEQe', Incyte Pharmaceuticals, Inc., Palo Alto, CA) daWbases. The signal sequence algoritbm computes a secretion signal score based on the cfzaractvr of the DNA nucleotides surrounding the first and optionally the second metltionine codon(s) (ATG) at the Y-e.r.d of :he sequence or sequence fragment under consideration. The nucleotides following the first A'i'G must code for at ieast 35 unambiguous amino acids without any stop codons. If the first = ATO has the required aznino acids, t:he second is not. examined, If neither nieets the requirement, the candidate sequence is not scored, in ordec- to dete,:.uine whetiier ttie EST sequence contains an authentic signal sequence, the DNA and corresponding amino acid sequences surrounding the ATG codon are scored using a set of seven sensors (evaluation pararr,eter.s) krlown to be associated with secretion signals. Use of this algorithm resulted in the identification of rLunerous poiypeptide-encoding nucleic acid sequences.
EXAMI'Lf: 4: Isolation of cDN A clones Lncotling flunzaz~P12O Polypentidps Using the techniques described in Examples 1 to 3 above, numerous full-length eDNA clones were identified as encoduig PRO ?r ol,ypeptides as disclosed herein. Tltese cDNAs were then deposited under the terms of the Budapest Treaty with the Ameri.can Type Culture Collection, 10801 University Blvd., Manassas, VA
20110-2209, USA (ATCC.".) as sHown in Table 7 below.

wu u2124umc PCT(CTSOy/27099 Table 7 Material ATCC 13ep. No. Degoset Date DNA16422-1209 209929 June 2, 1998 DNA19902-1669 203454 November 3, 1998 DNA21624-1391 209917 June 2, 1998 DNA34387-1138 209260 September 16, 1997 DNA358801160 209379 October 16, 1997 DNA39984-1221 209435 l~Tovember 7, 1997 DNA44189-1322 209699 March 26, 1998 DNA48303-2829 PTA-1342 February 8, 2000 DNA48320-1433 209904 May 27, 1998 DNA56049-2543 203662 February 9, 1999 DNA57694-1341 203017 Jtu1e 23, 1998 DNA59208-1373 209881 May 20, 1998 DNA59214-1449 203046 Juiy 1, 1998 DN,A.59485-1336 203015 June 23, 1998 DNA64966-1575 203575 Januaty 12, 1999 DNA 82403-2959 PTA-2317 August 1, 2000 DNA83505-2606 PTA-132 May 25, 1999 DNA84927-2585 203865 March 23, 1999 DNA92264-2616 203969 Apri127, 1999 DNA94713-2561 2.03835 March 9, 1999 DNA96869-2673 PTA-255 June 22, 1999 DNA96881-2699 PTA-553 August 17, 1999 DNA96889-2641 P'T'A-119 May 25, 1999 DNA96898-2640 PJ['A-122 May 25, 1999 DNA97003-2649 PTA-43 May 11, 1999 DNA98565-2701 PTA-481 August 3, 1999 DNA102846-2742 PTA-545 August 17, 1999 DNA102847-2726 PTA-517 Aigust 10, 1999 DNA 102880-2689 PT'A-383 July 20, 1999 DNA105782-2683 P'f t',-387 July 20, 1999 0NA108912-2680 PTA-124 May, 25, 1999 DNA115253-2757 PTA-612 August 31, 1999 DNA119302-2737 PTA-520 August 10, 1999 DIv'A119536-2752 PTA-551 August 17, 1999 DNA119542-2754 i''S'A-619 August 31, 1999 WO {)2i24888 PCTIUS01127099 Table 7 (cont') ~7osit Date MttErW ATCC Ueo. No ep DNA143498-2824 PTA-1263 Pebrnary 2, 2000 DNA145583-2820 PTA-1179 Jantrary 11, 2000 pNA161000-2896 PTA-1731 April 18, 2000 DNA 161005-2943 PTA-2243 June 27, 2000 DNA 170245-3053 PTA-2952 January 23, 2001 DNA171771-2919 PTA-1902 May 23, 2000 DNA173157-2981 PTA-2388 August 8, 2000 DNA175734-2985 PTA-2455 September 12, 2000 DNA176108-3040 PTA-2824 Dccember 19, 2000 DNA190710-3028 PTA-2822 December 19, 2000 i1NA190803-3019 PTA-2785 December 12, 2000 DNA191064-3069 PTA-3016 February 6, 2001 DNA 194909-3013 PTA-2779 December 12, 2000 DNA203532-3029 1''1'A-2823 17ecember 19, 2000 17NA213858-3060 PT.A-2958 January 23, 2001 DNA216676-3083 7'TA-31.57 March 6, 2001 DNA222653-3104 PTA-3330 April 24, 2001 I.)NA96897-2688 PTA-379 Ju y 20, 1999 DNA142917-3081 PTA-3155 March 6, 2001 17NA1.42930-'?914 F'TA-1901 May 23, 2000 DNA147253-2983 PTA--2405 Augnst 22, 2000 DNA149927-2887 PTA-1782 Aprt125, 2000 These deposits were made under the provisions of the Budapest Treaty on the Tnteraationa112ecootiotx of the Deposit of Nlicroorgaxtisms for ihe Purpose of Patent Procedure and the Regulations thereunder (Budapost Treaty). This assures maiz7renance of a viable culture of the deposit for 30 years from the date of deposit. I91e deposit.s will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Tnc. and ATCC, which aSsures porma.-sent and uauestrieted availability of the progeny of the culture of the deposit to tbe public upon issuance of the pertinent patent or upon laying open to the public of any pateat appliaation, whichever. comes first, and assures availability of the progeny to one determined by the Commissioner of Patents to be entitled thereto.

'1'he assigiiee of the present application has agreed that it a culture of the materials an deposit should d7e or be lost or destroyed when cuitivated under suitable conditions, the niaterials wilt be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a iicxme to practice the invention in contravention of the rights granted under the authority of any government in WO 02124888 PC'I'lUSO1/27099 accordance wilh its patent laws.

EXAMPI.E 5: Use of PRO as a hybridization probe The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe.
DNA conzprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PRO) in human tissue cDNA libraries or humaa tissae genomic libraries.
Hybridization aiid washiuig of filters containing either library DNAs is performed under the following high stringency eonditi.ons. Iiybridization of radiolabeled PRO-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1 1o SDS, fl.?% sodiuin pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42 C for 20 hours.
Washing of the f'ilters is performed in an aqueous solution of (). ix SSC and 0.1% SDS at 42 C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO can then be identified using standard techniques lrnown in the art.

FXAMPLE 6: Expression of PRO in F. coli This example illustrates preparation of an imglycosylated form of PRO by recombinant expression in E.
coli.
The DNA sequence encoding PRO is initially amplified using selected PCR
primers. The primers should contain restriction enzyme sites which correspond to the restriction enzymae sites on the selected expression vector.
A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E.
coli; see T3olivar et a.t., Ceize, 2:95 (1977)) which contains geiles for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR
amplified sequences are then ligated into Ehe vector. The vector will preferably include sequences wYricli encode for an antibiotic resistance gene, a trp promoter, a polyhis Ieader (inclu(iing the first six STII codoYLs, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU
gene.
The ligation mixture is then used to transform a selected E. coli strain usiutg the methods described in Sambrook et al., Wr._a. Transformauts are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. i=lasinid DNA cazi be isolated a_nd confirmed by restriction analysis and DNA
sequencing.
Selected clones cai be grown overnight in liquid culture medium such as LB
broth supplemented with antibiotics. The overnigi:t cultiare r.iay subsequently be used to inoculate a larger scale culttue. The cells are then grown to a desired optical density, during whicb the expression promoter is turned on.
After culturing the cells for se=yeral more hours, the celis can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized lising various agents Irnown in the art, and the solubilized PRO protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PRO may be expressed in E. colf in a poly-His tagged form, using the following procedure. The DNA

WO 02124888 PCT/[TS01/27099 encoding PRO is initially aniplified using selected PCR primers. The prinzers will contain rest.riction enzyme sites whic:n correspond tc the restriction enzyme sites on the selected expressicn vector, and other useful sequences providing for efficient anfl reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W31 10 fuUA(tonA) Ion galE
rpoHts(htpRts) cipP(laclq). Tza.nsformants are first grown in LB contaitutzg 50 mg/ml carbenicillin at 30 C with shalcing until an O.D.600 of 3-5 is reached. ru.lttu=es are tnen diluted 50-100 fold into CRAP media (prepared by mixing 3.si'7 g(N'.f-T4),Sdn, 0.71 g sodium citrate=2H2O, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mZ, water, as well as i.10 nu'vi MPOS, pIl 7.3, 0.55% (w/v) glucose and 7 mM
3vIgS0k) and grown. for approximately 20-30 hours at 30 C with shaking.
Samples are removed to verify expression by SDS-P.4.Gr ao.alysis, aud the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
E. coli paste from 0,5 to 1 L fermentations (6-10 g pellets) is resuspended 'ui 10 volumes (w/v) in 7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid soditun, sulfite and sodium tetratltionate is added to make final concentrations oi 0.I.1VJ[ and 0.02 M, respectively, and the solution is stirred overnight at 4 C. This step results in a denatured protein witli till cystei7te residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman F Iltracentifuge for 30 min. 'The supernatant is diluted with 3-5 volumes of nietal chelate column buffer (6 M guanicliz=.e, 20 mM Tris, p?-I 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen IVi-NITA metal chclate colutnn eqsilibrated in the metal chelate coltumt buffer. The coluina is washed with additicnal buffer containing 50 mM
inudazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractious containing the desired protein are pooled and stored at 4= C.. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its antino acid sequence.
The proteins Lre refolded by dilE+.ting the sarnple slowly into freshly prepared refolding buffer consisting of: 20 mlvl Tris, pI-18.6, 0.3 M NaCl, 2.5 H4 urea, 5 mM cysteine, 20 mM
glycine and 1 mM EDTA. Refolding volumes are chosen so tiiat the zir,al protein concentration is between 50 to 100 niicrogramslrbl. The refolding solution is stirred geutly at 4 C,' for 12-36 hours. TYie refolding *eaction is quenclied by the addition of TFA to a final concentration of 0.4%, (p13 of approximately 3). Before further purification of the protein, the solutioa is filtered througb a 0.22 inioron filter and acetonit"'=ile is added to 2-10%
final coiacentration. The refolded protein is chromatograpited on a Poros 12.1/H reve.rsed nhase coluznn using a mobile buffer of 0.1% TFA with elution with a gradient af acetonitrile fron7 10 to 80%. .A.liquots of fractions widi A280 absorbance are analyzed on SDS polyacryl<unide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of rnost proteias are eluted at the lowest concentrations of acetonitrile since those species are the most compact witth their hydrophobic interiors shielded from interaction witb the reversed phase resin.
Aggregated species are usuaily eIuted at higher acetonitrile concentrations.
In addition to resolving misfolded fortrts of proteins from tl.te desired form, ttze teversed phase stel) also reinoves endotoxin from the samples.
Fractions containing the desired folded PRO taoly3)eptide are pooled and the acetonitrile removed using a gentle streavn of n.itrogun direc [ed at the solution. f'roteins are formulated into 20mM Hepes, pH 6.8 with 0.14 M sodium chloricle and 4~l znannitol by dialysis or by gel fzttration using 025 Superfi.ue (Pharmacia) resins equiiibrated in ttie forrnulation buffer aud sterile faltered.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
BX.AMPLE 7: Expression of PRO in mammalian cells This example illustrates preparation of a potentially g?ycosylated form of PRO
by recombinant expression in mammalian cellse Tlie vector, pRK5 (see EP 307,247, publislaed March 15, 1989), is employed as the expression vector, Optionally, the PRO DN.A is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO
DNA using ligation F-nethods such as described in Sambrook et al., supra. The resulting vector is called pRK5-PRO.
In one embadiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tiss~ae culture plates in medium such as DMEM
supplermented with fetal calf seram and optionally, nutrient eonrponents andlor antibiotics. About 10 g pRK5-PRO DNA
is mixed with about 1jug DNA
encoding the VA RNA gene [Thirnmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 pl of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 Ei.1 of 50 mM HEPES (pH 7.35), 280 mM NaCl11.5 mM Nal'O4and a precipitate is allowed to form for 10 niinutes at 25 C. The precipitate is suspended and added to the 293 cells a.nd allowed to settle for about four hours at 37 C. The cutture medium is aspirated off and 2 ml of 20 o glycerol in PBS is added for 30 seconds. The 293 cells are then waslied with serum free medium, fresh me3iuzn is added and the cells are incubated for about 5 days.
Approximately 24 hc,urs after the transfections, the culture medium is removed and replaced with culture medium (alone) ar cYilture rzedium contaii;ing 200 ;uCi/ml''S-cysteine and 200 Ci(ml 3SS-methionine. After a 12 hour incubation, the conditioned medivni is collected, concentrated on a spin filter, and loaded onto a 15%
SDS gel. Tite processed gel znay be drisd arzcl exposed to film for a selected period of time to reveal the presence of PRO polypeptide. The cultures contairdng transfected ceils may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In ar: alternative te.cltnique, 1=ftO may be introduced into 293 cells transiently using the dextran sulfate methofl described by Somparyrac et al., Proc. iVati. Acad. Sci., 12;7575 (1981). 293 cells are grown to maxiinal density in a spinner flask and 700 pg pRIC5-1'RO DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated witlt 20% glycercl for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containhtg tissue culture medium, 5 p.glml bovine insulin and 0.1 glml bovine transferrin. After about four days, the conditioned niedia is centrifuged and filtered to remove cells and debris.
The sample containing expressed PRO can then be concentrated and purified by any selected method, such as dialysis and/or coltunrt c:nro.r,utography.
In another embod.ianent, PRO can be expressed in CHO cells. The p12K5-PRO can be tt'ansfected into CHO cells using knowr; reagents such as CaPO4 or DEAE-dextraa. As descrioed above, the cell cultu.res can be incubated, and the nieclium replaced witj~ culture medium (alone) or medium containing a radiolabel such as 355 lwCl VJG'iS,1V.lV GVVJ-'V4- c.v VVLI tt2f24888 PCT/US41/27099 rrtethlonihe. After dete=.kting tl;e p}eserice of Pl?.O polypeptide, the culture tr.editttn may be replaced with serttm free mediunt. Preferablv, the cultures are in;::bated for about 6 days, and then the con.ditioned medium is harvested, '1'hp meditirl coutaii,ing the expressed PRO can then be concentrated and purified by any selsated cnethod.
;Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be suboloned out of the pR,TC5 vector. The sulelone insert can undergo PCR to fuse in frante with a selected epitope tag such as a poly-hie3 tag into a 13aculovirus expr.ecs:on vector, The pcly-his tagged PRO insert can then be subeloned into a SV40 driven vector containing a sele;tion U.arker auch as DHFR for selection of stable clones. Finally, the CHO cells can be transfec:ted (as described above) with the SV40 d. iven vector.
Labeling may be perfortned, as desaribed above, to verify expr.essic>n. The m.ilture medium containing the expressed poly-His tagged PRO can then be wnceziltrated and parified b+, any selected method, such as by Niz+-chelate affinity ctzromatography.
PRO may also be (-xprtissc.d in CHO andlor COS cells by a transient expression procedure or in CIIO
cells by arotlier stable expressiora procedure.
Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an TgG constivct (i mmunoadh.esin), in vrhich the coding sequences for the solable forms (e,g. extraeellular domains) of the respective prote'v7s are fused to an 1gCr1 constant region secluence corataining the hinge, CH2 and CH2 domains andlor Is a poly-1Tis tagged forrn, Followirtg PCR a_mplif'icat:oti, the respective DNAs are subcloned in a CH0 expression vector using stardard tec;~nicjaes as dascriUed in Ausubel et al. ,~urrent Prato pls of lYlolecular 13iolo , Unit 3.16, 7obrz Wiley and Sor.s (1997). CHP expression vectors are constructed to liave compatible restricon sites 5' and 3' of the 1>NA. of interest ?o allow the convenSerrt shuttli-ig of 014A's. The veotor usui expression in CHO cells is as described ira Y uca.s et al., Nstcl_ cids Res. 714:9 (1774-1779 (1996), and uses the S'V40 early promoter/enhancer to drive expression of tize oBNA of interest and c!ibydrofolate rede:etase (DHFR). DT-1P'R. expression perm4ts selection for stable zraai.utenanee oz the plastnid following transfectiory .
Twelve micrograms of the desired plasrnid DNA is introduced into approximately 10 million CH.O cells using comrne:cially avallab~.e transffsction reagents Superfect' (Qiagen), Dosper or Fugene (Boehringet Nlacuu'aeim). The cells are g:orva as described in Lucas ev al., su r.
Approxisnately 3 x 10' cells are frcxzen In an ampule Por ftuttter grunrtia and productiot ? as described below.
Tiae atnuult,s ~=Saining tne pla,aaud DNA are thawed by placement into water bath and mixed by vortexing. T n.(.- c-ontents are pipett-A into ;t cetttrifuge tube containing 10 ml.s of ntedia and centrifuged at 1000 zpm for 5 minutes. "!'he scCerntttant is aspirated and Ghe cells are resuspended in 10 mT, of selective media (0,2 ;lsn filtereci PS20 witi~ 5% 0.21<m. diafiltered fetal bovine serum), The cells are then aliquoted into a 100 mL
spinner containing 90 tul, of seleciive nledia, After 1-2 days, the cel)s are transferred into a 250 ml., spinner fWed + wzth i S0 mi seleeti e gro wib znediu,n ~tfl frcitbated ar 37 C. After another 23 days, 250 ml., 500 mL and?A00 ml, spinners are seeled with 3 x 105 cel;sl:.nL. '1'ho cell tnedia is excbanged with fresh media by centrifugation and resuspemion in praductit,g medium. Although au.y suitable CHO media may be employed, a prodtxCtiou medium. deswribad in U.S. Patent No, 5,122,469, isstued.fuoe 16, 1992 may actually be used. A 3L product9.on spinzer is scccled at 1.2 I06 celU>ImL. On day 0, the cell number pH ie determir.ed. On day 1, the spinner is c1 e9, ai a 'Y '';.

WO 02/24888 PCT/USOl/27099 sampled and sparging with t"dtered a1 is commenced. On day 2, the spinner is satupled, the temperature shifted to 33'C, and 30 rn,L af 50J g/L glucose and 0.6 zr.L of 10% antifoarn (e.g., 35 % polydimethylsiloxana emulsion, Dow Corning 3651VIedical Grade L;tnulsiort) tal;en. '1'hroughotit the production, the pH is adjusted as necessary to keep it at around 7,2. After 10 days, or imtil the viability dropped below 70%, the cell ctiÃture is barvested by centrifugation and filtering through a 0.22 ,u,m filter. The filtrate was either stored at 4 C or immediately loaded onto coluiuns for puriiicz.tion.
For the poly-flis tagged cor,s'ructs, t~h;, proteins are pur=ified using a Ni-N'i:'A coltunn (Qiagen). Before purification, i.:.idazole is added to the conditioned media to a coneentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equi'=ibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a flow rate of 4-5 rn /mi.n. at 4 C. After loading, the column is washed with additional equilibration bnffer &)d the nrotein eluted with equilibration buffer containing 0.25 M iuudazole. The laighly purified protein is subserauently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4%
mannitol, pH 6.8, with a 25 int G25 SuperPine (Pharmacia) column and stored at -80 C.
Immunoadhesin (Fe-containing) constructs are purified from the conditioned media as follows. The conditioned mediiun is pumped onto a 5 ml Protein A colhunn (Pharmacia) which had been equilibrated in 20 mM
Na phospliate buffer, pi-I 6.8. After loading, the column is washed extensively with equilibration buffer before elution with. 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecti.ng 1 ml fractions into ttsbes containing 275 uI. of. I M'I'ris buffer, pH 9. 'T'he highly purified protein is subsequently desalted into storage buffer as described above for tlre poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylainide gels aii;i by N-terminal amino acid sequencimg by Edman degradation.
Many of tlie PRO polypeptides disclosed herein were successftill.y expressed as described above.
XAIvIPLE 8: B.,xpressiclq of,PRO in'Yeast Tlte following met9;od descripes recombii-;ant expression of PRO in yeast.
I{irst, yeast expression vectors are constrncted for intracellular production or secretion of PRO from the A.DH21GAPD13 pron-toter, DNA eiicoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selec=:ed p;asm:id to direcct intracellular expression of PRO. For secretiorõ DNA encoding PRO can be cloned into the selected l;lasrnid, together with DNA encoding the A17H2'GAPDH
promoter, a native PRO signal peptide or otner mamsnalian signual peptide, or, for example, a yeast alplta-factor or invertase secretory signal/leader sequence, and. tiziker sequences (it' needed) for expression of PRO.
Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultiued in selected fermantation media. The tranaformed yeast supernatants can be analyzed by precipitation witb 10% trici3'oroacetic acid an<1 separation by SDS-PAGE, followed by staining of the gels with Coomassie Blu,~ stain..
Recombinant PRO can subsec~~asntly be isolated and purified by removing the yeast cells from the fermentation mediurn by centxifugatiori and then concentrating the medium using selected cartridge filtezs. The concentrate containing PRO may furttxer ba purified using selected column chromatography resins.
Many of the PRO i:olypeptides disclosed herein were successfully expressed as described above.

WO 02/24888 PCTftJStll/27099 BXAMFLE ): Exuression of PI:tO i. Bac~t?ovu-us-Infected Insect. Cells Tlie following rneLhod desoribes recombinant expressio,i of PRO in Baculovirus-infected insect cells.
The sequence coding for PRO is fused upstream of an epitope tag contained within a baculovirus expression vector. 5uch er:tope tags zr.clude poly-his tags and immunoglobulin tags (like Fe regions of IgG).
A variety of plasnaids *nay be em.ployed, including plasnrids derived from commercially available plasnuds such as pVL1393 (.Novageu). Cirie"y, the sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence er_~ccdizrg thc ek irstcellula* dc<naiz of a t*-ansznambrane protein or the sequence encoding the mature prctain if ihe protein is extr.acelt.ula.r is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer rLiay incorporate flanking (selected) restriction enzyrr;e sites. The product is then digested with those selectetl restriction enzymes and subcioned into the expression vector.
Recombinant baculovirus is generated by co-transfecCing the above plasmid and BaculoGold7' virus DNA
(Pharmingen) into Spodoplera f't~ugiperda ("S9") cells (ATCC CRI, 1711) nsing lipofectin (commercially available from GIBCO-FsRL). Aiter 4 - 5 days of incubation at :28 C, the released viruses are harvested and used for further amplifications. Viral irifection and protein expression are performed as described by O'Reilley et al., Baculovirus exTression veciors: A Laboratory Manual, Oxford: Oxford University Press (1994), Expressed poly-his tagged PRO cart thcn be purified, for example, by NP-chelate affi.nity chromatography as follows. Extracts are prepared from recombinant vir'as-i.mfected Sf9 cells as described by Rupert et al., NaturE, 362:175-179 (1993). Briefly, Sf9 czl.s are washed, resuspended insonicatlonbuffer (25 mL Hepes, pH 7.9; 12.5 mM IvlgClz; 0.1 tnNi EDTA; 10% glycerol; 0.1% NP-40;
0.4 M KCl), and sonicated twice for 20 seconds on ice. 'Ihe sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in. loading buffer (50 cnM phosphate, 300 nrM NaCI, 10 f glycerol, pH 7.8) and filtered through aØ45 ret .filter.
A Ni2*-NyA agarose column (corninercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equiiibrated witlr 25 mL of loading buffer. The filtered cell extract is loaded onto the coluinn at 0.5 m~., per rnixstite. I'he coltuuta is washed to baseline A?,õ with loading buffer, at which point fraction collection is started. Next, t1.e colutnn is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCI, i0 J glycerol, p~l. 6.0), wLich elutes nonspeci':icali,y bound protein. After reaching AZao baseline again, the column is develorze;l witli a 0 to 500 rnM Iminazoie gradient in the secoiidary wash buffer. One mL
fractions a:e collected arid ar:.d.yzed by SDI;:-PAGE and silver staining or VJestern blotwith Ni3j'-NTA-conjugated to alkaline phosphatase (viagen;i_ Hiacfiions contairdng the cluted !-Iislo tagged PRO are pooled and dialyzed against loading buffer.
Alternatively, purification of t1.e TgG tagged (or Fc tagged) PRO can be performed using lanown clrromatograpl's,y techn<:qnes, including for instance, Proteir. A or protein G column chromatography.
Many of the PRO polypeptides cii-~clcsed herein were successfully expressed as described above.
EXEIMPI.,T: 10: F'rwationof Antibodies tita~,-I3ind ~0 This examp,e illustxates preparation of monoelonal, antibodies which can specifically bind PRO.
Techniques for producing the moneclonal antibodies are known in the art and are described, for instance, in Godiug, supr I:nrn'õnogens that may be employed include purified PRO, fusion proteins containing PRO, WO 02/24888 PC'I'/US0I/27099 and cells expressing recor3hiziar_.t PRO oi) tl:a celi surface. Selection of the immunogen can be made by the sldlled artisan without undue experinieniation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified izt complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an ainount from 1-100 micrograrns. Alternatively, the 'smmunogen is entulsified in ivIPI_-TDM adjuvan.t (Ribi Imanunochemicai Research, Hamilton, MT) and injected into the animaI's hind foot pads. Tlie immuatized txkice are then boosted 10 to 12 days later witli additional immunogen emulsified in the selected adiuvap.t. ' i'hereafter, for several weeks, the mice may also be boosted with additional ir ccxuzzi.zatiazr injecti.ons. Seram san"Ãples may be periodically obtained fxom the mice by retro-orbital bleeding for testing in ELISA asSays to detect anti-PRO antibodies.
After a suit:-able antibody titer lias been detected, the attixzials "positive" for antibodies can be injected with a final intravenous injection of PRO. Three, to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (issing 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. l, availaf)le from A'T'CC, No. CRL 1597. 'Cbe fusions generate hybridoma cells which can then be plated in 96 well tissue culttu-e plates containinb HAT
(hypoxanthine, aminopterin, and thymidine) medium to in.hibit proliferation of non-fizsed cells, myeloma hybrids, and spleen cell hybrids.
The :rzybridoana cells wiil be screened in an ELISA for reactivity against PRO. Deternunatioa of "positive." hybridoma cells secreti%, the clesire(I rnorioclonal autibodies against PRO is within the skill in the art.
'I'he positive hybrido:na cells can be L-tjected intraperitoneally into syngeneic Balb/c mice to produce ascites caritau)ing t;e anti-1'if Ormtaoclozial autibodies. Alternatively, the hybridoma cells can be grown intissue culture flasks or roiler bot-:les, Purification of the r,nonoclonal antibodies produced in the ascites can be accomplished using aminot::;ilin sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity etiromatography based upon binding of antibody to protein A or protein G can be employed.

BXAMPLE I l; Purification of PRO Polv e iidgs lrsine Speciiic ~Ztibodies Native or recombir.ant PRO polypeptides may be purified by a variety of standard techniques in the art of protein pGUific.ation. For example, pro-PRO polypeptide, mature PRO
polypeptide, or pre-PRO polypeptide is purified by nnmunoaffazrry caror.natography using antibodies specific for the PRO polypeptide of interest. In general, an irnmunoaffinity volozn..n is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated clu=oma.tograp hic resui.
Polycional unmunogiobui_n.s ar=e prepared from mtmune sera either by precipitation witli arnnionium sulfate or by pu: ificatzon on immobilized Proteizt.t~L (Pharmacia LKB
Biotechnology, Piscataway, N.J.). Likewise, rnonoclonal antibodies are prepareci froau mouse ascites f.Iuid by ammonium sulfate precipitation or chromatography on inunnoil'rLed f7rote.ir A. :='arc;aliy- purified ittununoglobulin is covalently attached to a chromatogral;hic resin st:cl3. ~;.s CnBr-activated S1+.PI-IAROSEr' (Pharmacia LKB Biotecimology). The antibody is coupled to the resiYi, tne rc,i=_a i.a blocked, and the derivative resin is washed according to the manufacturer's instrUCLlons, Such an immunoai'fhiity coluurm is utilized itt the purification of PRO
polypeptide by preparing a fraction from cells :;ontairing 1'I2t,~> t=,o; mejttide in a solible form. This preparation is derived by solubilization of the WO 02/74888 PCT/CiSOI/27099 whole cell or of a su v cellular fraction obtained v;a ciifferential centrifugation by the addition of detergent or by other methods well IcJtov:-,i in u're art. Alternatively, soluble PRO
polypeptide containing a signal sequence may be secreted in useful qLantiii, intn the in::d iwm in which the cells are grown.
A soluble PRO poi}roeptide-cozttairaing preparation is passed over the irmnwioaffinity column, and the column is washed under coaditions tliat. allow thc preferential absorbance of PRO polypeptide (e.g., high ionic strength buffer.s in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO poiypeptide binding a sonv _r1T ?_~t:ffer sueh as approximately pH 2-3, or a bigh concentration of a chaotrcpe ,ueh as urea or tfliocyanate ion7, and PRO polypeptide is collected.

EXAMPLE 12; Dru Scre~nin~
This invention is pan7cula=rly useful for screening compounds by using PRO
polypeptides or binding fxagment tbereof in any of a variety of drug screening techniques. The PRO
polypeptide or fraginent employed in such a test rz3ay either be free in solution, affnced to a solid support, borne on a cell surface, or located intracellularly. One method of drug screening utilizes eu-karyotic or prolcaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transfornied cc ls ia competitive b'rt.ding z.ssays. Such cells, either in. viable or fixed form, can be used for standard binding assays. One may nicasure, for example, the formation of complexes between PRO
polypeptide or a fragruent attci the agent being tested. Alternatively, one can examine the diminution in complex formation betwee;z the PRO poiypeptide and its target cell or target receptors caused by the agent being tested.
i hus, t:he present inveiztion provides 7uethocis of screening for drugs or any other agents whicb can affeet a PRO polypeptide-associatc.d d:sease or disorder. I'hese methods comprise contacting such an agent with an PRO
polypeptide or fragment the:ieof and assaying (1) for the presence of a complex between the agent and the PRO
polypeptide or fragment, or (ii) for the presence of a complex between the PRO
polypeptide or fragment and the cell, by methods well known in the art, in sucit competitive binding assays, the PRO polypeptide or fragment is typically labeled. After suitable incubatiorr, free PRO polypeptide or fragment is separated from that present in bound fornx, and Tlx amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polyTeptide or to in-terfere witii tie PRO poiypeptide/cell complex.
Anotfier techr,icrue for ~rug screening prov,'.des higli thronghput screening for compounds having suitable binding affinity to a pol3j>ep66e a.n3 is described in detail in WO 84/03564, published on September 13, 1984.
Briefly stated, large nunitfers of d:ff'erent srr!all. peptide test comnounds are synthesized on a solid substrate, such as plastic pins or some otue.r sur,'ace. As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well luown in the art.
Purified PRO polyp ptide can also be coated directly onto plates for use in the aforeinentioned drug screeniag techniques. In adaition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid suppoit.
This inventio:a also contemplates the use of conipetitzve davg screening assays in wliich neutralizing antibodies capable of bi;iding'PRO potypeptide specificaliy ca:azpete with a test compound for binding to PRO
polypeptide or fragments thereof. in this zriaimer, the antibedies can be used to detect the presence of auy peptide which shares one or more antigenic deteruninants with PRO polypeptide.

EXAMPLE 13: Rational Dt-~a I1esi The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i.e., a PRO polypeptide) or of small molecules with which they interact, e.g., agonists, antagonists, or inhibitors. Any of these exanil les cari be used to fashion drugs which are more active or stable forms of the PRO
polypeptide or whieh enhance or iuiterfere witil the function of the PRO
polypeptide fta vivo (c.f., Hodgson, BiolTechnoF,QLN, 2: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO
polypeptide-inhibitor compiex, is determined tiy x-ray crystallography, by coniputer niodeling or, most typically, by a combination of the two approaches. Both the stiape and charges of the PRO
polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule.
Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins.
In both cases, relevant structural inforno.ation is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors, Useful examples of rational drug design may include molecules which have improved activity or stability as sbown by Braxton and Wells,l3ioc e istry, 3I:7796-7801(1992) or which act as inlr.ibitors, agonists, or aniagonists of native peptides as shown by Athauda et at., J.
Biochetn., 113:742-746 (1993).
It is also possible ro isolate a target-specific antibody, selected by fanctional assay, as described above, and then to solve its crystal structure. This approac7, in principle, yields a pharmaeore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypie antibodies (anti-ids) to a functional, pharmacologically active a:.itibody. As a mirror image of a mirror image, the binding site of the anti-ids wauld be expected to be an analog o-i the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemicaily or biologically produced peptides. The isolated peptides would then act as ttie pharmacore.
By virtue ot' the present invention, sufficietat amounts of tlie PRO
polypeptide may be made available to perform such analytical studies as X-ray crystal<ography. In addition, lanowledge of the PRO polypeptide amino acid sequence provided iaer,~in will provide guidance to those employing cotnputer modeling techniques in place of or in addition to x-ray crystallography.

EXAMPLE 14: Abitity of pR.O l'oiyt~eUtides to Sti;nulate tbe Release of Proteok;lvcans from CartiIage (Assax The ability ot'vrious .E'RO polypeptides to stimulate the release of proteoglycans from cartilage tissue was tested as follows.
The metacarphophalangeal joint of 4-6 inorAth old pigs was aseptically dissected, and articular cartilage was removed by free hand .l:oialg being caref~!l to avoid the tuiderlyin.g bone. The cartilage was minced and eultured in bu?1c for 24 hc tirs in a humidified atinosprere of 95% air, 5%
COz in sero.m free (SF) media (DME1F121;1) with 0.1 % BSA and 100Ulml penicillin and 100liglmi streptomycin.
After washing three times, approximately 100 nag of articular cartiiage was at iquoted into znicronics tubes and incubated for an additional 24 izours in the a:~ove SF media. 1''RO polypeptides were then added at 1%
either alone or in combination with 18 ng/znl uiterleulcin-1a, a known stimolator of proteoglyczut release from cartilage tissue. The supernatant was then harvested and assayed for the ax-ncun.t of proteoglycans using the 1,9-dimethyi-rnetllylene blue (.UMB) colorinietric assay (Fatizdale and k3uttle, Biochem. Biophys. Acta 883:173-177 (1985)). A positive result in this assay indicates that tlze ~est po7.ypeptide will find use, for example, in the treatment of sports-related joint problems, : rl:icruar cartilage; defects, osteoartliritis or rheumatoid arthritis.
When various PRO pol,,Teptides v,ere tested in the above assay, the polypeptides demonstrated a marked ability to stirz-1ate release of proteoglycans froxn cartilage tissue both basally and after stimulation with interleukin-la and at 24 and 72 hours after treatment, thereby indicating that these PRO polypeptides are useful for stimulating proteoglycan release from cartilage tissue. As such, these PRO
polypeptides are useful for the treatment of sports-+_=elated joint pr(;blems, articular cartilage defects, osteoarthritis or rheumatoid arthritis.
PRO6018 polypeptide testing positive in this assay.

MPI,F~iS: )Ilinjan Micravascular I?ndotlielial Cell Proliferation Assay 146 This assay is design~~d to determine whether PRO polypeptides of the present invention show the ability to induce proliferation of l.uman rzticrovascular endothelial cells in culture and, therefore, function as useful growth factors.
Or, day 0, bumim n:;icrova.scular endothelial cells were plated i.n 96-well plates at 1000 cells/well per 100 microliter and incubatcd overnight in complete media [EBM-2 growth media, plus supplements: IGF-1; ascorbic acid; VP;GF; i7ECrF,; I~I Cx ; tzyci:=ocortisone, gentaezicin (GA-lOCO), and fetal bovine serum (FBS, Clonetics)].
On day 1, coniplete media was replaced by basal media [EBM-2 plus 1% FBS,j and addition of PRO polypeptides at 1%, 0.1 % and 0.01 %, On day 7, an assessment of cell proliferation was performed using the ViaLight HS
lcit [ATP/lucifera5e Lurnitechl, Results are expressed as % of tiie cell growth observed with control buffer.
Tite fullowing P:,G polypeptides stimulated human raiarovascular endothelial cell proliferation in this assay: PRG1313, z'R020050, and PR021383.
The following PRO polypeptides inhibited human microvascular endothelial cell proliferation in this assay: PRO6071, PRO448 I; ancl PRO6906.

EXAMt?LB l~i: 109icroazr~ Analysis to L)etsct t~verexpressicrn of P O
Polvnentides in Cancerous Tumors Nucieic acid microarrays, ofteza containing thousands of gene sequences, are use:Cul for identifying differentially expressed genes in diseased tissues as compared to ilieir normal counterparts. Using nucleic acid microarrays, test and control zr.P.tJA samples from test and control tissue samples are reverse transcribed and labeled to generate ci-3NA probes. 1'lie cr)NA. probes are th.c;n hybridized to an array of nucleic acids immobilized on a solid support. The array :s :.onfigured such that the sequence and position of each member of the array is lmown. For example, a sclection of genes known to be expressed 'ui certain disease states may be arrayed on a solid support. llybri<iizltiorl or a labeled probe witlz a particzalar array member indicates that the sample from which the probe was dQrived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sanipie is greater thzn h;vbridizatiun signa?, of a probe from a control (normal tissue) sample, the gene or genes \...t1 VG!'G-LVJV lVV.J- VG--GV
WO 02/24888 PCTlUSOI/27099 overexpressed in the disease tissue are identified. The implication of tlt,is result is that an overexpressed protein in a disease.d tissue is useful not only as a d'xagnostic marker for the presence of the disease condition, but also as a tl,erapautic target for treatsnent of the disease eondition.
The melIodoloU of hybridization of nucleie acids and raicroarray tecitnology is well known in the art;
In the present examplc, tbe specific preparation of nucleic acids for hybridization and, probes, slides, and hybridization coaditions are all detailad in U U.S. Provisionai Patent Application Serial No, 60/193,767, filed on March 31, 2000.
In the present e:tample, oarxerous turaors derived from various human tissues were studied for PRO
potypepifde--encoding getie expression relat'rv~e to non-cancerous human tissue in an attempt to identify those PRO
polypentides ~vhich are overeapressed in carr.ernus umors. Cancerous htunan tumor tissue from any of avariety of different btjman t=ar=.iors was ol~tained and compared tc a"universal"
epithelial control sample which was prep4red by pooiiny non-cancerous b~aixtan tsst?es of epithelial origin, including liver, kidney, and lung. mRNA
isolatcd from the pooled tissues represents a mixture of expressed gene products from these different tissues.
Microarray hybridization exporiments using the pooled control samples generated a linear plot In a 2-color analysis, The slope of tPie ii.ne generated in a 2-color analysis was then used to norrnalize the ratios of (test:control detectictiu) witl?in cach cxperimerit. The norxr.a.lized ratios from various experinients were then conipared andused to identify clustering of gene expression, Tbus, Lie pooled "universal control" sample not only allowed effective rela'ivc gene expression deterininatio,*ts in a simple 2-sample comparison, it also allowed multi-sample comparisons across several experiments.
lr. the presen~ experiments, nucleic acid probes derived from the herein described PRO polypeptide-en.codir.g nucieic aciL sequences tvere used in the creation of the znicroarray'and RNA from a panel of mine different tumor tissues (listed below) were used for the hybridiaation thereto. A value based upon the normalized ratio:experimeW9I ratio was designated as a"cutoff' ratid', Only values that were above this cutoff ratio were determined to be siguHicant. Table 8 below shows tbe results of these experiments, demonstrating that various PRO polypeptides o, the present invention are significantly nverexpressed in various hurnan tumor tissues, as conapared to a rton-cancerous h.uman tissue control or otber numan tumor lissues. As described above, these data demon.5!sate that tz e f'P.t) polypep!ides of the present invention are useful not onzy as diagnostic rnarkers for the preseace of one or :~,,ore c:,ncerous tumois, but also serve as rherapeutic targets for the treatment of those tumors.
TAST,.B
Molecnle ir vve4x -xl'eSet?~ir,: as com.pared tn normal controI:
k'R0240 oreast :un or universal normal control 1'1tU21110 Iung t:un~ior univer.sal normal control P.uOt56 colou =.u~,I,or universai normai control PetO'u56 i;ang ru;nor universal normal control 1'R0256 bt-east twnor universal normal control PR0305 :oloa tusnor universal normal wntrol P'?.C~=06 :-X-Ig 'u~or universal nor-xnal control :3G

TA13LB 8õf,ont' , Molecule ~s cserax~esse3 in: as egmp,ared to normal control;
PR0540 luvg tranor universal normal control PR0540 colon tumor universal normal control PR0773 breast tLunor universal nornial control PR0773 colon tsn:or iuriversal normal control PR0698 Colon turnor universal normal control PR0698 breast tumor mji.versal normal control PR0698 lur.g tumor uiiiversal normal control PR0698 prostate Wunor unlversal normal control PR0698 rectal tunxor universal normal control PR03567 colon tumor universal normal control PR03567 :;rcast t,~~or universal normal control PR03567 ;ufag tu!.tor universal normal control PR0826 -a?on Luror uaiversal normal control PR0826 l11uõ tixtrior universal normal control PR0826 breast turaor universal normal control PR0826 rc::tal t.r:r r uiuve,sl normal control PRfl826 'iver :uirar universal normal control PR01002 uuiverszJ normal control PR01002 Lnr trLmor Asniversa?. riorniai control PRO1068 K_ _'o nor utii.versal nortual control PRO1068 :iniveFsal normal control PRO1030 colon ts.imor tmiversal normal control PR01030 :>rc<:,t t_r,or u.nzversal normal control PRO1030 tr; :~i+.r,or universal normal control PRO1030 x.outatr "umor nniversal normal control PRO 1030 c ;;t~l i.inlc- universal normal control PRO4397 co'or< rur,or universal normal contral Pl'.04397 un'rve~sa'. normal control PR04344 eol.or: tuanor universal nornial control PR04344 tnng ttrfr(or universal normal control Pr?04344 ;Itmor universal normal control PR04407 universal normal control P204407 urlversal normal control PR04407 u.niversal normal control PR04407 i. c t~.;~ cr universal normal control PR04407 t nivezsal ncrma? contro'.

PR04316 rc'r.,~,. :-11or unilmrsa_l normal control PRO4315 :;; r= t T~,~or univer:sal norznal control PR05775 ca1.o11 A3111or itnivr.-rs ,_ ncrmai control PR06016 ;?: '. ~~_ Tnivcrs {l .ncrr~al control WO 02124888 PCTIlJS01/27099 TAI3'LE 8 fcont') olecule is overexpressed in: as co vared to normal control:
PR04980 breast tum.or universal normal control PR04980 colon tumor universal nozmal control PR04980 inng tiunor uni.versal normal control PR06018 cololi tumor universal normal control PR07168 colon turaor universal normal control PR06000 colon tumor universal normal control PRO6006 colon tumor universal normal control PR05800 co"'c?n tumor universal nornial control PR05800 br~east turnor universal normal control PR05800 luml; tumor uiuversal normal control PRO5800 tumor universal normal control PR07476 tiolo:_ uruor universal normal control PIZ010268 colo,~ tumor universal normal control PR06496 c.cli;m. turnor -aniversal normal control PR06496 breast tumor universal n.ormal control PR06496 lunj; tumor universal normal control PR07422 ~'x, tunc r u uve.rsal no:mal control PR07431 .;>,=,~ tumor universal normal control PRO28633 colon tumor universal norinal control PR028633 l.ung nunor miiversal normal control PR028633 liver tumor universal normal control PR021485 colon tuanor universal normal control PRO28700 ci as r.aa?or tlniversal normal control PR028700 >:ztx er universal normal control PR028700 colon tiunor universai. normal control PR034012 co'on ttimor universal normal control PR034012 1i-.ra 'urnor tmivorsa.l norsnal control PR034003 l tu..rr.or universal normal control PRrJ34003 1-_o, ~}mor universal normal control PR034001 colon tumor universal normal control PR034009 colon tumor universal nornzal control PR034009 _: a tctmor universal normal control PR034GC9 universal normal control PR034009 r<>.ctal tumor universal normal control PR034192 ;s~~nor universal normal control WU 02/248t38 PCTICJS01127099 'I'ABLE 8 (coni:') olecuie is overe~ressed in: as cnMared to normal control:
PR034564 colon tinnor universal nonnal control PR035444 colon tumor universal normal control PR05998 cclon tumor universal normal control PR05998 1ur; tcurlor universal normal control PR05998 k7duray t-lazn.or universal normal control PR019651 colon t-mYnor universal normal control PRU20221 liver tismor universal normal control PR021434 liver tumor universal normal control EXAMPLE 17: Fetal Hemoglobin Induction in an Erythroblastic Cell Line (Assay 107) This assay is useful ~,r screening PRO polypeptides for the ability to induce the switcli from adult hemoglobin to fetal hemoglobin in an erythroblastic cell line. Molecules testing positive in tltis assay are expected to be useful for therapeutically treating various mammalian hemoglobin-associated disorders such as the various thalassemias. 7'he assay is performed as follows. Frythroblastic cells are plated in standard growth medium at 1000 cells/well in a 96 we'I fernaat. PRO polypeptides are added to the growth mediuni at a concentration of 0,27o or 2% and the cells are incubated for 5 days at 37 C. As a positive control, cells are treated with I04uM
hemin and as a negative control, the cells are untreated. After 5 days, cell lysates are prepared and analyzed for the expression of gamma globin (a fetal marker). A positive in the assay is a gamma globin level at least 2-fold above ttze negative control.
PR020080 polypeptide tested positive in this assay.

EXAMPLE 18: Microarrav Analysis to Detect Overexpression of PRO Polypeptides in HUVEC Cells Treated witl'i Gro)e;Rh Factors This assay is designed to determ3zae whetlter PRO polypeptides of the present invention show the ability to induce angiogenesis by sti;nuiating endotheliai cell tube formation in I-IUVEC cells.
IVucleic acid microarrays, often containing thousands of gene sequences, are useitil for identifying differentially expressed genes in tissues exposed to various stimuli (e.g., grovc*,.h factors) as compared to their normal, unexposed counterparts. Using nucleic acid microarrays, test and contzol mRNA samples from test and control tissue samples are raverse transcribed and labeled to generate cDNA
probes. The eDNA probes are then hybridized to an array of mtcleic aeids immobilized on a solid support. The array is configured such that the sequence and position of each rctem.ber of the array is Irnown. Hybridization of a labeled probe with a particular array member indicates that the sa-mple from which the probe was dexived expresses that gene. If the hybridization signal of a probe from a test (exposed tissue) sample is greater than hybridization signal of a probe from a control (normal, unexposed tissue) sampie; the gene or genes overexpressed in the exposed tisstie are identified. The WO 02,'24888 PC3'/i3S0.tt27099 implication of this result is that an overoxpressed protein in an exposed tissue may be involved in the functional changes within the tissue following exposure to tlte stim.uli (e.g., tube for7nation).
The methodoiogy of hybridization of nucleic acids and microarray technology is well imown in the art.
In the present example, the specific preparation of nuoleic acids for hybridixation and probes, slides, and ktybrid'azation eortdit:ioits are all detailed in U.S. Provisional Patent Application Seria113o. 60/193,767, filed on March 31, 2000.
In the present example, HUVEC cells grown in eitlier collagen gels or fibrin gels were induced to form tubes by thA addition of various growth factors. Specifically, coIIagen gels were prepared as described previously in Yang et at., Aznerica;z T. Patizalogy, 1999, 155(3):887-895 and Xin et af.,Americruc J. f'atH.alogy, 2001, 158(3):1111-1120. po?towir:g gelation of the HWEC cells, IX basal med'ntrtt containing M199 supplemexxted with 1%TtBS, I;: T'I'S, 2 rtM Z.-glutamiate, 50 tagCnll ascorbic acid, 26.5 mM
NaHCO3, 100C7!ni1 penicillin and 100 CJimi streptom,ycEu was added. 'pube formation was elicited by the inclusion in the culture media of eithet a ntixture of phorbol myrsitate acetate (50 nM), vascular eudothelial cell growth factor (40 nglznl) and bas9c fibroblast growth factar (40 ngfml) ("PMA growth factor mix") or hepatocyte growth factor (40 ngJml) and vascular endrn?aelial ce;li growth factor (40 nglml) (140plVHCrP mix) for the indicated period of time, Pibrin Cr is wexe prepared by susnending Huvec (4 x I05 cellslmi) iu iVl199 containing 1%
fetal bovine serum (IIyclone) and human fibrinogen (2.5nighml). Thrombin (50IJJm1) was then added to the fibrpnogen sirspension at a ratio of 1 nart thrombin solution:30 parts fibrinogen suspension. T;1e solution was then layered onto 10 era tissue aulture plates (total volume: 15 i:tliplatr=) and allowed to solid'rfy at 37"C for 20 min. Tissue culture media (10 ml of BM
containir.g PMA (50 aM), bpCF (40ngi tnl) and V f!Giz (40 ng/ml)) was thezt added and the cells incubated at 37 C
irr 5%CO, in air for the :.ndicated period of time, Total RNA wns extracted from the ALT'V;'SC cells incubated for 0, 4, 8, 24, 40 and 50 hours in the different matrix and iyiedYa combinations using a TRTzol extraction followed by a second purification using RNAeasy Mini Kit (Qiagen). The total RNA was used to prepare cRNA, which was then hybridized to the rnicroarrays.
In the present experiments, nucleic acid probes derived from the herein described PRO polypeptide-encoding nucleic acid sequences were used in the creation of the rnicroarray and RNA from the HUVEC cells described above -wern used for the hybridization thereto, Pairwise comparisons were made using time 0 chips as a baseline. Thret. repticate samples we;e a.tzalyxed for each experimental condition and tirne. Hence thexe were 3 tizne 0 samples for eao,a tceatment and 3 replicates of each successive time point. Therefore, a 3 by 3 comparison was perfomaed for eaal: time point conlpnred against eacla time 0 point. Tbis resulted in 9 comparisons per tiniP poiiit. Only those genes that had increased c.xpression in all three non-time-0 repticatcs In each of the different -matrix and media combinations as compared to any of the three time zero replicates Wex6 considered positive, A2G:coagla this stringent method of data analysis does allow for false negatives, it minumizes fa?se positives.
PRO2,81, PiZC 156C, PiZ~,7189, PR04499, PTtO630$.1'R.O6000, PR010275,1'IL021207, I'R020933,aud PC?034274 tested nosit:v. 'sn tltis assay.

i j WtP W124nntt PCT/US01/27099 I;XAMPLE 12: Ttr.nlcz_ Vervis IlqrmaI Ditferentiat Tissue Expx ession Distribution Oligonucleotide probes were constructed from some of the PRO polypeptide-encoding nucleotide sequences slzav,+n in the accompanying ntow=es for use in quantitative PCR
amplification reactions. The oligonucleotide pr.=obes were c;3osen so as to give an approximately 200-600 base pair amplified fragment from the 3' end of its associated. template in a standard PCR reaction. 7'lte oligonucleotide probes were employed in standard quantitative PCR amplification r.eactions with cDNA libraries iso2ated frarn different hirman turnor and normal human tissue samples ancl analyzed by agarose gel electrophoresis so as to obtain a quantitative determination of tEze level of expression of tlte 1'RO polypeptide-encoding uueleic acid in the various tumor and normal tissues tested. P-acti:t was i:sed as a can.trol to assnre enat equivalent amounts of nucleic acid was used in each reaction. Identificatit?n of the differential expression of the PRO
polypeptide-encoding nucleic acid in one or more tumor tissues as conapared to one or u.are normal Gssues of the sanie tissue type renders the nzolecule usefu.l diagncstically for the deterniu.ation of the presettce or absence of ht.mor in a subject suspected of possessing a tumor as well as t.tierapeutically as a target for the treatznent of a tumor in a subject possessing such a tumor.
These assays provideci the fc1 c,wing results:
(1) DNA161005-2943 molec.r e is very highly expressed in tauman wnblilical vein endothelial cells (HUVEC), substantia niagra, hippocampus tud deudrocytes; hignly expressed in lymphoblasts; expressed in spleen, prostate, uterus and macrophages; a.trl is weal:ly expressed in cartilage and heart, Among a panel of normal and tumor tissues examined, it is expressed in esophageal tn??aoy:, and is not expressed in normal esophagus, normal stomach, stomach turnor, n.ormal kldr.:ev, kidney tun:orõ nc-rmall lung, lu.ng tumor, normal rectum, rectal tumor, normal liver and, liver tanror, (2) DNA170245-3053 inolecule is highty expressed in castilage, testis, adrenal gland, and uterus, and not expressed in 1-1tJVEC, colon tun-.tor, heart, placenta, bone marrow, spleen and aortic endothelial cells. I.n a panel of tumor and normat tissue samples examined, the DNA170245-3053 molecirte was found to be expressed in normal esophugus and esop'rtagtal rtttnor, expressed in noru.al stomacli and in stomacll tumor, not expressed in normal kidney, but expressed in kidney tumor, not expressed in nornial lung, but expressed in lung tumor, not expressed in normal rectum tior in rectal t,jrnor, and not expressed in normal liver, but is expressed in liver tumor.
(3) DNA173157-2981 r,u;;.e;;ulc is significantly expressed in the followfmg tissues: cartilage, testis, HUVEC, heart, placeiiia, bone tmrrcw, adrenaJ. gland, prostate, spleen, aortic endotlzelial cells, and uterus. When these assays were conducted oi2 a t:; nor tissue panel, it was fouaid that the DNA173157-79$1 molecule is significantly expressed inthe following tjssues: norznai esophagus aYid esaphagial tumor, rormal stomacIZ and stomach tumor, normal ?cidrtey a:ad kidney normal lung and lung tumor, normal rectum and rectal tttmor, normal liver and liver tumor, and colon tumor.
(4) DNA175734-2985 in.alecule is sigiiificantly express+;d in the adrenal gland and the uterus. The DNA175734-2985 molecule is not signiticautly expressed intlie following tissues: cartilage, testis, HUVEC, colon tnmor, lieart, placenta, bone ma:-row, prostate, spl;:en and aortic endothelial cells, Screening of a tumor panel revealed that DNAl'75734==2.985 is signific:antly expressed i-a normal esophagus but not in esophagial tumor.
SimilarIS,, while highly exp;. in nornaal rec.turr, DNA175734 2985 is exi)ressed to a lesser extent in rectal WU 02124888 PCT/IJSO1f27099 tumor. DNA175734-2985 is expressed eaually in normal stomach and stomach tumor as well as normal liver and liver tunlor. While not expressed in normal iddney, DNA175734-2985 is highly expressed in kidney tumor.
(5) DNA176108-3040 molecule is highly expressed in prostate and uterus, expressed in cartilage, testis, heart, placenta, bone marrow, adrenal gland and spleen, and not significantly expressed in HiJVLC, colon tumor, and aortic endothelial cells. In a panel of tumor and normal tissue samples examined, the DNA176108-3040 molecule was found to be liiglaly expressed in normal esophagus, but expressed at lower levels in esophagial tumor, highly expressed in normal stomach, and expressed at a lower level in stoniach tumor, expressed in Iddney and in kidney tumor, expressed in nortn,al rectam and at a lower level in rectal tumor, and expressed in normal liver and not expressed in liver tumor.
(6) DNA191064-3069 rnolecule is significantly expressed in the following tissues: cartilage, testis, HUVEC, heart, placenta, bone marrow, adrenal gland, prostate, spleen, aortic endothelial cells, and uterus and not significantly expressed in colon tumor. In a panel of tumor and normal tissue samples, the DNA191064-3069 molecule was found to be expressed in normal esophagus and in esopliagial tumors, expressed. in normal stomach and in stomach tumors, expressed in normal Iddney and in kidney tumors, expressed in normal lung and in lung tumors, expressed in normal rectum and in rectal tumors, expressed in normal liver and in liver tumors.
(7) DNA194909-3013 znolecule is highly expressed in placenta, and expressed in cartilage, testis, HUVEC, colon tumor, heart, bone marrow, adrenal gland, prostate, spleen, aortic endothelial cells and uterus. In a panel of tumor aiid normal tissue samples examined, the DNA194909-3013 molecule was found to be expressed in normal esophagus and expressed at a lovrer level in esopliagial tumor, not expressed in normal stomach nor stomach tumor, expressed in normal kidney and kidney tutnor, expressed in normal lung and lung tumor, expressed in nonnal rectum and rectal tumor, and not expressed in normal liver, but is expressed in liver tiunor.
(8) The PR034009 encoding genes of the invention (DNA203532-3029) were screened in normal tissues and the following primary tumors and the resulting values are reported below.
'l-amor Panel:
PR034009 encoding genes were expressed 39.3 fold higher in lung tumor tiaan normal lung. It is expressed 9.5 fold higher in esophagial tumors than normal esophagus. It is expressed 6.7 fold higher in Iddney tumor than normal lddney. It is expressed 4.0 fold higher in colon tumor than nornial colon. It is expressed 2.7 fold higher in stomach tumoz than normal stomach. It is expressed at siniilar levels in normal rectum and rectal turnor, nornral liver a,.zd liver tumor, normal uterus and uterine tumor.
Normal Panel:
For the normal tissue values, the normal tissue with the Iughest expression, in this case normal thymus, was given a value of 1 and atl other normal tissues were given a value of less than 1,and described as expressed, weakly expressed or not expressed, based on their ex ression relative to thymus. PR034009 encoding P genes were expressed in normal thymus. It is weakly expressed in lymphoblast, spleeu, heart, fetal limb, fetal lung, placenta, HUVEC, testis, fetal Icidney, uterus, prostate, macrophage, substantia nigra, hippocampus, liver, skiiz, esophagus, stomach, rectum, lcidney, thyroid, skeletal muscle, or fetal articutar cartilage.
It is not expressed in bone marrow, fetal liver, colon, lung or dendr=ocytes.
(9) DNA213858-3060 ntolecule is not significantly expressed in cartilage, te.stis, HUVEC, colon tumor, heart, WO 02/24888 PC"IY'(7S01/27099 placenta, bone marrow, adrenal gland, prostate, spleen, aortic endothelial cells or uterus. In a panel of tumor and nornzal tissue samples examined, the DNA213858-3060 molecule was found to be expressed in normal esophagus and esopliagial tumor, expressed in nornial stomaclt and in stomach tumor, expressed in normal kidney and and kidney tumor, expressed in normal lung and in lung tuanor, expressed in normal rectum and in rectal tumor, and expressed in normal liver and in liver tunzor.
(10) DNA216676-3083 molecule is significantly expressed in the following tissues: testis, heart, bone marrow, and uterus, and not significantly expressed in the following tissues:
cartilage, HUVEC, colon tumor, placenta, adrenal giand, prostate, spleen, or aortic endothelial cells In a panei of tumor and normal tissues samples exarnined, the DNA216676-3083 molecule was fotwd to be expressed in normal esophagus and esophagial tumor, not expressed in normal stomach, but isexpressed in stomach t.umor, not expressed in normal Iddney nor in kidney tittnor, not expressed in nornial lung, bat is expressed in hmg tumor, not expressed in normal rectum, but is expressed in rectal tumor, and not expres'sed ir, jiorznal liver nor in liver tumor.
(11) DNA222653-3104 nt.olecule is significantly expressed testis, and not significantly expressed in cartilage, HUVEC, colon tumor, heart, placenta, bone marrow, adrenal gland, prostate, spleen, aortic endothelial cells and uterus. In a panel of tumor and normal tissue sarnples examined, the DNA22653-3104 niolecule was not expressed in nornial esophagus, esophagial tumor, norraal stomach, stomach tumor, normal kidney, lddney tumor, normal liuag, lung tumor, normal rectum, rectal tumor, normal liver and liver tumor.

EXAMPI.E 20; Guinea 1' Vascular Leak Assay -n This assay is designed to determine whether PRO polypeptides of the present invention show the ability to induce vascular permeabi.lity. Polypeptides testing positive in this assay are expected to be useful for the therapeutic treatment of conditions wl?ich woirld benefit from enhanced vascular perLteability including, for example, conditions which may benefit froin enhanced local immtnte system cell in~ltration.
Hairless guinea pigs weighing 350 grams or more were anestbetized with Ketamine (75-80 mg/kg) and 5 mg/kg Xylazine intramuscularly. Test saniples containing the PRO
polypepti(ie or a physiological buffer without the test polypeptide are injected into slcin on tlte back of the test animals with 100 1-cl per injection site intradermally. There were approximately 16-24 htjection sites per animal. One trJ. of Evans blue dye (1% in PBS) is then injected intracardialle. Skin vascutar permeability responses to the compounds fi.e., blernisbe's at the injection sites of injection) are visually scored by xneasuring the diameter (in mm) of blue-colored leaks from the site of zujection at 1 and 6 ho:zrs post adntinistration of the test materials, The mm diameter of blueness at the site of hijection is observed and recorded as well as ttte severity of the vascular leakage. Blemishes of at least S mm in diameter are considered positive for the assay when testing purified proteins, being indicative of the ability to induce vascular leakage or permeability. A response greater than 7 ruan diameter is considered positive for conditioned media samples. Iiu.man VEGF at 0.1 yg1100 k4l is used as a positive control, inducing a response of 15-23 mm diaineter.
PRO19822 polypeptides tested positive in tfiis assay.

WO 02/24888 PCTYt1St)1127099 EKAIbIPL?i. 21: Skin Vascular Permeabilitv Assav (Assav 64), This assay shows that certain polypeptides of the invention stimulate an immune response and induce inflammation by inducing mononuclear cell, eosinophil and PMN infiltration at thz site of injection of the animal.
Compounds which stimulate an immune response are useful therapeutically where stimulation of an irnmune response is beneficial. This sldn vascular permeability assay is conducted as follows. Hairless guinea pigs weighing 350 grams or znore are anesthetized with ketamine (75-80 nzg/Kg) and 5 rnglKg xylazine intramuscularly (IM). A sample of purified polypeptide of the invention or a conditioned media test sample is injected iutradermally onto the backs of the test animals with 100 l per injection site. It is possible to have about 10-30, preferably about 16-24, injection sites per animal. One l of }svans blue dye (1 % in physiologic buffered saline) is injected 'urtracardially. Blemishes at the injection sites are tlien measured (mm diameter) at 1 hr and 6 hr post injection. Anibnals were sacrificed at 6 hrs after injection. Each skin injection site is biopsied and fixed in formalir,. The skins are then prepared for histopathologic evaluation. Each site is evaluated for inflammatory cell infiltration into the slr'n. Sites with visibAe iaflanunatory cell inflammation are scored as positive.
Zuflammatozy cells may be neutropbilic, eosinophil'zc, tb.onocytic or lymphocytic. At least a mininial perivascular infiltrate at the izajectian site is scored as positive, no infiltrate at the site of injection is scored as negative.
P12O19822 poiypeptide tested positive izz this assa.y..

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Claims

1. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:112, wherein the nucleic acid is overexpressed in liver tumor.

2. Isolated nucleic acid having at least 80%, nucleic acid sequence identity to a nucleotide sequence shown in SEQ ID NO:111, wherein the nucleic acid is overexpressed in liver tumor.

3. Isolated nucleic acid having at least 80% nucleic acid sequence identity the full-length coding sequence of the nucleotide sequence shown in SEQ ID NO:111, wherein the nucleic acid is overexpressed in liver tumor.

4. A vector comprising the nucleic acid of Claim 1.

5. A host cell comprising the vector of Claim 4.

6. The host cell of Claim 5, wherein said cell is a CHO cell.

7. The host cell of Claim 5, wherein said cell is an E. coli.

8. The host Cell of Claim 5, wherein said cell is a yeast cell.

9. A process for producing a PRO21434 polypeptide of SEQ ID NO:112 comprising culturing the host cell of Claim 5 under conditions suitable for expression of said PRO
polypeptide and recovering said PRO polypeptide from the cell culture.

10. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence shown in SEQ ID NO:112, wherein nucleic acid encoding the polypeptide is overexpressed in liver tumor.

11. A chimeric molecule comprising a polypeptide according to Claim 10 fused to a heterologous amino acid sequence.

12. The chimeric molecule of Claim 11, wherein said heterologous amino acid sequence is an epitope tag sequence.

13. The chimeric molecule of Claim 11, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin.

14. An antibody which specifically binds to a polypeptide according to Claim 10.

15. The antibody of Claim 14, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.

16. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:112, lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in SEQ ID NO:112, with its associated signal peptide; or (c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in, SEQ ID NO:112, lacking its associated signal peptide, wherein the nucleic acid is overexpressed in colon tumor.

17. An isolated polypeptide having at least 80% amino acid sequence identity to:
(a) an amino acid sequence of the polypeptide shown in SEQ ID NO:112, lacking its associated signal peptide;
(b) an amine acid sequence of an extracellular domain of the polypeptide shown in SEQ
ID NO:112, with its associated signal peptide; or (c) an amino acid sequence of an extracellular domain of the polypeptide shown in SEQ
ID NO:112, lacking its associated signal peptide, wherein nucleic acid encoding the polypeptide is overexpressed in colon tumor.

18. A method for detecting the presence of tumor in a mammal, said method comprising comparing the level of expression of PRO21434 polypeptide shown in SEQ ID
NO:112 in (a) a test sample of cells taken from said mammal and (b) a control sample of normal cells of the same cell type, wherein a higher level of expression of said PRO
polypeptide in the test sample as compared to the control sample is indicative of the presence of tumor in said mammal.

19. The method of Claim 18, wherein said tumor is liver tumor.

20. An oligonucleotide probe derived from and specific for the nucleotide sequences shown in SEQ ID NO:111.