CA2534018A1

CA2534018A1 - Secreted and transmembrane polypeptides and nucleic acids encoding the same

Info

Publication number: CA2534018A1
Application number: CA002534018A
Authority: CA
Inventors: Kevin P. Baker; Jian Chen; Luc Desnoyers; Audrey Goddard; Paul J. Godowski; Austin L. Gurney; James Pan; Victoria Smith; Colin K. Watanabe; William I. Wood; Zemin Zhang
Original assignee: Individual
Current assignee: Genentech Inc
Priority date: 2000-03-01
Filing date: 2001-02-28
Publication date: 2001-09-20
Also published as: CA2531917A1; WO2001068848A2; WO2001068848A3; CA2533903A1; AU6802801A; AU2001268028A1; CA2534391A1; JP2004508805A; CA2401448A1; CA2534186A1; EP1259614A2; JP2008301822A; CA2534030A1; CA2533831A1; CA2533991A1; JP2006081548A; CA2539214A1

Abstract

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Description

LA PI2ESENTE P:~RTIE DE CETTE DEiYL~NDF OLr CE DRE~%~TS
COyIPREND PLUS D'U~I' TOLYIE.
CECI EST LE TOiYIE ~ DE
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadian des Brevets.
~JIVI~~ ~:'~'L~~C~.TI~I~IS / PATENTS
THIS SECTION OF THE APPLICATION I PATEN T COr~f'I'AiNS l~iO~E
THAN ONE VOLUME.
THIS IS VOLUyIE OF
NOTE: For additional volumes please contact the Canadian Patent Office.

WO 01/fi8848 PCT1US01J06520 SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE
SAME
FIELD 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 important 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 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. These secreted polypeptides or signaling 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, interleukins, erythropoietins, colony stimulating factors, and various other cytoldnes, are secretory proteins.
Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts are being undertaken by both industry and academia to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secretedproteins.. Examplesof screenin$methods and techniquesrye described in the lieratuzve~se~, for-.
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Patent No. 5,536,637)].
Membrane-bound proteins and receptors can play important 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 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, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation 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 WO 01Ifi8848 PCT/US01/Ofi520 nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor immunoadhesins, 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 receptorlligand interaction.
Efforts are being undertaken by both industry and academia to identify new, native receptor or membrane-bound proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel 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 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 % 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% 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, 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 full-length amino acid sequence as disclosed herein; an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular 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, 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 % 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% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternatively at least about 94 WO 01/G8848 PCT/USOIlOG520 nucleic acid sequence 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 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 amino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
Tn a further 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 % 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, alternatively at least about 96 % nucleic acid sequence identity, alternatively at least about 97 %
nucleic acid sequence identity, akternatively 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 sequence encoding a PRO polypeptide which is either iransmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domains) 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 may find 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 length, usually at least about 10 nucleotides in alternatively at least about 15 nucleotides in length, alternativelylength, at least about 20 nucleotides in alternatively at least about 30 nucleotides in length, alternativelylength, at least about 40 nucleotides in alternatively at least about 50 nucleotides in length, alternativelylength, at least about 60 nucleotides in alternatively at least about 70 nucleotides in length, length, alternatively at least about 80 nucleotides in alternatively at least about 90 nucleotides in length, alternativelylength, at least about 100 nucleotides in alternatively at least about 110 nucleotides in length, length, alternatively at least about 120 nucleotides in 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, alternatively at least about 170 nucleotides in length, alternatively 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, alternatively at least about 500 nucleotides in length, alternatively 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 length. It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO pokypeptide-encoding nucleotide sequence with other laiown nucleotide sequences using any of a number of well Irnown sequence alignment programs and determining which PRO
polypeptide-encoding nucleotide sequence fragments) are novel. All of such PRO polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the PRO pokypeptide 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 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, alternatively at least about 82% amino acid sequence identity, alternatively 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% amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular 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, the 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, alternatively at least about 82 % amino acid sequence identity, alternatively 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 % amino acid sequence identity to an amino acid sequence encoded by any of the human protein IO cDNAs deposited with the ATCC as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing 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.
Another aspect the invention provides an isolated PRO polypeptide which is either transmembrane 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 the 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.
In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO
polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
In a still further embodiment, the invention concerns a composition of matter comprising a PRO
polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically 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 medicament useful in the treatment of a condition which is responsive to the PRO
polypepdde, an agonist or antagonist thereof or an anti-PRO antibody.
In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cell comprising 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 polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence.
Example of such chimeric molecules comprise any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below descn'bed polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligomicleotide probes which may be useful for isolating genomic and cDNA 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 NO:1) of a native sequence PR0276 cDNA, wherein SEQ ID NO:1 is a clone designated herein as "DNA16435-1208".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding sequence of SEQ ID
NO:1 shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID N0:3) of a native sequence PR0284 cDNA, wherein SEQ ID N0:3 is a clone designated herein as "DNA23318-1211".
Figure 4 shows the amino acid sequence (SEQ ID N0:4) derived from the coding sequence of SEQ ID
N0:3 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID NO:S) of a native sequence PR0193 cDNA, wherein SEQ ID NO:S is a clone designated herein as "DNA23322-1393".
Figure 6 shows the amino acid sequence (SEQ 1D N0:6) derived from the coding sequence of SEQ ID
NO:S shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID N0:7) of a native sequence PR0190 cDNA, wherein SEQ ID N0:7 is a clone designated herein as "DNA23334-1392".
Figure 8 shows the amino acid sequence (SEQ ID N0:8) derived from the coding sequence of SEQ ID
N0:7 shown in Figure 7.
Figure 9 shows a nucleotide sequence (SEQ ID N0:9) of a native sequence PR0180 cDNA, wherein SEQ ID N0:9 is a clone designated herein as "DNA26843-1389".
Figure 10 shows the amino acid sequence (SEQ ID NO:10) derived from the coding sequence of SEQ
ID N0:9 shown in Figure 9.

Figure 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequence PR0194 cDNA, wherein SEQ ID NO:11 is a clone designated herein as "DNA26844-1394".
Figure 12 shows the amino acid sequence (SEQ ID N0:12) derived from the coding sequence of SEQ
ID NO:l l shown in Pigure 11.
Figure 13 shows a nucleotide sequence (SEQ ID N0:13) of a native sequence PR0218 cDNA, wherein SEQ ID N0:13 is a clone designated herein as "DNA30867-1335".
Figure 14 shows the amino acid sequence (SEQ ID N0:14) derived from the coding sequence of SEQ
ID N0:13 shown in Figure 13.
Figure 15 shows a nucleotide sequence (SEQ ID N0:15) of a native sequence PR0260 cDNA, wherein SEQ ID N0:15 is a clone designated herein as "DNA33470-1175".
Figure 16 shows the amino acid sequence (SEQ ID N0:16) derived from the coding sequence of SEQ
ID N0:15 shown in Figure 15.
Figure 17 shows a nucleotide sequence (SEQ ID N0:17) of a native sequence PR0233 cDNA, wherein SEQ ID N0:17 is a clone designated herein as "DNA34436-1238".
Figure 18 shows the amino acid sequence (SEQ ID N0:18) derived from the coding sequence of SEQ
ID N0:17 shown in Figure 17.
Figure 19 shows a nucleotide sequence (SEQ )D N0:19) of a native sequence PR0234 cDNA, wherein SEQ ID N0:19 is a clone designated herein as "DNA35557-1137".
Figure 20 shows the amino acid sequence (SEQ ID N0:20) dexived from the coding sequence of SEQ
ID N0:19 shown in Figure 19.
Figure 21 shows a nucleotide sequence (SEQ ID N0:21) of a native sequence PR0236 cDNA, wherein SEQ ID N0:21 is a clone designated herein as "DNA35599-1168".
Figure 22 shows the amino acid sequence (SEQ ID N0:22) derived from the coding sequence of SEQ
ID N0:21 shown in Figure 21.
Figure 23 shows a nucleotide sequence (SEQ ID N0:23) of a native sequence PR0244 cDNA, wherein SEQ ID N0:23 is a clone designated herein as "DNA35668-1171".
Figure 24 shows the amino acid sequence (SEQ ID N0:24) derived from the coding sequence of SEQ
ID N0:23 shown in Pigure 23.
Figure 25 shows a nucleotide sequence (SEQ ID N0:25) of a native sequence PR0262 cDNA, wherein SEQ ID N0:25 is a clone designated herein as "DNA36992-1168".
Figure 26 shows the amino acid sequence (SEQ ID N0:26) derived from the coding sequence of SEQ
1D N0:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID N0:27) of a native sequence PR0271 cDNA, wherein SEQ ID N0:27 is a clone designated herein as "DNA39423-1182" .
Figure 28 shows the amino acid sequence (SEQ ID N0:28) derived from the coding sequence of SEQ
ID N0:27 shown in Figure 27.
Figure 29 shows a nucleotide sequence (SEQ ID N0:29) of a native sequence PR0268 cDNA, wherein SEQ ID N0:29 is a clone designated herein as "DNA39427-1179".

Figure 30 shows the amino acid sequence (SEQ ID N0:30) derived from the coding sequence of SEQ
ID N0:29 shown in Figure 29.
Figure 31 shows a nucleotide sequence (SEQ 1D N0:31) of a native sequence PR0270 cDNA, wherein SEQ ID N0:31 is a clone designated herein as "DNA39510-1181".
Figure 32 shows the amino acid sequence (SEQ ID N0:32) derived from the coding sequence of SEQ
ID N0:31 shown in Figure 31.
Figure 33 shows a nucleotide sequence (SEQ ID N0;33) of a native sequence PR0355 cDNA, wherein SEQ ID N0:33 is a clone designated herein as "DNA39518-1247".
Figure 34 shows the amino acid sequence (SEQ ID N0:34) derived from the coding sequence of SEQ
ID N0:33 shown in Figute 33.
Figure 35 shows a nucleotide sequence (SEQ ID N0:35) of a native sequence PR0298 cDNA, wherein SEQ m N0:35 is a clone designated herein as "DNA39975-1210".
Figure 36 shows the amino acid sequence (SEQ ID N0:36) derived from the coding sequence of SEQ
ID N0:35 shown in Figure 35.
Figure 37 shows a nucleotide sequence (SEQ ID N0:37) of a native sequence PR0299 cDNA, wherein SEQ ID N0:37 is a clone designated herein as "DNA39976-1215".
Figure 38 shows the amino acid sequence (SEQ ID N0:38) derived from the coding sequence of SEQ
ID N0:37 shown in Figure 37.
Figure 39 shows a nucleotide sequence (SEQ ID N0:39) of a native sequence PR0296 cDNA, wherein SEQ ID N0:39 is a clone designated herein as "DNA39979-1213".
Figure 40 shows the amino acid sequence (SEQ ID N0:40) derived from the coding sequence of SEQ
ID N0:39 shown in Figure 39.
Figute 41 shows a nucleotide sequence (SEQ ID N0;41) of a native sequence PR0329 cDNA, wherein SEQ ID N0:41 is a clone designated herein as "DNA40594-1233".
Figure 42 shows the amino acid sequence (SEQ ID N0:42) derived from the coding sequence of SEQ
ID N0:41 shown in Figure 41.
Figure 43 shows a nucleotide sequence (SEQ ID N0:43) of a native sequence PR0330 cDNA, wherein SEQ ID N0:43 is a clone designated herein as "DNA40603-1232".
Figure 44 shows the amino acid sequence (SEQ >D N0:44) derived from the coding sequence of SEQ
ID N0:43 shown in Figure 43.
Figure 45 shows a nucleotide sequence (SEQ ID N0:45) of a native sequence PR0294 cDNA, wherein SEQ ID N0:45 is a clone designated herein as "DNA40604-1187".
Figure 46 shows the amino acid sequence (SEQ 117 N0:46) derived from the coding sequence of SEQ
ID N0:45 shown in Figure 45.
Figure 47 shows a nucleotide sequence (SEQ ID N0:47) of a native sequence PR0300 cDNA, wherein SEQ ID N0:47 is a clone designated herein as "DNA40625-1189".
Figure 48 shows the amino acid sequence (SEQ ID N0:48) derived from the coding sequence of SEQ
ID N0:47 shown in Figure 47.

Figure 49 shows a nucleotide sequence (SEQ 1D N0:49) of a native sequence PR0307 cDNA, wherein SEQ ID N0:49 is a clone designated herein as "DNA41225-1217".
Figure 50 shows the amino acid sequence (SEQ ID N0:50) derived from the coding sequence of SEQ
ll~ N0:49 shown in Figure 49.
Figure 51 shows a nucleotide sequence (SEQ ID N0:51) of a native sequence PR0334 cDNA, wherein SEQ ID N0:51 is a clone designated herein as "DNA41379-1236".
Figure 52 shows the amino acid sequence (SEQ ID N0:52) derived from the coding sequence of SEQ
1D N0:51 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ ID N0:53) of a native sequence PR0352 cDNA, wherein SEQ ID N0:53 is a clone designated herein as "DNA41386-1316".
Figure 54 shows the amino acid sequence (SEQ 1D N0:54) derived from the coding sequence of SEQ
ID N0:53 shown in Figure 53.
Figure 55 shows a nucleotide sequence (SEQ ID N0:55) of a native sequence PR0710 cDNA, wherein SEQ 1D N0:55 is a clone designated herein as "DNA44161-1434".
Figure 56 shows the amino acid sequence (SEQ ID N0:56) derived from the coding sequence of SEQ
ID N0:55 shown in Figure 55.
Figure 57 shows a nucleotide sequence (SEQ ID N0:57) of a native sequence PR0873 cDNA, wherein SEQ ID N0:57 is a clone designated herein as "DNA44179-1362".
Figure 58 shows the amino acid sequence (SEQ ID N0:58) derived from the coding sequence of SEQ
ID N0:5'7 shown in Figure 57.
Figure 59 shows a nucleotide sequence (SEQ ID N0:59) of a native sequence PR0354 cDNA, wherein SEQ ID N0:59 is a clone designated herein as "DNA44192-1246".
Figure 60 shows the amino acid sequence (SEQ ID N0:60) derived from the coding sequence of SEQ
ID N0:59 shown in Figure 59.
Figure 61 shows a nucleotide sequence (SEQ ID N0:61) of a native sequence PR01151 cDNA, wherein SEQ ID N0:61 is a clone designated herein as "DNA44694-1500".
Figure 62 shows the amino acid sequence (SEQ ID N0:62) derived from the coding sequence of SEQ
ID N0:61 shown in Figure 61.
Figure 63 shows a nucleotide sequence (SEQ ID N0:63) of a native sequence PR0382 cDNA, wherein SEQ ID N0:63 is a clone designated herein as "DNA45234-1277".
Figure 64 shows the amino acid sequence (SEQ )D N0;64) derived from the coding sequence of SEQ
ID N0:63 shown in Figure 63.
Figure 65 shows a nucleotide sequence (SEQ ID N0:65) of a native sequence PR01864 cDNA, wherein SEQ D7 N0:65 is a clone designated herein as "DNA45409-2511".
Figure 66 shows the amino acid sequence (SEQ ID N0:66) derived from the coding sequence of SEQ
ID N0:65 shown in Figure 65.
Figure 67 shows a nucleotide sequence (SEQ ID N0:67) of a native sequence PR0386 cDNA, wherein SEQ ID N0:67 is a clone designated herein as "DNA45415-1318".

Figure 68 shows the amino acid sequence (SEQ ID N0:68) derived from the coding sequence of SEQ
ID N0:67 shown in Figure 67.
Figure 69 shows a nucleotide sequence (SEQ 1D N0:69) of a native sequence PROS41 cDNA, wherein SEQ ID N0:69 is a clone designated herein as "DNA45417-1432".
Figure 70 shows the amino acid sequence (SEQ ID N0:70) derived from the coding sequence of SEQ
ID N0:69 shown in Figure 69.
Figure 71 shows a nucleotide sequence (SEQ ID N0:71) of a native sequence PR08S2 cDNA, wherein SEQ ID N0:71 is a clone designated herein as "DNA4S493-1349".
Figure 72 shows the amino acid sequence (SEQ ID N0:72) derived from the coding sequence of SEQ
1D N0:71 shown in Figure 71, Figure 73 shows a nucleotide sequence (SEQ ID N0:73) of a native sequence PR0700 cDNA, wherein SEQ ID N0:73 is a clone designated herein as "DNA46776-1284".
Figure 74 shows the amino acid sequence (SEQ ID N0:74) derived from the coding sequence of SEQ
ID N0:73 shown in Figure 73.
Figures 75A-75B show a nucleotide sequence (SEQ ID N0:75) of a native sequence PR0708 cDNA, wherein SEQ ID N0:75 is a clone designated herein as "DNA48296-1292".
Figure 76 shows the amino acid sequence (SEQ ID N0:76) derived from the coding sequence of SEQ
ID N0:75 shown in Figures 75A-75B.
Figure 77 shows a nucleotide sequence (SEQ 1D N0:77) of a native sequence PR0707 cDNA, wherein SEQ ID N0:77 is a clone designated herein as "DNA48306-1291".
Figure 78 shows the amino acid sequence (SEQ ID N0:78) derived from the coding sequence of SEQ
ID N0:77 shown in Figure 77.
Figure 79 shows a nucleotide sequence (SEQ ID N0:79) of a native sequence PR0864 cDNA, wherein SEQ ID N0:79 is a clone designated herein as "DNA48328-1355".
Figure 80 shows the amino acid sequence (SEQ ID N0:80) derived from the coding sequence of SEQ
2$ ID N0:79 shown in Figure 79.
Figure 81 shows a nucleotide sequence (SEQ ID N0:81) of a native sequence PR0706 cDNA, wherein SEQ ID N0:81 is a clone designated herein as "DNA48329-1290".
Figure 82 shows the amino acid sequence (SEQ ID N0:82) derived from the coding sequence of SEQ
ID N0:81 shown in Figure 81.
Figure 83 shows a nucleotide sequence (SfiQ ID N0:83) of a native sequence PR0732 cDNA, wherein SEQ ID N0:83 is a clone designated herein as "DNA48334-1435".
Figure 84 shows the amino acid sequence (SEQ ID N0:84) derived from the coding sequence of SEQ
ID N0:83 shown in Figure 83.
Figure 85 shows a nucleotide sequence (SEQ ID N0:85) of a native sequence PR0537 cDNA, wherein SEQ II7 N0:85 is a clone designated herein as "DNA49141-1431".
Figure 86 shows the amino acid sequence (SEQ ID N0:86) derived from the coding sequence of SEQ
ID N0:85 shown in Figure 8S.

Figure 87 shows a nucleotide sequence (SEQ ID N0:87) of a native sequence PR0545 cDNA, wherein SEQ ID N0:87 is a clone designated herein as "DNA49624-1279".
Figure 88 shows the amino acid sequence (SEQ ID N0:88) derived from the coding sequence of SEQ
ID N0:87 shown in Figure 87.
Figure 89 shows a nucleotide sequence (SEQ ID N0:89) of a native sequence PR0718 cDNA, wherein SEQ ID N0:89 is a clone designated herein as "DNA49647-1398".
Figure 90 shows the amino acid sequence (SEQ ID N0:90) derived from the coding sequence of SEQ
ID N0:89 shown in Figure 89.
Figure 91 shows a nucleotide sequence (SEQ ID N0:91) of a native sequence PR0872 cDNA, wherein SEQ ID N0:91 is a clone designated herein as "DNA49819-1439".
Figure 92 shows the amino acid sequence (SEQ ID N0:92) derived from the coding sequence of SEQ
ID N0:91 shown in Figure 91.
Figure 93 shows a nucleotide sequence (SEQ ID N0:93) of a native sequence PR0704 cDNA, wherein SEQ ID N0:93 is a clone designated herein as "DNA50911-1288".
Figure 94 shows the amino acid sequence (SEQ ID N0:94) derived from the coding sequence of SEQ
ID N0:93 shown in Figure 93.
Figure 95 shows a nucleotide sequence (SEQ ID N0:95) of a native sequence PR0705 cDNA, wherein SEQ ID N0:95 is a clone designated herein as "DNA50914-1289".
Figure 96 shows the amino acid sequence (SEQ ID N0:96) derived from the coding sequence of SEQ
ID N0:95 shown in Figure 95.
Figure 97 shows a nucleotide sequence (SEQ ID N0:97) of a native sequence PR0871 cDNA, wherein SEQ ID N0:97 is a clone designated herein as "DNA50919-1361".
Figure 98 shows the amino acid sequence (SEQ ID N0:98) derived from the coding sequence of SEQ
ID N0:97 shown in Figure 97.
Figure 99 shows a nucleotide sequence (SEQ ID N0:99) of a native sequence PR0702 cDNA, wherein SEQ ID N0:99 is a clone designated herein as "DNA50980-1286".
Figure 100 shows the amino acid sequence (SEQ ID NO:100) derived from the coding sequence of SEQ
ID N0:99 shown in Figure 99.
Figure 101 shows a nucleotide sequence (SEQ ID NO:101) of a native sequence PR0944 cDNA, wherein SEQ ID NO:101 is a clone designated herein as "DNA52185-1370".
Figure 102 shows the amino acid sequence (SEQ ID N0:102) derived from the coding sequence of SEQ
ID NO:101 shown in Pigure 101.
Figure 103 shows a nucleotide sequence (SEQ ID N0:103) of a native sequence PR0739 cDNA, wherein SEQ ID N0:103 is a clone designated herein as "DNA52756".
Figure 104 shows the amino acid sequence (SEQ ID N0:104) derived from the coding sequence of SEQ
ID N0:103 shown in Figure 103.
Figure 105 shows a nucleotide sequence (SEQ ID N0:105) of a native sequence PR0941 cDNA, wherein SEQ ID N0:105 is a clone designated herein as "DNA53906-1368".

Figure 106 shows the amino acid sequence (SEQ ID N0:106) derived from the coding sequence of SEQ
ID NO:105 shown in Figure 105.
Figure 107 shows a nucleotide sequence (SEQ ID N0:107) of a native sequence PR01082 cDNA, wherein SEQ )D N0:107 is a clone designated herein as "DNA53912-1457".
Figure 108 shows the amino acid sequence (SEQ ID N0:108) derived from the coding sequence of SEQ
ID N0:107 shown in Figure 107.
Figure 109 shows a nucleotide sequence (SEQ ID N0:109) of a native sequence PR01133 cDNA, wherein SEQ 117 N0:109 is a clone designated herein as "DNA53913-1490".
Figure 110 shows the amino acid sequence (SEQ ID NO:110) derived from the coding sequence of SEQ
ID N0:109 shown in Figure 109.
Figure 111 shows anucleotide sequence (SEQ TD NO:111) of a native sequence PR0983 cDNA, wherein SEQ ID NO:111 is a clone designated herein as "DNA53977-1371".
Figure 112 shows the amino acid sequence (SEQ ID N0:112) derived from the coding sequence of SEQ
ID NO:111 shown in Figure 111.
Figure 113 shows a nucleotide sequence (SEQ ID N0:113) of a native sequence PR0784 cDNA, wherein SEQ ID N0:113 is a clone designated herein as "DNA53978-1443".
Figure 114 shows the amino acid sequence (SEQ ID N0:114) derived from the coding sequence of SEQ
ID N0:113 shown in Figure 113.
Figure 115 shows a nucleotide sequence (SEQ ID NO:115) of a native sequence PR0783 cDNA, wherein SEQ ID NO:115 is a clone designated herein as "DNA53996-1442".
Figure 116 shows the amino acid sequence (SEQ ID N0:116) derived from the coding sequence of SEQ
ID NO:115 shown in Figure 115.
Figure 117 shows a nucleotide sequence (SEQ ID N0:117) of a native sequence PR0940 cDNA, wherein SEQ ID N0:117 is a clone designated herein as "DNA54002-1367".
Figure 118 shows the amino acid sequence (SEQ ID N0:118) derived from the coding sequence of SEQ
ID N0:117 shown in Figure 117.
Figure 119 shows a nucleotide sequence (SEQ ID N0:119) of a native sequence PR0768 cDNA, wherein SEQ ID N0:119 is a clone designated herein as "DNA55737-1345".
Figure 120 shows the amino acid sequence (SEQ ID N0:120) derived from the coding sequence of SEQ
ID N0:119 shown in Figure 119.
Figure 121 shows a nucleotide sequence (SEQ ID N0:121) of a native sequence PR01079 cDNA, wherein SEQ ID N0:121 is a clone designated herein as "DNA56050-1455".
Figure 122 shows the amino acid sequence (SEQ ID N0:122) derived from the coding sequence of SEQ
ID N0:121 shown in Figure 121.
Figure 123 shows a nucleotide sequence (SEQ ID N0:123) of a native sequence PR01078 cDNA, wherein SEQ ID N0:123 is a clone designated herein as "DNA56052-1454".
Figure 124 shows the amino acid sequence (SEQ ID N0:124) derived from the coding sequence of SEQ
ID N0:123 shown in Figure 123.

Figure 125 shows a nucleotide sequence (SEQ ID N0:125) of a native sequence PR01018 cDNA, wherein SEQ ID N0:125 is a clone designated herein as "DNA56107-1415".
Figure 126 shows the amino acid sequence (SEQ ID N0:126) derived from the coding sequence of SEQ
ID N0:125 shown in Figure 125.
Figure 127 shows a nucleotide sequence (SEQ ID N0:127) of a native sequence PR0793 cDNA, wherein SEQ ID N0:127 is a clone designated herein as "DNA56110-1437".
Figure 128 shows the amino acid sequence (SEQ ID N0:128) derived from the coding sequence of SEQ
ID N0:127 shown in Figure 127.
Figure 129 shows a nucleotide sequence (SEQ ID N0:129) of a native sequence PR01773 cDNA, wherein SEQ ID N0:129 is a clone designated herein as "DNA56406-1704".
Figure 130 shows the amino acid sequence (SEQ ID N0:130) derived from the coding sequence of SEQ
ID N0:129 shown in Figure 129.
Figure 131 shows a nucleotide sequence (SEQ ID N0:131) of a native sequence PR01014 cDNA, wherein SEQ ID N0:131 is a clone designated herein as "DNA56409-1377".
Figure 132 shows the amino acid sequence (SEQ ID N0:132) derived from the coding sequence of SEQ
ID N0:131 shown in Figure 131.
Figure 133 shows a nucleotide sequence (SEQ ID N0:133) of a native sequence PR01013 cDNA, wherein SEQ ID N0:133 is a clone designated herein as "DNA56410-1414".
Figure 134 shows the amino acid sequence (SEQ ID N0:134) derived from the coding sequence of SEQ
ID N0:133 shown in Figure 133.
Figure 135 shows a nucleotide sequence (SEQ ID N0:135) of a native sequence PR0937 cDNA, wherein SEQ ID N0:135 is a clone designated herein as "DNA56436-1448".
Figure 136 shows the amino acid sequence (SEQ ID N0:136) derived from the coding sequence of SEQ
ID N0:135 shown in Figure 135.
Figure 137 shows a nucleotide sequence (SEQ ID N0:137) of a native sequence PR01477 cDNA, wherein SEQ ID NO:I37 is a clone designated herein as "DNA56529-1647".
Figure 138 shows the amino acid sequence (SEQ ID N0:138) derived fmm the coding sequence of SEQ
ID N0:137 shown in Figure 137.
Figure 139 shows a nucleotide sequence (SEQ ID N0:139) of a native sequence PR0842 cDNA, wherein SEQ 1D N0:139 is a clone designated herein as "DNA56855-1447".
Figure 140 shows the amino acid sequence (SEQ ID N0:140) derived from the.
coding sequence of SEQ
ID NO:I39 shown in Figure 139.
Figure 141 shows a nucleotide sequence (SEQ ID N0:141) of a native sequence PR0839 cDNA, wherein SEQ ID N0:141 is a clone designated herein as "DNA56859-1445".
Figure 142 shows the amino acid sequence (SfiQ ID N0:142) derived from the coding sequence of SEQ
ID N0:141 shown in Figure 141.
Figure 143 shows a nucleotide sequence (SEQ ID N0:143) of a native sequence PR01180 cDNA, wherein SEQ 1D N0:143 is a clone designated herein as "DNA56860-1510".

Figure 144 shows the amino acid sequence (SEQ ID N0:144) derived from the coding sequence of SEQ
ID N0:143 shown in Figure 143.
Figure 145 shows a nucleotide sequence (SEQ ID N0:145) of a native sequence PR01134 cDNA, wherein SEQ ID N0:145 is a clone designated herein as "DNA5686S-1491".
Figure 146 shows the amino acid sequence (SEQ ID N0:146) derived from the coding sequence of SEQ
ID N0:145 shown in Figure 145.
Figure 147 shows a nucleotide sequence (SEQ ID N0:147) of a native sequence PR01115 cDNA, wherein SEQ ID N0:147 is a clone designated herein as "DNA56868-1478".
Figure 148 shows the amino acid sequence (SEQ ID N0:148) derived from the coding sequence of SEQ
ID N0:147 shown in Figure 147.
Figure 149 shows a nucleotide sequence (SEQ ID N0:149) of a native sequence PR01277 cDNA, wherein SEQ ID N0:149 is a clone designated herein as "DNA56869-1545".
Figure 150 shows the amino acid sequence (SEQ ID N0:150) derived from the coding sequence of SEQ
ID N0:149 shown in Figure 149.
Figure 151 shows a nucleotide sequence (SEQ ID N0:151) of a native sequence PR01135 cDNA, 1S wherein SEQ ID N0:151 is a clone designated herein as "DNA56870-1492".
Figure 152 shows the amino acid sequence (SEQ ID N0:152) derived from the coding sequence of SEQ
ID NO:1S1 shown in Figure 151.
Figure 153 shows a nucleotide sequence (SEQ ID N0:153) of a native sequence PR0827 cDNA, wherein SEQ ID N0:153 is a clone designated herein as "DNA57039-1402".
Figure 154 shows the amino acid sequence (SEQ ID N0:154) derived from the coding sequence of SEQ
ID N0:153 shown in Figure 153.
Figure 155 shows a nucleotide sequence (SEQ 1D N0:155) of a native sequence PR01057 cDNA, wherein SEQ ID N0:155 is a clone designated herein as "DNA57253-1382".
Figure 156 shows the amino acid sequence (SEQ ID N0:156) derived from the coding sequence of SEQ
1D NO:I55 shown in Figure I55.
Figure 157 shows a nucleotide sequence (SEQ ID N0:157) of a native sequence PR01113 cDNA, wherein SEQ ID N0:157 is a clone designated hereia as "DNA57254-1477".
Figure 158 shows the amino acid sequence (SEQ ID N0:158) derived from the coding sequence of SEQ
ID N0:157 shown in Figure 157.
Figure 159 shows a nucleotide sequence (SEQ ID N0:159) of a native sequence PR01006 cDNA, wherein SEQ 1D N0:159 is a clone designated herein as "DNA57699-1412" .
Figure 160 shows the amino acid sequence (SEQ ID N0:160) derived from the coding sequence of SEQ
ID N0:159 shown in Figure 159.
Figure 161 shows a nucleotide sequence (SEQ ID N0:161) of a native sequence PR01074 cDNA, wherein SEQ ID N0:161 is a clone designated herein as "DNA57704-1452".
Figure 162 shows the amino acid sequence (SEQ ID N0:162) derived from the coding sequence of SEQ
1D N0:161 shown in Figure 161 Figure 163 shows a nucleotide sequence (SEQ ID N0:163) of a native sequence PR01073 cDNA, wherein SEQ ID N0:163 is a clone designated hexein as "DNA57710-1451".
Figure 164 shows the amino acid sequence (SEQ ID N0:164) derived from the coding sequence of SEQ
ID N0:163 shown in Figure 163.
Figure 165 shows a nucleotide sequence (SEQ ID N0:165) of a native sequence PR01136 cDNA, wherein SEQ ID N0:165 is a clone designated herein as "DNA57827-1493".
Figure 166 shows the amino acid sequence (SEQ ID N0:166) derived from the coding sequence of SEQ
ID N0:165 shown in Figure 165.
Figure 167 shows a nucleotide sequence (SEQ ID N0:167) of a native sequence PR01004 cDNA, wherein SEQ ID N0:167 is a clone designated herein as "DNA57844-1410" .
Figure 168 shows the amino acid sequence (SEQ ID N0:168) derived from the coding sequence of SEQ
ID N0:167 shown in Figure 167.
Figure 169 shows a nucleotide sequence (SEQ ID N0:169) of a native sequence PR01344 cDNA, wherein SEQ ID N0:169 is a clone designated herein as "DNA58723-1588".
Figure 170 shows the amino acid sequence (SEQ ID N0:170) derived from the coding sequence of SEQ
ID N0:169 shown in Figure 169.
Figure x71 shows a nucleotide sequence (SEQ ID N0:171) of a native sequence PRO1110 cDNA, wherein SEQ ID N0:171 is a clone designated herein as "DNA58727-1474".
Figure 172 shows the amino acid sequence (SEQ ID N0:172) derived from the coding sequence of SEQ
ID N0:171 shown in Figure 171.
Figure 173 shows a nucleotide sequence (SEQ ID N0:173) of a native sequence PR01378 cDNA, wherein SEQ ID N0:173 is a clone designated herein as "DNA58730-1607".
Figure 174 shows the amino acid sequence (SEQ ID N0:174) derived from the coding sequence of SEQ
ID N0:173 shown in Figure 173.
Figure 175 shows a nucleotide sequence (SEQ ID N0:175) of a native sequence PR01481 cDNA, wherein SEQ ID N0:175 is a clone designated herein as "DNA58732-1650".
Figure 176 shows the amino acid sequence (SEQ ID N0:176) derived from the coding sequence of SEQ
ID N0:175 shown in Figure 175.
Figure 177 shows a nucleotide sequence (SEQ ID N0:177) of a native sequence PR01109 cDNA, wherein SEQ ID N0:177 is a clone designated herein as "DNA58737-1473".
Figure 178 shows the amino acid sequence (SEQ ID N0:178) derived from the coding sequence of SEQ
ID N0:177 shown in Figure 177.
Figure 179 shows a nucleotide sequence (SEQ ID N0:179) of a native sequence PR01383 cDNA, wherein SEQ ID N0:179 is a clone designated herein as "DNA58743-1609".
Figure 180 shows the amino acid sequence (SEQ ID N0:180) derived from the coding sequence of SEQ
ID N0:179 shown in Figure 179.
Figure 181 shows a nucleotide sequence (SEQ ID N0:181) of a native sequence PR01072 cDNA, wherein SEQ ID N0:181 is a clone designated herein as "DNA58747-1384".

Figure 182 shows the amino acid sequence (SEQ ID N0:182) derived from the coding sequence of SEQ
ID N0:181 shown in Figure 181.
Figure 183 shows a nucleotide sequence (SEQ ID N0:183) of a native sequence PR01189 cDNA, wherein SEQ ID N0:183 is a clone designated herein as "DNAS8828-1519".
Figure 184 shows the amino acid sequence (SEQ ID N0:184) derived from the coding sequence of SEQ
1D N0:183 shown in Figure 183.
Figure 185 shows a nucleotide sequence (SEQ ID N0:185) of a native sequence PR01003 cDNA, wherein SEQ ID N0:185 is a clone designated herein as "DNA58846-1409".
Figure 186 shows the amino acid sequence (SEQ ID N0:186) derived from the coding sequence of SEQ
ID N0:185 shown in Figure 185.
Figure 187 shows a nucleotide sequence (SEQ ID N0:187) of a native sequence PR01108 cDNA, wherein SEQ ID N0:187 is a clone designated herein as "DNA58848-1472".
Figure 188 shows the amino acid sequence (SEQ ID N0:188) derived from the coding sequence of SEQ
ID N0:187 shown in Figure 187.
Figure 189 shows a nucleotide sequence (SEQ ID N0:189) of a native sequence PR01137 eDNA, wherein SEQ ID N0:189 is a clone designated herein as "DNA58849-1494".
Figure 190 shows the amino acid sequence (SEQ ID N0:190) derived from the coding sequence of SEQ
ID N0:189 shown in Figure 189.
Figure 191 shows a nucleotide sequence (SEQ ID N0:191) of a native sequence PR01138 cDNA, wherein SEQ ID N0:191 is a clone designated herein as "DNA58850-1495".
Figure 192 shows the amino acid sequence (SEQ ID N0:192) derived from the coding sequence of SEQ
ID N0:191 shown in Figure 191.
Figure 193 shows a nucleotide sequence (SEQ 1D N0:193) of a native sequence PR01415 cDNA, wherein SEQ ID N0:193 is a clone designated herein as "DNAS8852-1637".
Figure 194 shows the amino acid sequence (SEQ ID N0:194) derived from the coding sequence of SEQ
ID N0:193 shown in Figure 193.
Figure 195 shows a nucleotide sequence (SEQ ID N0:195) of a native sequence PR01054 cDNA, wherein SEQ ID N0:195 is a clone designated herein as "DNA58853-1423".
Figure 196 shows the amino acid sequence (SEQ lD N0:196) derived from the coding sequence of SEQ
ID N0:195 shown in Figure 195.
Figure 197 shows a nucleotide sequence (SEQ 1D NO:197) of a native sequence PR0994 cDNA, wherein SEQ ID N0:197 is a clone designated herein as "DNA58855-1422".
Figure 198 shows the amino acid sequence (SEQ ID N0:198) derived from the coding sequence of SEQ
ID N0:197 shown in Figure 197.
Figure 199 shows a nucleotide sequence (SEQ ID N0:199) of a native sequence PR01069 cDNA, wherein SEQ ID N0:199 is a clone designated herein as "DNAS9211-1450".
Figure 200 shows the amino acid sequence (SEQ ID N0:200) derived from the coding sequence of SEQ
ID N0:199 shown in Figure 199.

Figure 201 shows a nucleotide sequence (SEQ ID N0:201) of a native sequence PR01411 cDNA, wherein SEQ 1D N0:201 is a clone designated herein as "DNA59212-1627".
Figure 202 shows the amino acid sequence (SEQ ID N0:202) derived from the coding sequence of SEQ
ID N0:201 shown in Figure 201.
Figure 203 shows a nucleotide sequence (SEQ ID N0:203) of a native sequence PR01129 cDNA, wherein SEQ ID N0:203 is a clone designated herein as "DNA59213-1487".
Figure 204 shows the amino acid sequence (SEQ ID N0:204) derived from the coding sequence of SEQ
)D N0:203 shown in Figure 203.
Figure 20S shows a nucleotide sequence (SEQ ID N0:205) of a native sequence PR01359 cDNA, wherein SEQ ID N0:205 is a clone designated herein as "DNA59219-1613".
Figure 206 shows the amino acid sequence (SEQ ID N0:206) derived from the coding sequence of SEQ
ID N0:205 shown in Figure 205.
Figure 207 shows a nucleotide sequence (SEQ ID N0:207) of a native sequence PR01139 cDNA, wherein SEQ ID N0:207 is a clone designated herein as "DNA59497-1496".
Figure 208 shows the amino acid sequence (SEQ ID N0:208) derived from the coding sequence of SEQ
ID N0:207 shown in Figure 207.
Figure 209 shows a nucleotide sequence (SEQ ID N0:209) of a native sequence PR01065 cDNA, wherein SEQ ID N0:209 is a clone designated herein as "DNA59602-1436".
Figure 210 shows the amino acid sequence (SEQ ID N0:210) derived from the coding sequence of SEQ
ID N0:209 shown in Figure 209.
Figure 211 shows a nucleotide sequence (SEQ ID N0:211) of a native sequence PR01028 cDNA, wherein SEQ ID N0:211 is a clone designated herein as "DNA59603-1419".
Figure 212 shows the amino acid sequence (SEQ ID N0:212) derived from the coding sequence of SEQ
ID N0:211 shown in Figure 211.
Figure 213 shows a nucleotide sequence (SEQ ID N0:2I3) of a native sequence PROI027 cDNA, wherein SEQ ID N0:213 is a clone designated herein as "DNA59605-1418".
Figure 214 shows the amino acid sequence (SEQ m N0:214) derived from the coding sequence of S8Q
ID N0:213 shown in Figure 213.
Pigure 215 shows a nucleotide sequence (SEQ ID N0:215) of a native sequence PR01140 cDNA, wherein SEQ ID N0:215 is a clone designated herein as "DNA59607-1497".
Figure 216 shows the amino acid sequence (SEQ ID N0:216) derived from the coding sequence of SEQ
ID N0:215 shown in Figure 215.
Figure 217 shows a nucleotide sequence (SEQ ID N0:217) of a native sequence PR01291 cDNA, wherein SEQ ID N0:217 is a clone designated herein as "DNA59610-1556".
Figure 218 shows the amino acid sequence (SEQ ID N0:218) derived from the coding sequence of SEQ
ID N0:217 shown in Figure 217.
Figure 219 shows a nucleotide sequence (SEQ ID N0:219) of a native sequence PR01105 cDNA, wherein SEQ ID N0:219 is a clone designated herein as "DNA59612-1466".

Figure 220 shows the amino acid sequence (SEQ ID N0:220) derived from the coding sequence of SEQ
ID N0:219 shown in Figure 219.
Figure 221 shows a nucleotide sequence (SEQ ID N0:221) of a native sequence PR01026 cDNA, wherein SEQ ID N0:221 is a clone designated herein as "DNA59613-1417".
Figure 222 shows the amino acid sequence (SEQ ID N0:222) derived from the coding sequence of SEQ
ID N0:221 shown in Figure 221.
Figure 223 shows a nucleotide sequence (SEQ ID N0:223) of a native sequence PR01104 cDNA, wherein SEQ ID N0:223 is a clone designated herein as "DNA59616-1465".
Figure 224 shows the amino acid sequence (SEQ ID N0:224) derived from the coding sequence of SEQ
ID N0:223 shown in Figure 223.
Figure 225 shows a nucleotide sequence (SEQ ID N0:225) of a native sequence PRO1100 cDNA, wherein SEQ ID N0:225 is a clone designated herein as "DNA59619-1464".
Figure 226 shows the amino acid sequence (SEQ ID N0:226) derived from the coding sequence of SEQ
ID N0:225 shown in Figure 225.
Figure 227 shows a nucleotide sequence (SEQ ID N0:227) of a native sequence PR01141 cDNA, wherein SEQ TD N0:227 is a clone designated herein as "DNA59625-1498".
Figure 228 shows the amino acid sequence (SEQ ID N0:228) derived from the coding sequence of SEQ
ID N0:227 shown in Figure 227.
Figure 229 shows a nucleotide sequence (SEQ ID N0:229) of a native sequence PR01772 cDNA, wherein SEQ ID N0:229 is a clone designated herein as "DNA59817-1703".
Figure 230 shows the amino acid sequence (SEQ ID N0:230) derived from the coding sequence of SEQ
ID N0:229 shown in Pigure 229.
Figure 231 shows a nucleotide sequence (SEQ ID N0:231) of a native sequence PR01064 cDNA, wherein SBQ ID N0:231 is a clone designated herein as "DNA59827-1426".
Figure 232 shows the amino acid sequence (SEQ ID N0:232) derived from the coding sequence of SEQ
ID N0:231 shown in Figure 231.
Figure 233 shows a nucleotide sequence (SEQ ID N0:233) of a native sequence PR01379 cDNA, wherein SEQ II7 N0:233 is a clone designated herein as "DNA59828-1608".
Figure 234 shows the amino acid sequence (SEQ ID N0:234) derived from the coding sequence of SEQ
ID N0:233 shown in Figure 233.
Figure 235 shows a nucleotide sequence (SEQ ID N0:235) of a native sequence PR03573 cDNA, wherein SEQ ID N0:235 is a clone designated herein as "DNA59837-2545".
Figure 236 shows the amino acid sequence (SEQ ID N0:236) derived from the coding sequence of SEQ
ID N0:235 shown in Figure 235.
Figure 237 shows a nucleotide sequence (SEQ ID N0:237) of a native sequence PR03566 cDNA, wherein SEQ ID N0:237 is a clone designated herein as "DNA59844-2542".
Figure 238 shows the amino acid sequence (SEQ ID N0:238) derived from the coding sequence of SEQ
ID N0:237 shown in Figure 237.

Figure 239 shows a nucleotide sequence (SEQ ID N0:239) of a native sequence PR01156 cDNA, wherein SEQ ID N0:239 is a clone designated herein as "DNA59853-1505".
Figure 240 shows the amino acid sequence (SEQ ID N0:240) derived from the coding sequence of SEQ
ID N0:239 shown in Figure 239.
Figure 241 shows a nucleotide sequence (SEQ ID N0:241) of a native sequence PR01098 cDNA, wherein SEQ ID N0:241 is a clone designated herein as "DNA59854-1459".
Figure 242 shows the amino acid sequence (SEQ ID N0:242) derived from the coding sequence of SEQ
ID N0:241 shown in Figure 241.
Figure 243 shows a nucleotide sequence (SEQ ID N0:243) of a native sequence PR01128 cDNA, wherein SEQ ID N0:243 is a clone designated herein as "DNA59855-1485".
Figure 244 shows the amino acid sequence (SEQ ID N0:244) derived from the coding sequence of SEQ
ID N0:243 shown in Figure 243.
Figure 245 shows a nucleotide sequence (SEQ ID N0:245) of a native sequence PR01248 cDNA, wherein SEQ ID N0:245 is a clone designated herein as "DNA60278-1530".
Figure 246 shows the amino acid sequence (SEQ ID N0:246) derived from the coding sequence of SEQ
ID N0:245 shown in Figure 245.
Figure 247 shows a nucleotide sequence (SEQ ID N0:247) of a native sequence PR01127 cDNA, wherein SEQ ID N0:247 is a clone designated herein as "DNA60283-1484".
Figure 248 shows the amino acid sequence (SEQ ID N0:248) derived from the coding sequence of SEQ
ID N0:247 shown in Figure 247.
Figure 249 shows a nucleotide sequence (SEQ ID N0:249) of a native sequence PR01316 cDNA, wherein SEQ ID N0:249 is a clone designated herein as "DNA60608-1577".
Figure 250 shows the amino acid sequence (SEQ ID N0:250) derived from the coding sequence of SEQ
ID N0:249 shown in Figure 249.
Figure 251 shows a nucleotide sequence (SEQ ID N0:251) of a native sequence PR01197 cDNA, wherein SEQ ID N0:251 is a clone designated herein as "DNA60611-1524".
Figure 252 shows the amino acid sequence (SEQ ID N0:252) derived from the coding sequence of SEQ
ID N0:251 shown in Figure 251.
Figure 253 shows a nucleotide sequence (SEQ ID N0:253) of a native sequence PR01125 cDNA, wherein SEQ ID N0:253 is a clone designated herein as "DNA60619-1482".
Figure 254 shows the amino acid sequence (SEQ ID N0:254) derived from the coding sequence of SEQ
ID N0:253 shown in Figure 253.
Figure 255 shows a nucleotide sequence (SEQ ID N0:255) of a native sequence PR01158 cDNA, wherein SEQ ID N0:255 is a clone designated herein as "DNA60625-1507".
Figure 256 shows the amino acid sequence (SEQ ID N0:256) derived from the coding sequence of SEQ
ID N0:255 shown in Figure 255.
Figure 257 shows a nucleotide sequence (SEQ ID N0:257) of a native sequence PR01124 cDNA, wherein SEQ ID N0:257 is a clone designated herein as "DNA60629-1481".

WO Ot/G8848 PCT/USO1/06520 Figure 258 shows the amino acid sequence (SEQ ID N0:258) derived from the coding sequence of SEQ
ID N0:257 shown in Figure 257.
Figure 259 shows a nucleotide sequence (SEQ ID N0:259) of a native sequence PR01380 cDNA, wherein SEQ ID N0:259 is a clone designated herein as "DNA60740-1615".
Figure 260 shows the amino acid sequence (SEQ ID N0:260) derived from the coding sequence of SEQ
ID N0:259 shown in Figure 259.
Figure 261 shows a nucleotide sequence (SEQ ID N0:261) of a native sequence PR01377 cDNA, wherein SEQ ID N0:261 is a clone designated herein as "DNA61608-1606".
Figure 262 shows the amino acid sequence (SEQ ID N0:262) derived from the coding sequence of SEQ
ID N0:261 shown in Figure 261.
Figure 263 shows a nucleotide sequence (SEQ ID N0:263) of a native sequence PR01287 cDNA, wherein SEQ ID N0:263 is a clone designated herein as "DNA61755-1554" .
Figure 264 shows the amino acid sequence (SEQ ID N0:264) derived from the coding sequence of SEQ
ID N0:263 shown in Figure 263.
Figure 265 shows a nucleotide sequence (SEQ ID N0:265) of a native sequence PR01249 cDNA, 1S wherein SEQ ID N0:265 is a clone designated herein as "DNA62809-1531".
Figure 266 shows the amino acid sequence (SEQ ID N0:266) derived from the coding sequence of SEQ
ID N0:265 shown in Figure 265.
Figure 267 shows a nucleotide sequence (SEQ ID N0:267) of a native sequence PR01335 cDNA, wherein SEQ ID N0:267 is a clone designated herein as "DNA62812-1594".
Figure 268 shows the amino acid sequence (SEQ ID N0:268) derived from the coding sequence of SEQ
ID N0:267 shown in Figure 267.
Figure 269 shows a nucleotide sequence (SEQ ID N0:269) of a native sequence PR03572 cDNA, wherein SEQ ID N0:269 is a clone designated herein as "DNA62813-2544".
Figure 270 shows the amino acid sequence (SEQ ID N0:270) derived from the coding sequence of SEQ
2S ID N0:269 shown in Figure 269.
Figure 271 shows a nucleotide sequence (SEQ ID N0:271) of a native sequence PR01599 cDNA, wherein SEQ ID N0:271 is a clone designated herein as "DNA62845-1684", Figure 272 shows the amino acid sequence (SEQ ID N0:272) derived from the coding sequence of SEQ
ID N0:271 shown in Figure 271.
Figure 273 shows a nucleotide sequence (SEQ ID N0:273) of a native sequence PR01374 cDNA, wherein SEQ ID N0:273 is a clone designated herein as "DNA64849-1604".
Figure 274 shows the amino acid sequence (SEQ ID N0:274) derived from the coding sequence of SEQ
ID N0:273 shown in Figure 273.
Figure 275 shows a nucleotide sequence (SEQ ID N0:275) of a native sequence PR01345 cDNA, wherein SEQ ID N0:275 is a clone designated herein as "DNA64852-1589".
Figure 276 shows the amino acid sequence (SEQ ID N0:276) derived from the coding sequence of SEQ
ID N0:275 shown in Figure 275.

Figure 277 shows a nucleotide sequence (SEQ ID N0:277) of a native sequence PR01311 cDNA, wherein SEQ ID N0:277 is a clone designated herein as "DNA64863-1573".
Figure 278 shows the amino acid sequence (SEQ ID N0:278) derived from the coding sequence of SEQ
ID N0:277 shown in Figure 277.
Figure 279 shows a nucleotide sequence (SEQ ID N0:279) of a native sequence PR01357 cDNA, wherein SEQ ID N0:279 is a clone designated herein as "DNA64881-1602".
Figure 280 shows the amino acid sequence (SEQ ID N0:280) derived from the coding sequence of SEQ
ID N0:279 shown in Figure 279.
Figure 281 shows a nucleotide sequence (SEQ ID N0:281) of a native sequence PR01557 cDNA, wherein SEQ ID N0:281 is a clone designated herein as "DNA64902-1667".
Figure 282 shows the amino acid sequence (SEQ ID N0:282) derived from the coding sequence of SEQ
ID N0:281 shown in Figure 281.
Figure 283 shows a nucleotide sequence (SEQ ID N0:283) of a native sequence PR01305 cDNA, wherein SEQ ID N0:283 is a clone designated herein as "DNA64952-1568".
Figure 284 shows the amino acid sequence (SEQ ID N0:284) derived from the coding sequence of SEQ
ID N0:283 shown in Figure 283.
Figure 28S shows a nucleotide sequence (SEQ ID N0:285) of a native sequence PR01302 cDNA, wherein SEQ ID N0:285 is a clone designated herein as "DNA65403-1565".
Figure 286 shows the amino acid sequence (SEQ ID N0:286) derived from the coding sequence of SEQ
ID N0:285 shown in Figure 285.
Figure 287 shows a nucleotide sequence (SEQ ID N0:287) of a native sequence PR01266 cDNA, wherein SEQ ID N0:287 is a clone designated herein as "DNA65413-1534".
Figure 288 shows the amino acid sequence (SEQ ID N0:288) derived from the coding sequence of SEQ
ID N0:287 shown in Figure 287.
Figures 289A-289B show a nucleotide sequence (SEQ ID N0:289) of a native sequence PR01336 cDNA, wherein SEQ ID N0:289 is a clone designated herein as "DNA65423-1595".
Figure 290 shows the amino acid sequence (SEQ ID N0:290) derived from the coding sequence of SEQ
ID N0:289 shown in Figures 289A-289B.
Figure 291 shows a nucleotide sequence (SEQ ID N0:291) of a native sequence PR01278 cDNA, wherein SEQ ID N0:291 is a clone designated herein as "DNA66304-1546".
Figure 292 shows the amino acid sequence (SEQ ID N0:292) derived from the coding sequence of SEQ
ID N0:291 shown in Figure 291.
Figure 293 shows a nucleotide sequence (SEQ ID N0:293) of a native sequence PR01270 cDNA, wherein SEQ ID N0:293 is a clone designated herein as "DNA66308-1537".
Figure 294 shows the amino acid sequence (SEQ ID N0:294) derived from the coding sequence of SEQ
ID N0:293 shown in Figure 293.
Figure 295 shows a nucleotide sequence (SEQ ID N0:295) of a native sequence PR01298 cDNA, wherein SEQ ID N0:295 is a clone designated herein as "DNA66511-1563".

Figure 296 shows the amino acid sequence (SEQ LD N0:296) derived from the coding sequence of SEQ
ID N0:295 shown in Figure 295.
Figure 297 shows a nucleotide sequence (SEQ ID N0:297) of a native sequence PR01301 cDNA, wherein SEQ ID N0:297 is a clone designated herein as "DNA66512-1564".
Figure 298 shows the amino acid sequence (SEQ ID N0:298) derived from the coding sequence of SEQ
ID N0:297 shown in Figure 297.
Figure 299 shows a nucleotide sequence (SEQ ID N0:299) of a native sequence PR01268 cDNA, wherein SEQ ID N0:299 is a clone designated herein as "DNA66519-1535".
Figure 300 shows the amino acid sequence (SEQ ID N0:300) derived from the coding sequence of SEQ
ID N0:299 shown in Figure 299.
Figure 301 shows a nucleotide sequence (SEQ ID N0:301) of a native sequence PR01327 cDNA, wherein SEQ m N0:301 is a clone designated herein as "DNA66521-1583".
Figure 302 shows the amino acid sequence (SEQ ID N0;302) derived from the coding sequence of SEQ
ID N0:301 shown in Figure 301.
Figure 303 shows a nucleotide sequence (SEQ ID N0:303) of a native sequence PR01328 cDNA, ZS wherein SEQ ID N0:303 is a clone designated herein as "DNA66658-1584".
Figure 304 shows the amino acid sequence (SEQ ID N0:304) derived from the coding sequence of SEQ
ID N0:303 shown in Figure 303.
Figure 305 shows a nucleotide sequence (SEQ ID N0:305) of a native sequence PR01329 cDNA, wherein SEQ ID N0:305 is a clone designated herein as "DNA66660-1585".
Figure 306 shows the amino acid sequence (SEQ ID N0:306) derived from the coding sequence of SEQ
ID N0:305 shown in Figure 305.
Figure 307 shows a nucleotide sequence (SEQ ID N0:307) of a native sequence PR01339 cDNA, wherein SEQ ID N0:307 is a clone designated herein as "DNA66669-1597".
Figure 308 shows the amino acid sequence (SEQ ID N0:308) derived from the coding sequence of SEQ
ID N0:307 shown in Figure 307.
Figure 309 shows a nucleotide sequence (SEQ ID N0:309) of a native sequence PR01342 cDNA, wherein SEQ 1D N0:309 is a clone designated herein as "DNA66674-1599".
Figure 310 shows the amino acid sequence (SEQ ID N0:310) derived from the coding sequence of SEQ
ID N0:309 shown in Figure 309.
Figures 311A-311B show a nucleotide sequence (SEQ ID N0:311) of a native sequence PR01487 cDNA, wherein SEQ ID N0:311 is a clone designated herein as "DNA68836-1656".
Figure 312 shows the amino acid sequence (SEQ ID N0:312) derived from the coding sequence of SEQ
ID N0:311 shown in Figures 311A-311B.
Figure 313 shows a nucleotide sequence (SEQ ID N0:313) of a native sequence PR03579 cDNA, wherein SEQ ID N0:313 is a clone designated herein as "DNA68862-2546".
Figure 314 shows the amino acid sequence (SEQ ID N0:314) derived from the coding sequence of SEQ
ID N0:313 shown in Figure 313.

Figure 315 shows a nucleotide sequence (SEQ ID N0:315) of a native sequence PR01472 cDNA, wherein SEQ ID N0:315 is a clone designated herein as "DNA68866-1644", Figure 316 shows the amino acid sequence (SEQ ID N0:316) derived from the coding sequence of SEQ
ID N0:315 shown in Figure 315.
Figure 317 shows a nucleotide sequence (SEQ ID N0:317) of a native sequence PR01385 cDNA, wherein SEQ ID N0:317 is a clone designated herein as "DNA68869-1610".
Pigure 318 shows the amino acid sequence (SEQ ID N0:318) derived from the coding sequence of SEQ
ID N0:317 shown in Figure 317.
Figure 319 shows a nucleotide sequence (SEQ ID N0:319) of a native sequence PR01461 cDNA, wherein SEQ ID N0:319 is a clone designated herein as "DNA68871-1638".
Figure 320 shows the amino acid sequence (SEQ ID N0:320) derived from the coding sequence of SEQ
ID N0:319 shown in Figure 319.
Figure 321 shows a nucleotide sequence (SEQ ID N0:321) of a native sequence PR01429 cDNA, wherein SEQ ID N0:321 is a clone designated herein as "DNA68879-1631".
Figure 322 shows the amino acid sequence (SEQ ID N0:322) derived from the coding sequence of SEQ
)D N0:321 shown in Figure 321.
Figure 323 shows a nucleotide sequence (SEQ ID N0:323) of a native sequence PR01568 cDNA, wherein SEQ ID N0:323 is a clone designated herein as "DNA68880-1676".
Figure 324 shows the amino acid sequence (SEQ ID N0:324) derived from the coding sequence of SEQ
ID N0:323 shown in Figure 323.
Figure 325 shows a nucleotide sequence (SEQ ID N0:325) of a native sequence PR01569 cDNA, wherein SEQ ID N0:325 is a clone designated herein as "DNA68882-1677".
Figure 326 shows the amino acid sequence (SEQ ID N0:326) derived from the coding sequence of SEQ
ID N0:325 shown in Figure 325.
Figure 327 shows a nucleotide sequence (SEQ ID N0:327) of a native sequence PR01753 cDNA, wherein SEQ ID N0:327 is a clone designated herein as "DNA68883-1691".
Figure 328 shows the amino acid sequence (SEQ ID N0:328) derived from the coding sequence of SEQ
ID N0:327 shown in Figure 327.
Figure 329 shows a nucleotide sequence (SEQ ID N0:329) of a native sequence PR01570 cDNA, wherein SEQ ID N0:329 is a clone designated herein as "DNA68885-1678".
Figure 330 shows the amino acid sequence (SEQ ID N0:330) derived from the coding sequence of SEQ
ID N0:329 shown in Figure 329.
Figure 331 shows a nucleotide sequence (SEQ ID N0:331) of a native sequence PR01559 cDNA, wherein SEQ ID N0:331 is a clone designated herein as "DNA68886".
Figure 332 shows the amino acid sequence (SEQ ID N0:332) derived from the coding sequence of SEQ
ID N0:331 shown in Figure 331.
Figure 333 shows a nucleotide sequence (SEQ ID N0:333) of a native sequence PR01486 cDNA, wherein SEQ ID N0:333 is a clone designated herein as "DNA71180-1655".

Figure 334 shows the amino acid sequence (SEQ TD N0:334) derived from the coding sequence of SEQ
ID N0:333 shown in Figure 333.
Figure 335 shows a nucleotide sequence (SEQ 1D N0:335) of a native sequence PR01433 cDNA, wherein SEQ ID N0:335 is a clone designated herein as "DNA71184-1634".
Figure 336 shows the amino acid sequence (SEQ ID N0:336) derived from the coding sequence of SEQ
ID N0:335 shown in Figure 335.
Figure 337 shows a nucleotide sequence (SEQ ID N0:337) of a native sequence PR01490 cDNA, wherein SEQ 1D N0:337 is a clone designated herein as "DNA71213-1659".
Figure 338 shows the amino acid sequence (SEQ ID N0:338) derived from the coding sequence of SEQ
ID N0:337 shown in Figure 337.
Figure 339 shows a nucleotide sequence (SEQ ID N0:339) of a native sequence PR01482 cDNA, wherein SEQ ID N0:339 is a clone designated herein as "DNA71234-1651".
Figure 340 shows the amino acid sequence (SEQ )D N0:340) derived from the coding sequence of SEQ
ID N0:339 shown in Figure 339.
Figure 341 shows a nucleotide sequence (SEQ ID N0:341) of a native sequence PR01449 cDNA, wherein SEQ ID N0:341 is a clone designated herein as "DNA71269-1621".
Figure 342 shows the amino acid sequence (SEQ ID N0:342) derived from the coding sequence of SEQ
ID N0:341 shown in Figure 341.
Figure 343 shows a nucleotide sequence (SEQ ID N0:343) of a native sequence PR01446 cDNA, wherein SEQ ID N0:343 is a clone designated herein as "DNA71277-1636".
Figure 344 shows the amino acid sequence (SEQ ID N0:344) derived from the coding sequence of SEQ
ID N0:343 shown in Figure 343.
Figure 345 shows a nucleotide sequence (SEQ ID N0:345) of a native sequence PR01604 cDNA, wherein SEQ ID N0:345 is a clone designated herein as "DNA71286-1687".
Figure 346 shows the amino acid sequence (SEQ ID N0:346) derived from the coding sequence of SEQ
ID N0:345 shown in Figure 345.
Figure 347 shows a nucleotide sequence (SEQ ID N0:347) of a native sequence PR01491 cDNA, wherein SEQ ID N0:347 is a clone designated herein as "DNA71883-1660".
Figure 348 shows the amino acid sequence (SEQ )D N0:348) derived from the coding sequence of SEQ
ID N0:347 shown in Figure 347.
Figure 349 shows a nucleotide sequence (SEQ ID N0:349) of a native sequence PR01431 cDNA, wherein SEQ )D N0:349 is a clone designated herein as "DNA73401-1633".
Figure 350 shows the amino acid sequence (SEQ ID N0:350) derived from the coding sequence of SEQ
ID N0:349 shown in Figure 349.
Figures 351A-351B show a nucleotide sequence (SEQ ID N0:351) of a native sequence PR01563 cDNA, wherein SEQ ID N0:351 is a clone designated herein as "DNA73492-1671".
Figure 3S2 shows the amino acid sequence (SEQ ID N0:352) derived from the coding sequence of SEQ
ID N0:351 shown in Figures 351A-351B.

Figure 353 shows a nucleotide sequence (SEQ ID N0:353) of a native sequence PR01571 eDNA, wherein SEQ ID N0:353 is a clone designated herein as "DNA73730-1679".
Figure 354 shows the amino acid sequence (SEQ ID N0:354) derived from the coding sequence of SEQ
ID N0:353 shown in Figure 353.
Figure 355 shows a nucleotide sequence (SEQ ID N0:355) of a native sequence PR01572 eDNA, wherein SEQ )D N0:35S is a clone designated herein as "DNA73734-1680".
Figure 356 shows the amino acid sequence (SEQ ID N0:356) derived from the coding sequence of SEQ
ID N0:355 shown in Figure 355.
Figure 357 shows a nucleotide sequence (SEQ ID N0:357) of a native sequence PR01573 eDNA, wherein SEQ ID N0:357 is a clone designated herein as "DNA73735-1681".
Figure 358 shows the amino acid sequence (SEQ ID N0:358) derived from the~coding sequence of SEQ
ID N0:357 shown in Figure 357.
Figure 359 shows a nucleotide sequence (SEQ ID N0:359) of a native sequence PR01508 eDNA, wherein SEQ ID N0:359 is a clone designated herein as "DNA73742-1662".
Figure 360 shows the amino acid sequence (SEQ ID N0:360) derived from the coding sequence of SEQ
ID N0:359 shown in Figure 359.
Figure 361 shows a nucleotide sequence (SEQ ID N0:361) of a native sequence PR0148S cDNA, wherein SEQ U~ N0:361 is a clone designated herein as "DNA73746-1654".
Figure 362 shows the amino acid sequence (SEQ ID N0:362) derived from the coding sequence of SEQ
ID N0:361 shown in Figure 361.
Figure 363 shows a nucleotide sequence (SEQ ID N0:363) of a native sequence PR01564 cDNA, wherein SEQ ID N0:363 is a clone designated herein as "DNA73760-1672".
Figure 364 shows the amino acid sequence (SEQ ID N0:364) derived from the coding sequence of SEQ
ID N0:363 shown in Figure 363.
Figure 365 shows a nucleotide sequence (SEQ ID N0:365) of a native sequence PR01550 cDNA, wherein SEQ ID N0:365 is a clone designated herein as "DNA76393-1664".
Figure 366 shows the amino acid sequence (SEQ ID N0:366) derived from the coding sequence of SEQ
ID N0:365 shown in Figure 365.
Figure 367 shows a nucleotide sequence (SEQ ID N0:367) of a native sequence PR01757 eDNA, wherein SEQ ID N0:367 is a clone designated herein as "DNA76398-1699".
Figure 368 shows the amino acid sequence (SEQ ID N0:368) derived from the coding sequence of SEQ
ID N0:367 shown in Figure 367.
Figure 369 shows a nucleotide sequence (SEQ ID N0:369) of a native sequence PR01758 cDNA, wherein SEQ ID N0:369 is a clone designated herein as "DNA76399-1700".
Figure 370 shows the amino acid sequence (SEQ ID N0:370) derived from the coding sequence of SEQ
ID N0:369 shown in Figure 369.
Figure 371 shows a nucleotide sequence (SEQ ID N0:371) of a native sequence PR01781 cDNA, wherein SEQ ID N0:371 is a clone designated herein as "DNA76522-2500".

Figure 372 shows the amino acid sequence (SEQ ID N0:372) derived from the coding sequence of SEQ
ID N0:371 shown in Figure 371.
Figure 373 shows a nucleotide sequence (SEQ ID N0:373) of a native sequence PR01606 cDNA, wherein SEQ ID N0:373 is a clone designated herein as "DNA76533-1689".
Figure 374 shows the amino acid sequence (SEQ ID N0:374) derived from the coding sequence of SEQ
ID N0:373 shown in Figure 373.
Figure 375 shows a nucleotide sequence (SEQ ID N0:375) of a native sequence PR01784 cDNA, wherein SEQ ID N0:375 is a clone designated herein as "DNA77303-2502".
Figure 376 shows the amino acid sequence (SEQ ID N0:376) derived from the coding sequence of SEQ
ID N0:375 shown in Figure 375.
Figure 377 shows a nucleotide sequence (SEQ ID N0:377) of a native sequence PR01774 cDNA, wherein SEQ )D N0:377 is a clone designated herein as "DNA77626-1705".
Figure 378 shows the amino acid sequence (SEQ ID N0:378) derived from the coding sequence of SEQ
ID N0:377 shown in Figure 377.
Figure 379 shows a nucleotide sequence (SEQ ID N0:379) of a native sequence PR01605 cDNA, wherein SEQ ID N0:379 is a clone designated herein as "DNA77648-1688".
Figure 380 shows the amino acid sequence (SEQ ID N0:380) derived from the coding sequence of SEQ
ID N0:379 shown in Figure 379.
Figure 381 shows a nucleotide sequence (SEQ ID N0:381) of a native sequence PR01928 cDNA, wherein SEQ ID N0:381 is a clone designated herein as "DNA81754-2532".
Figure 382 shows the amino acid sequence (SEQ )D N0:382) derived from the coding sequence of SEQ
ID N0:381 shown in Figure 381.
Figure 383 shows a nucleotide sequence (SEQ ID N0:383) of a native sequence PR01865 cDNA, wherein SEQ ID N0:383 is a clone designated herein as "DNA81757-2512".
Figure 384 shows the amino acid sequence (SEQ ID N0:384) derived from the coding sequence of SEQ
ID N0:383 shown in Figure 383.
Figure 385 shows a nucleotide sequence (SEQ ID N0:385) of a native sequence PR01925 cDNA, wherein SEQ ID N0:385 is a clone designated herein as "DNA82302-2529".
Figure 386 shows the amino acid sequence (SEQ ID N0:386) derived from the coding sequence of SEQ
ID N0:385 shown in Figure 385.
Figure 387 shows a nucleotide sequence (SEQ ID N0:387) of a native sequence PR01926 cDNA, wherein SEQ ID N0:387 is a clone designated herein as "DNA82340-2530" .
Figure 388 shows the amino acid sequence (SEQ ID N0:388) derived from the coding sequence of SEQ
ID N0:387 shown in Figure 387.
Figure 389 shows a nucleotide sequence (SEQ ID N0:389) of a native sequence PR02630 cDNA, wherein SEQ ID N0:389 is a clone designated herein as "DNA83551".
Figure 390 shows the amino acid sequence (SEQ ID N0:390) derived from the coding sequence of SEQ
ID N0:389 shown in Figure 389.

Figure 391 shows a nucleotide sequence (SEQ ID N0:391) of a native sequence PR03443 cDNA, wherein SEQ ID N0:391 is a clone designated herein as "DNA87991-2540".
Figure 392 shows the amino acid sequence (SEQ ID N0:392) derived from the coding sequence of SEQ
)D N0:391 shown in Figure 391.
Figure 393 shows a nucleotide sequence (SEQ ID N0:393) of a native sequence PR03301 cDNA, wherein SEQ ID N0:393 is a clone designated herein as "DNA88002".
Figure 394 shows the amino acid sequence (SEQ ID N0:394) derived from the coding sequence of SEQ
ID N0:393 shown in Figure 393.
Figure 395 shows a nucleotide sequence (SEQ ID N0:395) of a native sequence PR03442 cDNA, wherein SEQ ID N0:395 is a clone designated herein as "DNA92238-2539".
Figure 396 shows the amino acid sequence (SEQ ID N0:396) derived from the coding sequence of SEQ
ID N0:395 shown in Figure 395.
Figure 397 shows a nucleotide sequence (SEQ ID N0:397) of a native sequence PR04978 cDNA, wherein SEQ ID N0:397 is a clone designated herein as "DNA95930".
Figure 398 shows the amino acid sequence (SEQ ID N0:398) derived from the coding sequence of SEQ
ID N0:397 shown in Figure 397.
Figure 399 shows a mxcleotide sequence (SEQ ID N0:399) of a native sequence PR05801 cDNA, wherein SEQ ID N0:399 is a clone designated herein as "DNA115291-2681".
Figure 400 shows the amino acid sequence (SEQ ID N0:400) derived from the coding sequence of SEQ
ID N0:399 shown in Figure 399.
Figure 401 shows a nucleotide sequence (SEQ ID N0:401) of a native sequence PR019630 cDNA, wherein SEQ ID N0:401 is a clone designated herein as "DNA23336-2861".
Figure 402 shows the amino acid sequence (SEQ ID N0:402) derived from Lhe coding sequence of SEQ
ID N0:401 shown in Figure 401.
Figure 403 shows a nucleotide sequence (SEQ ID N0:403) of a native sequence PR0203 cDNA, wherein SEQ )D N0:403 is a clone designated herein as "DNA30862-1396".
Figure 404 shows the amino acid sequence (SEQ ID N0:404) derived from the coding sequence of SEQ
ID N0:403 shown in Figure 403.
Figure 405 shows a nucleotide sequence (SEQ ID N0:405) of a native sequence PR0204 cDNA, wherein SEQ ID N0:405 is a clone designated herein as "DNA30871-1157".
Figure 406 shows the amino acid sequence (SEQ ID N0:406) derived from the coding sequence of SEQ
ID N0:405 shown in Pigure 405.
Figure 407 shows a nucleotide sequence (SEQ ID N0:407) of a native sequence PR0210 cDNA, wherein SEQ ID N0:407 is a clone designated herein as "DNA32279-1131".
Figure 408 shows the amino acid sequence (SEQ ID N0:408) derived from the coding sequence of SEQ
ID N0:407 shown in Figure 407.
Figure 409 shows a nucleotide sequence (SEQ ID N0:409) of a native sequence PR0223 cDNA, wherein SEQ ID N0:409 is a clone designated herein as "DNA33206-1165".

Figure 410 shows the amino acid sequence (SEQ 1D N0:410) derived from the coding sequence of SEQ
ID N0:409 shown in Figure 409.
Figure 411 shows a nucleotide sequence (SEQ ID N0:411) of a native sequence PR0247 cDNA, wherein SEQ ID N0:411 is a clone designated herein as "DNA35673-1201".
Figure 412 shows the amino acid sequence (SEQ ID N0:412) derived from the coding sequence of SEQ
S ID N0:411 shown in Figure 411.
Figure 413 shows a nucleotide sequence (SEQ ID N0:413) of a native sequence PR0358 cDNA, wherein SEQ ID N0:413 is a clone designated herein as "DNA47361-1154-2".
Figure 414 shows the amino acid sequence (SEQ ID N0:414) derived from the coding sequence of SEQ
ID N0:413 shown in Figure 413.
Figure 415 shows a nucleotide sequence (SEQ ID N0:415) of a native sequence PR0724 cDNA, wherein SEQ ID N0:415 is a clone designated herein as "DNA49631-1328".
Figure 416 shows the amino acid sequence (SEQ ID N0:416) derived from the coding sequence of SEQ
ID N0:415 shown in Figure 415.
Figure 417 shows a nucleotide sequence (SEQ ID N0:417) of a native sequence PR0868 cDNA, wherein SEQ ID N0:417 is a clone designated herein as "DNA52594-1270".
Figure 418 shows the amino acid sequence (SEQ ID N0:418) derived from the coding sequence of SEQ
ID N0:417 shown in Figure 417.
Figure 419 shows a nucleotide sequence (SEQ ID N0:419) of a native sequence PR0740 cDNA, wherein SEQ ID N0:419 is a clone designated herein as "DNA55800-1263".
Figure 420 shows the amino acid sequence (SEQ ID N0:420) derived from the coding sequence of SEQ
ID N0:419 shown in Figure 419.
Figure 421 shows a nucleotide sequence (SEQ ID N0:421) of a native sequence PR01478 cDNA, wherein SEQ ID N0:421 is a clone designated herein as "DNA56531-1648".
Figure 422 shows the amino acid sequence (SEQ ID N0:422) derived from the coding sequence of SEQ
B7 N0:421 shown in Figure 421.
Figvre 423 shows a nucleotide sequence (SEQ ID N0:423) of a native sequence PR0162 cDNA, wherein SEQ ID N0:423 is a clone designated herein as "DNA56965-1356".
Figure 424 shows the amino acid sequence (SEQ ID N0:424) derived from the coding sequence of SEQ
ID N0:423 shown in Figure 423.
Figure 42S shows a nucleotide sequence (SEQ ID N0:425) of a native sequence PR0828 cDNA, wherein SEQ ID N0:425 is a clone designated herein as "DNA57037-1444".
Figure 426 shows the amino acid sequence (SEQ ID N0:426) derived from the coding sequence of SEQ
ID N0:425 shown in Figure 425.
Figure 427 shows a nucleotide sequence (SEQ ID N0:427) of a native sequence PR0819 cDNA, wherein SEQ ID N0:427 is a clone designated herein as "DNA57695-1340".
Figure 428 shows the amino acid sequence (SEQ ID N0:428) derived from the coding sequence of SEQ
ID N0:427 shown in Figure 427.

Figure 429 shows a nucleotide sequence (SEQ ID N0:429) of a native sequence PR0813 cDNA, wherein SEQ ID N0:429 is a clone designated herein as "DNA57834-1339".
Figure 430 shows the amino acid sequence (SEQ ID N0:430) derived from the coding sequence of SEQ
ID N0:429 shown in Figure 429.
Figure 431 shows a nucleotide sequence (SEQ ID N0:431) of a native sequence PR01194 cDNA, wherein SEQ ID N0:431 is a clone designated herein as "DNA57841-1522".
Figure 432 shows the amino acid sequence (SEQ ID N0:432) derived from the coding sequence of SEQ
ID N0:431 shown in Figure 431.
Figure 433 shows a nucleotide sequence (SEQ ID N0:433) of a native sequence PR0887 cDNA, wherein SEQ ID N0:433 is a clone designated herein as "DNA58130".
Figure 434 shows the amino acid sequence (SEQ ID N0:434) derived from the coding sequence of SEQ
ID N0:433 shown in Figure 433.
Figure 435 shows a nucleotide sequence (SEQ ID N0:435) of a native sequence PR01071 cDNA, wherein SEQ ID N0:435 is a clone designated herein as "DNA58847-1383".
Figure 436 shows the amino acid sequence (SEQ ID N0:436) derived from the coding sequence of SEQ
ID N0:435 shown in Figure 435.
Figure 437 shows a nucleotide sequence (SEQ ID N0:437) of a native sequence PR01029 cDNA, wherein SEQ ID N0:437 is a clone designated herein as "DNA59493-1420".
Figure 438 shows the amino acid sequence (SEQ ID N0:438) derived from the coding sequence of SEQ
ID N0:437 shown in Figure 437.
Figure 439 shows a nucleotide sequence (SEQ ID N0:439) of a native sequence PR01190 cDNA, wherein SEQ ID N0:439 is a clone designated herein as "DNA59586-1520".
Figure 440 shows the amino acid sequence (SEQ ID N0:440) derived from the coding sequence of SEQ
ID N0:439 shown in Figure 439.
Figure 441 shows a nucleotide sequence (SEQ ID N0:441) of a native sequence PR04334 cDNA, wherein SEQ ID N0:441 is a clone designated herein as "DNA59608-2577".
Figure 442 shows the amino acid sequence (SEQ )I7 N0:442) derived from the coding sequence of SEQ
ID N0:441 shown in Figure 441.
Figure 443 shows a nucleotide sequence (SEQ ID N0:443) of a native sequence PR01155 cDNA, wherein SEQ iD N0:443 is a clone designated herein as "DNA59849-1504".
Figure 444 shows the amino acid sequence (SEQ ID N0:444) derived from the coding sequence of SEQ
ID N0:443 shown in Figure 443.
Figure 445 shows a nucleotide sequence (SEQ ID N0:445) of a native sequence PR01157 cDNA, wherein SEQ ID N0:445 is a clone designated herein as "DNA60292-1506".
Figure 446 shows the amino acid sequence (SEQ ID N0:446) derived from the coding sequence of SEQ
ID N0:445 shown in Figure 445.
Figure 447 shows a nucleotide sequence (SEQ 1D N0:447) of a native sequence PROI122 cDNA, wherein SEQ ID N0:447 is a clone designated herein as "DNA62377-1381-1".

Figure 448 shows the amino acid sequence (SEQ ID N0:448) derived from the coding sequence of SEQ
ID N0:447 shown in Figure 447.
Figure 449 shows a nucleotide sequence (SEQ ID N0:449) of a native sequence PR01183 cDNA, wherein SEQ ID N0:449 is a clone designated herein as "DNA62880-1513".
Figure 450 shows the amino acid sequence (SEQ ID N0:450) derived from the coding sequence of SEQ
ID N0:449 shown in Figure 449.
Figure 451 shows a nucleotide sequence (SEQ ID NO_:451) of a native sequence PR01337 cDNA, wherein SEQ 1D N0:451 is a clone designated herein as "DNA66672-1586".
Figure 452 shows the amino acid sequence (SEQ ID N0:452) derived from the coding sequence of SEQ
ID N0:451 shown in Figure 451.
Figure 453 shows a nucleotide sequence (SEQ ID N0:453) of a native sequence PR01480 cDNA, wherein SEQ m N0:453 is a clone designated herein as "DNA67962-1649".
Figure 454 shows the amino acid sequence (SEQ ID N0:454) derived from the coding sequence of SEQ
ID N0:453 shown in Figure 453.
Figure 45S shows a nucleotide sequence (SEQ ID N0:455) of a native sequence PR019645 cDNA, wherein SEQ ID N0:455 is a clone designated herein as "DNA69555-2867".
Figure 456 shows the amino acid sequence (SEQ 1D N0:456) derived from the coding sequence of SEQ
ID N0:455 shown in Figure 455.
Figure 457 shows a nucleotide sequence (SEQ ID N0:457) of a native sequence PR09782 cDNA, wherein SEQ ID N0:457 is a clone designated herein as "DNA71162-2764" .
Figure 458 shows the amino acid sequence (SEQ ID N0:458) derived from the coding sequence of SEQ
ID N0:457 shown in Figure 457.
Figure 459 shows a nucleotide sequence (SEQ ID N0:459) of a native sequence PR01419 cDNA, wherein SEQ ID N0:459 is a clone designated herein as "DNA71290-1630".
Figure 460 shows the amino acid sequence (SEQ ID N0:460) derived from the coding sequence of SEQ
ID N0:459 shown in Figure 459.
Figure 4b1 shows a nucleotide sequence (SEQ ID N0:461) of a native sequence PR01575 cDNA, wherein SEQ ID N0:461 is a clone designated herein as "DNA76401-1683".
Figure 462 shows the amino acid sequence (SEQ ID N0:462) derived from the coding sequence of SEQ
ID N0:461 shown in Figure 461.
Figure 463 shows a nucleotide sequence (SEQ ID N0:463) of a native sequence PR01567 cDNA, wherein SEQ ID N0:463 is a clone designated herein as "DNA76541-1675".
Figure 464 shows the amino acid sequence (SEQ ID N0:464) derived from the coding sequence of SEQ
ID N0:463 shown in Figure 463.
Figure 465 shows a nucleotide sequence (SEQ ID N0:465) of a native sequence PR01891 cDNA, wherein SEQ 1D N0:465 is a clone designated herein as "DNA76788-2526".
Figure 466 shows the amino acid sequence (SEQ ID N0:466) derived from the coding sequence of SEQ
)D N0:465 shown in Figure 465, WO 01/68848 PCT/US01J06i20 Figure 467 shows a nucleotide sequence (SEQ ID N0:467) of a native sequence PR01889 cDNA, wherein SEQ ID N0:467 is a clone designated herein as "DNA77623-2524".
Figure 468 shows the amino acid sequence (SEQ ID N0:468) derived from the coding sequence of SEQ
ID N0:467 shown in Figure 467.
Figure 469 shows a nucleotide sequence (SEQ ID N0:469) of a native sequence PR01785 cDNA, wherein SEQ TD N0:469 is a clone designated herein as "DNA80136-2503".
Figure 470 shows the amino acid sequence (SEQ ID N0:470) derived from the coding sequence of SEQ
ID N0:469 shown in Figure 469.
Figure 471 shows a nucleotide sequence (SEQ ID N0:471) of a native sequence PR06003 cDNA, wherein SEQ 1D N0:471 is a clone designated herein as ~DNA83568-2692".
Figure 472 shows the amino acid sequence (SEQ ID N0:472) derived from the coding sequence of SEQ
ID N0:471 shown in Figure 471.
Figure 473 shows a nucleotide sequence (SEQ ID N0:473) of a native sequence PR04333 cDNA, wherein SEQ ID N0:473 is a clone designated herein as "DNA84210-2576".
Figure 474 shows the amino acid sequence (SEQ ID N0:474) derived from the coding sequence of SEQ
ID N0:473 shown in Figure 473.
Figure 475 shows a nucleotide sequence (SEQ ID N0:475) of a native sequence PR04356 cDNA, wherein SEQ 1D N0:475 is a clone designated herein as "DNA86576-2595".
Figure 476 shows the amino acid sequence (SEQ ID N0:476) derived from the coding sequence of SEQ
ID N0:475 shown in Figure 475.
Figure 477 shows a nucleotide sequence (SEQ ID N0:477) of a native sequence PR04352 cDNA, wherein SEQ ID N0:477 is a clone designated herein as ~DNA87976-2593".
Figure 478 shows the amino acid sequence (SEQ ID N0:478) derived from the coding sequence of SEQ
ID N0:477 shown in Figure 477.
Figure 479 shows a nucleotide sequence (SEQ ID N0:479) of a native sequence PR04354 cDNA, wherein SEQ ID N0:479 is a clone designated herein as "DNA92256-2596".
Figure 480 shows the amino acid sequence (SEQ ID N0:480) derived from the coding sequence of SEQ
ID N0:479 shown in Figure 479.
Figure 48I shows a nucleotide sequence (SEQ ID N0:481) of a native sequence PR04369 cDNA, wherein SEQ ID N0:481 is a clone designated herein as "DNA92289-2598".
Figure 482 shows the amino acid sequence (SEQ ID N0:482) derived from the coding sequence of SEQ
ID N0:481 shown in Figure 481.
Figure 483 shows a nucleotide sequence (SEQ ID N0:483) of a native sequence PR06030 cDNA, wherein SEQ ID N0:483 is a clone designated herein as "DNA96850-2705".
Figure 484 shows the amino acid sequence (SEQ ID N0:484) derived from the coding sequence of SEQ
ID N0:483 shown in Figure 483.
Figure 485 shows a nucleotide sequence (SEQ ID N0:485) of a native sequence PR04433 cDNA, wherein SEQ ID N0:485 is a clone designated herein as "DNA96855-2629".

Figure 486 shows the amino acid sequence (SEQ ID N0:486) derived from the coding sequence of SEQ
ID N0:485 shown in Figure 485.
Figure 487 shows a nucleotide sequence (SEQ ID N0:487) of a native sequence PR04424 cDNA, wherein SEQ ID N0:487 is a clone designated herein as "DNA96857-2636".
Figure 488 shows the amino acid sequence (SEQ >D N0:488) derived from the coding sequence of SEQ
ID N0:487 shown in Figure 487.
Figure 489 shows a nucleotide sequence (SEQ ID N0:489) of a native sequence PR06017 cDNA, wherein SEQ ID N0:489 is a clone designated herein as "DNA96860-2700".
Figure 490 shows the amino acid sequence (SEQ ID N0:490) derived from the coding sequence of SEQ
ID N0:489 shown in Figure 489.
Figure 491 shows a nucleotide sequence (SEQ ID N0:491) of a native sequence PR019563 cDNA, wherein SEQ ID N0:491 is a clone designated herein as "DNA96861-2844".
Figure 492 shows the amino acid sequence (SEQ ID N0:492) derived from the coding sequence of SEQ
ID N0:491 shown in Figure 491.
Figure 493 shows a nucleotide sequence (SEQ ID N0:493) of a native sequence PR06015 cDNA, wherein SEQ ID N0:493 is a clone designated herein as "DNA96866-2698".
Figure 494 shows the amino acid sequence (SEQ ID N0:494) derived from the coding sequence of SEQ
ID N0:493 shown in Figure 493.
Figure 495 shows a nucleotide sequence (SEQ ID N0:495) of a native sequence PR05779 cDNA, wherein SEQ ID N0:495 is a clone designated herein as "DNA96870-2676".
Figure 496 shows the amino acid sequence (SEQ ID N0:496) derived from the coding sequence of SEQ
ID N0:495 shown in Figure 495.
Figure 497 shows a nucleotide sequence (SEQ ID N0:497) of a native sequence PR05776 cDNA, wherein SEQ ID N0:497 is a clone designated herein as "DNA96872-2674".
Figure 498 shows the amino acid sequence (SEQ ID N0:498) derived from the coding sequence of SEQ
ID N0:497 shown in Figure 497.
Figure 499 shows a nucleotide sequence (SEQ ID N0:499) of a native sequence PR04430 cDNA, wherein SEQ ID N0:499 is a clone designated herein as "DNA96878-2626".
Figure 500 shows the amino acid sequence (SEQ ID N0:500) derived from the coding sequence of SEQ
ID N0:499 shown in Figure 499.
Figure 501 shows a nucleotide sequence (SEQ ID N0:501) of a native sequence PR04421 cDNA, wherein SEQ ID N0:501 is a clone designated herein as "DNA96879-2619".
Figure 502 shows the amino acid sequence (SEQ ID N0:502) derived from the coding sequence of SEQ
ID N0:501 shown in Figure 501.
Figure 503 shows a nucleotide sequence (SEQ ID N0:503) of a native sequence PR04499 cDNA, wherein SEQ ID N0:503 is a clone designated herein as "DNA96889-2641".
Figure 504 shows the amino acid sequence (SEQ ID N0:504) derived from the coding sequence of SEQ
ID N0:503 shown in Figure 503.

Figure SOS shows a nucleotide sequence (SEQ ID N0:505) of a native sequence PR04423 cDNA, wherein SEQ ID N0:505 is a clone designated herein as "DNA96893-2621".
Figure 506 shows the amino acid sequence (SEQ ID N0:506) derived from the coding sequence of SEQ
ID NO:SOS shown in Figure 505.
Figure 507 shows a nucleotide sequence (SEQ TD N0:507) of a native sequence PR05998 cDNA, wherein SEQ ID N0:507 is a clone designated herein as "DNA96897-2688".
Figure 508 shows the amino acid sequence (SEQ ID N0:508) derived from the coding sequence of SEQ
ID N0:507 shown in Figure 507.
Figure S09 shows a nucleotide sequence (SEQ ID N0:509) of a native sequence PR04501 cDNA, wherein SEQ 1D N0:509 is a clone designated herein as "DNA98564-2643".
Figure 510 shows the amino acid sequence (SEQ ID NO:S10) derived from the coding sequence of SEQ
ID N0:509 shown in Figure 509.
Figure 511 shows a nucleotide sequence (SEQ ID N0:511) of a native sequence PR06240 cDNA, wherein SEQ ID NO:S11 is a clone designated herein as "DNA107443-2718".
Figure 512 shows the amino acid sequence (SEQ ID N0:512) derived from the coding sequence of SEQ
ID N0:511 shown in Figure 511.
Figure SI3 shows a nucleotide sequence (SEQ ID N0:513) of a native sequence PR06245 cDNA, wherein SEQ 1D N0:513 is a clone designated herein as "DNA107786-2723".
Fignre S14 shows the amino acid sequence (SEQ ID N0:514) derived from the coding sequence of SEQ
ID N0:513 shown in Figure 513.
Figure S15 shows a nucleotide sequence (SEQ 1D N0:515) of a native sequence PR06175 cDNA, wherein SEQ ID N0:515 is a clone designated herein as "DNA108682-2712".
Figure 516 shows the amino acid sequence (SEQ ID NO:S16) derived from the coding sequence of SEQ
ID N0:51S shown in Figure 515.
Figure 517 shows a nucleotide sequence (SEQ 1D N0:517) of a native sequence PR09742 cDNA, wherein SEQ ID N0:517 is a clone designated herein as "DNA108684-2761".
Figure 518 shows the amino acid sequence (SEQ ID N0:518) derived from the coding sequence of SEQ
ID NO:SI7 shown in Figure 517.
Figure 519 shows a nucleotide sequence (SEQ 1D N0:519) of a native sequence PR07179 cDNA, wherein SEQ ID N0:519 is a clone designated herein as ~DNA108701-2749".
Figure 520 shows the amino acid sequence (SEQ ID N0:520) derived from the coding sequence of SEQ
ID N0:519 shown in Figure 519.
Figure 521 shows a nucleotide sequence (SEQ ID N0:521) of a native sequence PR06239 cDNA, wherein SEQ ID N0:521 is a clone designated herein as "DNA108720-2717".
Figure 522 shows the amino acid sequence (SEQ ID N0:522) derived from the coding sequence of SEQ
ID N0:521 shown in Figure 521.
Figure 523 shows a nucleotide sequence (SEQ ID N0:523) of a native sequence PR06493 cDNA, wherein SEQ 1D N0:523 is a clone designated herein as "DNA108726-2729".

Figure 524 shows the amino acid sequence (SEQ ID N0:524) derived from the coding sequence of SEQ
ID N0:523 shown in Figure 523.
Figures 525A-525B show a nucleotide sequence (SEQ 1D N0:525) of a native sequence PR09741 cDNA, wherein SEQ 1D N0:525 is a clone designated herein as "DNA108728-2760".
Figure 526 shows the amino acid sequence (SEQ ID N0:526) derived from the coding sequence of SEQ
ID N0:525 shown in Figures 525A-525B.
Figure 527 shows a nucleotide sequence (SEQ ID N0:527) of a native sequence PR09822 cDNA, wherein SEQ ID N0:527 is a clone designated herein as "DNA108738-2767".
Figure 528 shows the amino acid sequence (SEQ 1D N0:528) derived from the coding sequence of SEQ
ID N0:527 shown in Figure 527.
Figure 529 shows a nucleotide sequence (SEQ ID N0:529) of a native sequence PR06244 cDNA, wherein SEQ ID N0:529 is a clone designated herein as "DNA108743-2722".
Figure 530 shows the amino acid sequence (SEQ ID N0:530) derived from the coding sequence of SEQ
ID N0:529 shown in Figure 529.
Figure 531 shows a nucleotide sequence (SEQ 1D N0:531) of a native sequence PR09740 cDNA, wherein SEQ ID N0:531 is a clone designated herein as "DNA108758-2759".
Figure 532 shows the amino acid sequence (SEQ ID N0:532) derived from the coding sequence of SEQ
ID N0:531 shown in Figure 53I.
Figure 533 shows a nucleotide sequence (SEQ ID N0:533) of a native sequence PR09739 eDNA, wherein SEQ ID N0:533 is a clone designated herein as "DNA108765-2758".
Figure 534 shows the amino acid sequence (SEQ ID N0:534) derived from the coding sequence of SEQ
ID N0:533 shown in Figure 533.
Figure 535 shows a nucleotide sequence (SEQ ID N0:535) of a native sequence PR07177 cDNA, wherein SEQ ID N0:535 is a clone designated herein as "DNA108783-2747".
Figure 536 shows the amino acid sequence (SEQ ID N0:536) derived from the coding sequence of SEQ
ID N0:535 shown in Figure 535.
Figure 537 shows a nucleotide sequence (SEQ ID N0:537) of a native sequence PR07178 cDNA, wherein SEQ ID N0:537 is a clone designated herein as "DNA108789-2748".
Figure 538 shows the amino acid sequence (SEQ ID N0:538) derived from the coding sequence of SEQ
m N0:537 shown in Figure 537.
Figure 539 shows a nucleotide sequence (SEQ ID N0:539) of a native sequence PR06246 eDNA, wherein SEQ ID N0:539 is a clone designated herein as "DNA108806-2724".
Figure 540 shows the amino acid sequence (SEQ ID N0:540) derived from the coding sequence of SEQ
ID N0:539 shown in Figure 539.
Figure 541 shows a mzcleotide sequence (SEQ ID N0:541) of a native sequence PR06241 eDNA, wherein SEQ ID N0:541 is a clone designated herein as "DNA108936-2719".
Figure 542 shows the amino acid sequence (SEQ ID N0:542) derived from the coding sequence of SEQ
1D N0:541 shown in Figure 541.

Figure 543 shows a nucleotide sequence (SEQ ID N0:543) of a native sequence PR09835 cDNA, wherein SEQ >D N0:543 is a clone designated herein as "DNA119510-2771".
Figure 544 shows the amino acid sequence (SEQ ID N0:544) derived from the coding sequence of SEQ
ID N0:543 shown in Figure 543.
Figure 545 shows a nucleotide sequence (SEQ ID N0:545) of a native sequence PR09857 cDNA, wherein SEQ ID N0:545 is a clone designated herein as "DNA119517-2778".
Figure 546 shows the amino acid sequence (SEQ ID N0:546) derived from the coding sequence of 5EQ
1D N0:545 shown in Figure 545.
Figure 547 shows a nucleotide sequence (SEQ ID N0:547) of a native sequence PR07436 cDNA, wherein SEQ ID N0:547 is a clone designated herein as "DNA119535-2756".
Figure 548 shows the amino acid sequence (SEQ ID N0:548) derived from the coding sequence of SEQ
ID NO:S47 shown in Figure 547.
Figure 549 shows a nucleotide sequence (SEQ ID N0:549) of a native sequence PR09856 cDNA, wherein SEQ ID N0:549 is a clone designated herein as "DNA119537-2777".
Figure 550 shows the amino acid sequence (SEQ ID N0:550) derived from the coding sequence of SEQ
ID N0:549 shown in Figure 549.
Figure 551 shows a nucleotide sequence (SEQ ID N0:551) of a native sequence PR019605 cDNA, wherein SEQ ID N0:551 is a clone designated herein as "DNA119714-2851".
Figure 552 shows the amino acid sequence (SEQ ID N0:552) derived from the coding sequence of SEQ
ID N0:551 shown in Figure 551.
Figure 553 shows a nucleotide sequence (SEQ ID N0:553) of a native sequence PR09859 cDNA, wherein SEQ ID N0:553 is a clone designated herein as "DNA125170-2780".
Figure 554 shows the amino acid sequence (SEQ ID N0:554) derived from the coding sequence of SEQ
ID NO:S53 shown in Figure 553.
Figure 555 shows a nucleotide sequence (SEQ ID N0:555) of a native sequence PR012970 cDNA, wherein SEQ ID N0:555 is a clone designated herein as "DNA129594-2841".
Figure S56 shows the amino acid sequence (SEQ ID N0:556) derived from the coding sequence of SEQ
ID N0:555 shown in Figure 555.
Figure 557 shows a nucleotide sequence (SEQ ID N0:557) of a native sequence PROI9626 cDNA, wherein SEQ ID N0:557 is a clone designated herein as "DNA129793-2857".
Figure 558 shows the amino acid sequence (SEQ ID N0:558) derived from the coding sequence of SEQ
ID N0:557 shown in Figure 557.
Figure 559 shows a nucleotide sequence (SEQ ID N0:559) of a native sequence PR09833 cDNA, wherein SEQ ID NO:S59 is a clone designated herein as "DNA130809-2769".
Figure 560 shows the amino acid sequence (SEQ ID NO:S60) derived from the coding sequence of SEQ
ID N0:559 shown in Figure 559.
Figure 561 shows a nucleotide sequence (SEQ ID N0:561) of a native sequence PR019670 cDNA, wherein SEQ 1D N0:561 is a clone designated herein as "DNA131639-2874".

Figure 562 shows the amino acid sequence (SEQ ID N0:562) derived from the coding sequence of SEQ
ID N0:561 shown in Figure 561.
Figure 563 shows a nucleotide sequence (SEQ ID N0:563) of a native sequence PR019624 cDNA, wherein SEQ ID N0:563 is a clone designated herein as "DNA131649-2855".
Figure 564 shows the amino acid sequence (SEQ ID N0:564) derived from the coding sequence of SEQ
ID N0:563 shown in Figure 563.
Figure 565 shows a nucleotide sequence (SEQ ID N0:565) of a native sequence PR019680 cDNA, wherein SEQ 117 N0:565 is a clone designated herein as "DNA131652-2876".
Figure 566 shows the amino acid sequence (SEQ ID N0:566) derived from the coding sequence of SEQ
ID N0:565 shown in Figure 565.
Figure 567 shows a nucleotide sequence (SEQ ID N0:567) of a native sequence PR019675 cDNA, wherein SEQ ID N0:567 is a clone designated herein as "DNA131658-2875".
Figure 568 shows the amino acid sequence (SEQ ID N0:568) derived from the coding sequence of SEQ
ID N0:567 shown in Figure 567.
Figure 569 shows a nucleotide sequence (SEQ ID N0:569) of a native sequence PR09834 cDNA, wherein SEQ B7 N0:569 is a clone designated herein as "DNA132162-2770".
Figure 570 shows the amino acid sequence (SEQ ID N0:570) derived from the coding sequence of SEQ
ID N0:569 shown in Figure 569.
Figure 571 shows a nucleotide sequence (SEQ ID N0:571) of a native sequence PR09744 cDNA, wherein SEQ ID N0:571 is a clone designated herein as "DNA136110-2763".
Figure 572 shows the amino acid sequence (SEQ ID N0:572) derived from the coding sequence of SEQ
TD N0:571 shown in Figure 571.
Figure 573 shows a nucleotide sequence (SEQ ID N0:573) of a native sequence PR019644 cDNA, wherein SEQ )D N0:573 is a clone designated herein as "DNA139592-2866".
Figure 574 shows the amino acid sequence (SEQ ID N0:574) derived from the coding sequence of SEQ
ID N0:573 shown in Figure 573.
Figure 575 shows a nucleotide sequence (SEQ ID N0:575) of a native sequence PR019625 cDNA, wherein SEQ ID N0:575 is a clone designated herein as "DNA139608-2856".
Figure 576 shows the amino acid sequence (SEQ ID N0:576) derived from the coding sequence of SEQ
ID N0:575 shown in Figure 575.
Figure 577 shows a nucleotide sequence (SEQ ID N0:577) of a native sequence PR019597 cDNA, wherein SEQ ID N0:577 is a clone designated herein as "DNA143292-2848".
Figure 578 shows the amino acid sequence (SEQ ID N0:578) derived from the coding sequence of SEQ
ID N0:577 shown in Figure 577.
Figure 579 shows a nucleotide sequence (SEQ ID N0:579) of a native sequence PR016090 cDNA, wherein SEQ ID N0:579 is a clone designated herein as "DNA144844-2843".
Figure 580 shows the amino acid sequence (SEQ ID N0:580) derived from the coding sequence of SEQ
ID N0:579 shown in Figure 579.

WO 01/68848 rc:~rmwnvb,iv Figure 581 shows a nucleotide sequence (SEQ ID N0:581) of a native sequence PR019576 cDNA, wherein SEQ ID N0:581 is a clone designated herein as "DNA144857-2845".
Figure 582 shows the amino acid sequence (SEQ ID N0:582) derived from the coding sequence of SEQ
1D NO:S81 shown in Figure 581.
Figure S83 shows a nucleotide sequence (SEQ ID N0:583) of a native sequence PR019646 cDNA, wherein SEQ U~ N0:583 is a clone designated herein as "DNA145841-2868".
Figure 584 shows the amino acid sequence (SEQ ID N0:584) derived from the coding sequence of SEQ
ID N0:583 shown in Figure 583.
Figure 585 shows a nucleotide sequence (SEQ ID N0:585) of a native sequence PR019814 cDNA, wherein SEQ ID NO:S85 is a clone designated herein as "DNA148004-2882".
Figure 586 shows the amino acid sequence (SEQ ID N0:586) derived from the coding sequence of SEQ
ID N0:585 shown in Figure 585.
Figure 587 shows a nucleotide sequence (SEQ ID N0:587) of a native sequence PR019669 cDNA, wherein SEQ ID N0:587 is a clone designated herein as "DNA149893-2873".
Figure 588 shows the amino acid sequence (SEQ ID N0:588) derived from the coding sequence of SEQ
ID N0:587 shown in Figure 587.
Figure 589 shows a nucleotide sequence (SEQ ID N0:589) of a native sequence PR019818 cDNA, wherein SEQ ID N0:589 is a clone designated herein as "DNA149930-2884".
Figure 590 shows the amino acid sequence (SEQ ID N0:590) derived from the coding sequence of SEQ
ID N0:589 shown in Figure 589.
Figure 591 shows a nucleotide sequence (SEQ iD N0:591) of a native sequence PR020088 cDNA, wherein SEQ ID N0:591 is a clone designated herein as "DNA150157-2898".
Figure 592 shows the amino acid sequence (SEQ ID N0:592) derived from the coding sequence of SEQ
ID N0:591 shown in Figure 591.
Figure 593 shows a nucleotide sequence (SEQ ID N0:593) of a native sequence PR016089 cDNA, wherein SEQ ID N0:593 is a clone designated herein as "DNA150163-2842".
Figure 594 shows the amino acid sequence (SEQ ID N0:594) derived from the coding sequence of SEQ
ID N0:593 shown in Figure 593.
Figure 595 shows a nucleotide sequence (SEQ ID N0:59S) of a native sequence PR020025 cDNA, wherein SEQ ID N0:595 is a clone designated herein as "DNA153579-2894".
Figure 596 shows the amino acid sequence (SEQ ID N0:596) derived from the coding sequence of SEQ
ID N0:595 shown in Figure 595.
Figure 597 shows a nucleotide sequence (SEQ ID N0:597) of a native sequence PR020040 cDNA, wherein SEQ ID N0:597 is a clone designated herein as "DNA164625-2890".
Figure 598 shows the amino acid sequence (SEQ ID N0:598) derived from the coding sequence of SEQ
ID N0:597 shown in Figure 597.
Figure 599 shows a nucleotide sequence (SEQ ID N0:599) of a native sequence PR0791 cDNA, wherein SEQ ID N0:599 is a clone designated herein as "DNA57838-1337".

Figure 600 shows the amino acid sequence (SEQ ID N0:600) derived from the coding sequence of SEQ
ID N0:599 shown in Figure 599.
Figure 601 shows a nucleotide sequence (SEQ ID N0:601) of a native sequence PR01131 cDNA, wherein SEQ ID N0:601 is a clone designated herein as "DNA59777-1480".
Figure 602 shows the amino acid sequence (SEQ ID N0:602) derived from the coding sequence of SEQ
ID N0:601 shown in Figure 601.
Figure 603 shows a nucleotide sequence (SEQ ID N0:603) of a native sequence PR01343 cDNA, wherein SEQ ID N0:603 is a clone designated herein as "DNA66675-1587".
Figure 604 shows the amino acid sequence (SEQ )D N0:604) derived from the coding sequence of SEQ
ID N0:603 shown in Figure 603.
Figure 605 shows a nucleotide sequence (SEQ ID N0:605) of a native sequence PR01760 cDNA, wherein SEQ ID N0:605 is a clone designated herein as "DNA76532-1702".
Figure 606 shows the amino acid sequence (SEQ ID N0:6(~ derived from the coding sequence of SEQ
ID N0:605 shown in Figure 605.
Figure 607 shows a nucleotide sequence (SEQ ID N0:607) of a native sequence PR06029 eDNA, wherein SEQ ID N0:607 is a clone designated herein as "DNA105849-2704".
Figure 608 shows the amino acid sequence (SEQ ID N0:608) derived from the coding sequence of SEQ
ID N0:607 shown in Figure 607.
Figure 609 shows a nucleotide sequence (SEQ ID N0:609) of a native sequence PR01801 cDNA, wherein SEQ ID N0:609 is a clone designated herein as "DNA83500-2506".
Figure 610 shows the amino acid sequence (SEQ DJ N0:610) derived from the coding sequence of SEQ
ID N0:609 shown in Figure 609.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed 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 terms "PRO/number polypeptide"
and "PRO/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term "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 individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term "PRO
polypeptide" also includes variants of the PRO/number polypeptides disclosed herein.
A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Sucli native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide"
specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g. , an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring 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 amino acids sequences shown in the accompanying figures. Start and stop codons are shown in bold font and underlined in the figures. However, while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 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 transmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5 % of such domains. It will 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 either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular 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 and/or the accompanying figures. It is noted, however, that the C-terminal 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. En~. 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 more than about 5 amino acids on either side of the C-terminal boundary of the signal pepride as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polygeptide as defined above or below having at least about 80% amino acid sequence identity with 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, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Such PRO polypeptide variants include, for instance, PRO

polypeptides wherein one or more amino 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 idemity, alternatively at least about 82% amino acid sequence identity, alternatively 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, S 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 9196 amino acid sequence identity, alternatively at least about 9296 amino acid sequence identity, alternatively at least about 93 96 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 abort 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 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, with or without the signal peptide, as disclosed herein or any other speciFcally 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 acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length, alternatively at least about 90 amino acids in 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 more.
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues is a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining 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 such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.OD. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino 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 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
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 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 of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO", wherein "PRO" represents the amino acid sequence of a hypothetical PRO polypeptide of interest, "Comparison Protein"
represents the amino acid sequence of a polypeptide against which the "PRO" polypeptide of interest is being compared, and "X, "Y" and "Z" each represent different hypothetical amino acid residues.
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, q6 amino acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzvmolo;~y 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST 2 is employed, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the PRO
polypeptide of interest having a sequence derived from the native PRO
polypeptide and the comparison amino acid sequence of interest (i.e., the sequence against which the PRO polypeptide of interest is being compared which 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 has or having at least 80% amino acid sequence identity to the amino acid sequence B", the amino acid sequence A is the comparison amino acid sequence of interest and the amina acid sequence B is the amino acid sequence of the PRO polypeptide of interest.
Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

National Institute of Health, 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 complexity length = 15/5, multi-pass e-value =
0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino 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 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
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 96 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 lacking 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 fragment 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 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 % 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, alternatively at least about 96 % nucleic acid sequence identity, alternatively at least about 97 To nucleic acid sequence identity, alternatively at least about 98 Yb 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 polypepdde sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.

Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length;
alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternatively at least about 240 nucleotides in length, alternatively at least about 270 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more.
"Percent (%) nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Ine. and the source code shown in Table 1 below has been tiled with user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1 below.
The ALIGN-2 program should be compiled for use on a UNTX operating system, preferably digital UNIX V4.OD.
All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment 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 sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. However, % nucleic acid sequence identity values may also be obtained as described below by using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO
polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO
polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (i.e., the sequence against which the PRO polypeptide-encoding nucleic acid molecule 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-encoding nucleic acid molecule of interest. For example, in the statement °an isolated nucleic acid molecule 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 nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO
polypeptide-encoding nucleic acid molecule of interest.
Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
25 In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment 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 sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the 9'o nucleic acid sequence identity of D to C.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active PRO
polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length PRO polypeptide as disclosed herein. PRO variant polypeptides may be those that are encoded by a PRO variant polynucleatide.
"Isolated, " when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 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 using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one 10 component of the PRO polypeptide natural environment will not be pzesent.
Ordinarily, however, 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 ordinarily associated in the natural source of the polypepdde-encoding nucleic acid. An isolated 15 polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature.
Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic 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 operator sequence, and a ribosome binding site. Eukaryotic cells are lmown to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional relationship with 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 preprotein that participates in the secretion of the polypeptide; a promoter or enhancer 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 linked"
means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligalion at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-PRO
monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments of anti-PRO antibodies (see below).
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially WO O1/188~8 PCT/USOI/0(s20 homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used.
As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biolo~y, Wiley Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that:
(1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 %
polyvinylpyrrolidone/SOmM sodium phosphate 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 (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 /cg/ml), 0.1 % SDS, and 10 % dextran sulfate at 42 °C, with washes at 42 °C in 0.2 x SSC (sodium chloride/sodium citrate) and SO % formamide 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 Sambrook et al., Molecular Cloning:
A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., 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%
formamide, 5 x SSC (I50 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.~, 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO
polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues {preferably, between about 10 and 20 amino acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding *-trademark 46 specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, 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 ~rafers to forms) 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 similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a PRO polypeptide may comprise contacting a PRO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide.
"Treatrnent" refers to both therapeutic treatment and prophylactic 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 administration of the agents) in a continuous mode as opposed to an acute mode, so as to maintain the initial 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, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
"Carriers" as used herein 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 carrier is an aqueous pH buffered solution.
Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid;
Iow molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poIyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENT''~, polyethylene glycol (PEG), and PLURONICST'"
"Antibody fragments" comprise 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 antibodies (Zapata et al., Protein Ena. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed 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 designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site.
This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL 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) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine residues) of the constant domains bear a free thiol group. F(ab'~ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also lmown.
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 amino 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, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and V~ domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacolog~of Monoclonal Antibodies, vol. II3, 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-chain variable domain (VH) connected to a light-chain variable domain (V,,) in the same polypeptide chain (VH V~. By using a linker 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 chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP
404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will 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 amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing 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 "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound or composition which 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 fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
By "solid phase" 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 the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.
A "liposome" is a small 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 liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.
An "effective amount" of a polypeptide disclosed herein or an agonist or antagonist thereof is an amount sufficient to carry out a specifically stated purpose. An "effective amount"
may be determined empirically and in a routine manner, in relation to the stated purpose.

Table 1 /*
* C-C
increased from to * Z
is average of EQ

* B
is average of ND

* match with stop is M;
stop-stop =
0;
J
(joker) match =

*/

#defineM -8 /* value of a match with a stop */

int day[26][26] _ {

/* _ 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
*/

7* { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, A 0,-2, 1, 1, 0, 0,-6, 0,-3, 0}, *l I* { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2 _M,-1, B 1, 0, 0, 0, 0,-2,-5, 0,-3, 1}, *1 /* {-2,-4,15,-5, 5,-4,-3,-3, 2, 0,-5,-6,-5,~, M,-3,-5, C 4, 0,-2, 0,-2,-8, 0, 0,-5}, */

/* { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1, D 2,-1, 0, 0, 0,-2,-7, 0,-4, 2}, *I

I* { 0, 2,-5, 3, 4; 5, 0, 1,-2, 0, 0,-3,-2, 1 =M,-1, E 2,-1, 0, 0, 0,-2, 7, 0,-4, 3}, */

/* {-4,-5,-4,-6,-5, 9; 5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, F 0,-1, 0, 0, 7,-5}, *!

/* { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M; 1,-1,-3, G 1, 0, 0,-1,-7, 0,-5, 0}, */

I* {-1, 1,-3, 1, 1, 2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, H 3, 2,-1,-1, 0,-2,-3, 0, 0, 2}, *l /* {-1, 2,-2,-2,-2, 1,-3,-2, 5, 0, 2, 2, 2,-2 -M,-2,-2, I 2,-1, 0, 0, 4,-5, 0,-1; 2}, */

/* { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, O, M, 0, J 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, */

/* {-1, 0,-5, 0, 0,-5, 2, 0,-2, 0, 5,-3, 0, 1 =M,-1, K 1, 3, 0, 0, 0, 2,-3, 0,-4, 0}, */

/* {-2,-3; 6, 4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3 =M,-3,-2,-3,-3,-1, L 0, 2,-2, 0,-1, 2}, *l /* {-1,-2,-5,3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, M 0,-2,-1, 0, 2,-4, 0,-2,-1}, */

/* { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1, N 1, 0, 1, 0, 0,-2,-4, 0,-2, 1}, */

/* M =M =M, M =M, M =M _M, 0 =M, M, M,_M =M =M,_M,_M,_M, O M -M}, */ ~M =M, M =M =M =M, /* _ P { 1,-1,-3,-1; 1,-5,-1, 0,-2, 0,-1,-3,-2,-1 -M, 6, *! 0, 0, 1, 0, 0,-1,-6, 0,-5, 0}, !* { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1; 2,-1, 1 =M, 0, Q 4, 1,-1,-1, 0,-2,-5, 0,-4, 3}, *1 /* {-2, 0,-4,-1; 1,-4,-3, 2,-2, 0, 3,-3, 0, 0, M, 0, R 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, *l /* { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M, 1,-1, S 0, 2, 1, 0,-1,-2, 0,-3, 0}, */

/* { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, O, M, 0,-1,-1, T 1, 3, 0, 0,-5, 0; 3, 0}, */

/* { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 =M, 0, U 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, *1 /* { 0,-2,-2, 2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2, M,-1,-2,-2,-1, V 0, 0, 4,-6, 0,-2,-2}, */

/* {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4 =M,-6,-5, W 2,-2,-5, 0,-6,17, 0, 0,-6}, */

/* { o, o, o, o, o, o, o, o, o, o, o, o, o, o =M, o, x o, o, o, o, o, o, o, o, o, o}, *l /* {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4;
Y 3,-3, 0, 2, 0, 0,10,-4}, *I

/* { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,-M, 0, Z 3, 0, 0, 0, 0,-2,-6, 0,-4, 4}
*/

};

Table 1 cont'Z
/*

*/

#include<stdio.h>

#include<
ctype.
h >

#defmeMAX1MP /* max jumps in a diag *!

#defmeMA?CGAP /* don't continue to penalize 24 gaps larger than this */

#defmeJMPS 1024 I * max jmps in an path *I

#defineMX 4 /* save if there's at least MX-1 bases since last jmp */

#defineDMAT 3 /* value of matching bases */

#defmeDMIS 0 /* penalty for mismatched bases */

#defmeDINSO8 /* penalty for a gap */

#defmeDINSI1 /* penalty per base */

1S #defmePINSO8 /* penalty for a gap */

#defmePINSl4 /* penalty per residue */

struct jmp {

shortn[MAXJMP];
/* size of jmp (neg for defy) */

unsigned short x[MA3CJMP];
/*
base no.
of jmp in seq x *!

}; /* limits seq to 2" 16 -1 */

strnct diag {

int score; /* score at last jmp */

2S long offset; l* offset of pxev block */

shortijmp; l* current jmp index */

struct /* list of jmps */
jmp jp;

struct path {

int spc; l* number of leading spaces *!

shortn[JMPS];/*of jmp (gap) *!
size int x[JMPS];
/* loc of jmp (last elem before gap) */

char *ofile; !* output file name *!

char *namex[2J;/* seq names: getseqs0 */

char *prog; /* prog name for err msgs */

char *seqx[2];/* seqs: getseqsQ */

int dmax; /* best diag: nw0 */

int dmax0; /* final diag */

int dna; ~ /* set if dna: main() */

int endgaps;/* set if penalizing end gaps */

int gapx, /* total gaps in seqs */
gapy;

int len0, /* seq lens */
lenl;

int ngapx, /* total size of gaps */
ngapy;

int smax; /* max score: nwp */

int *xbm; /* bitmap for matching */

long offset; /* current offset in jmp file */

SO structdiag *dx; /* holds diagonals */

structpath pp[2]; /* holds path for seqs */

char *callocQ, *mallocQ, *index0, *strcpyp;

char *getseqQ, *g calloc0;

WO 01/68848 PCTlUS01/06520 Table 1 (cont'1 /* Needleman-Wunsch alignment program * usage: progs filel filet * where filel and filet are two dna or two protein sequences.
S * 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 unsigned short x in the jmp struct) * A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a mtp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 *!
#include "nw.h"
1S #include "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 j;
static -pbval[26] _ {
1, 2~(1< <('D'-'A'))~(1< <('N'-'A')), 4, 8, 16, 32, 64, 128, 256, OxFFFFFFF, 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'))I(1«('Q'-'A')) };
main(ac, aV) lrialn int ac;
char *avp;
{
grog = av[O];
~(~!=3){
fprintf(stderr,"usage: %s filet filet\n", prog);
fprintf(stderr, "where filet and filet are two dna or two protein sequences.\n");
fprintf(stderr, "The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignored\n");
fprintf(stderr,"Output is in the file 1"align.out\"\n");
exit(1);
namex[0] = av[1];
namex[1] = av[2];
seqx[0] = getseq(namex[0], &len0);
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : ~bval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; - /* output file */
S0 nwQ; /* fill in the matrix, get the possible jmps */
readjmps0; /* get the actual jmps */
print(); !* print state, alignment */
cleanup(0); /* unlink any tmp files */

Table l~cont') /* do the alignment, return best score: maim * dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983 * pro: PAM 250 values * When scores are equal, we prefer mismatches to any gap, prefer * a new gap to extending an ongoing gap, and prefer a gap in seqx * to a gap in seq y.
*!
nw() nW
{
char *px, *py; /* seqs and ptrs *I
int *ndely, *dely; /* keep track of dely */
int ndelx, deli; /* keep track of deli */
int *tmp; l* for swapping row0, cowl *!
int mis; /* score for each type */
int ins0, insl; I* insertion penalties *I
register id; /* diagonal index */
register ij; /* jmp index */
register *col0, *coll; /* score for curr, last row */
register xx, yy; /* index into seqs */
dx = (struct diag *)g_calloc("to get diags", len0+lenl+ 1, sizeof(struct diag));
ndely = (int *)g calloc("to get ndely", lenl+1, sizeof(int));
dely = (int *)g calloc("to get dely", lenl+1, sizeof(int));
col0 = (int *)g caltoc("to get col0", lenl+1, sizeof(int));
coll = (int *)g calloc("to get coil", lenl+1, sizeof(int));
ins0 -_ (dna)? DINSO : PINSO;
insl = (dna)? DINSl : PINSl;
smax = -10000;
if (endgaps) {
for (col0[0] = defy[0] _ -ins0, yy = 1; yy < = lenl; yy-i-+) {
col0[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yy] = yy;
col0[0] = 0; /* Waterman Bull Math Biol 84 */

else for (yy = 1; yy < = lent; yy++) dely[yy] _ -~;
/* fill in match matrix */
for (px = seqx[0], xx = 1; xx < = len0; px++, xx++) {
/* initialize first entry in col */
if (endgaps) {
~(~ _= 1) toll[0] = deli = -(ins0+insl);
else col l [0] = deli = col0[0] - ins 1;
ndelx = xx;
else {
coil[o] = o;
delx = -ins0;
ndelx = 0;

Table 1 (cony) for (py = seqx[l], yy = 1; yy <= lenl; py++, yy++) {

mis = col0[yy-1];

if (dna) S mis += (xbm[*px-'A']&xbm[*py-'A'])? DMAT :
DMIS;

else mis += day[*px-'A'][*py-'A'];

/* update penalty for del in x seq;

* favor ~w del over ongong del * ignore MAXGAP if weighting endgaps */

if (endgaps ~ ~ ndely[yy] < MAXGAP) {

if (col0[yy] - ins0 > = defy[yy]) {

defy[yy] = col0[yy] - (ins0+insl);

ndely[yy] = 1;

} ~e {

defy[yy] -= insl;

ndely[yy]++;

}

} else {

if (col0[yy] - (ins0+insl) >'= dely[yy]) {

dely[yy] = colOjyy] - (ins0+insl);

ndely[yy] = 1;

} else ndely[yy]++;

}

/* update penalty for del in y seq;

* favor new del over ongong del */

if (endgaps ~ ~ ndelx < MAXGAP) {

if (coll(yy-1] - ins0 > = deli) {

deli = coll(yy-1] - (ins0+insl);

ndelx = 1;

} else {

deli -= insl;

ndelx++;

}

} else {

if (coll[yy-1] - (ins0+insl) > = deli) {

delx = coll[yy-1] - (ins0+insl);

ndelx = 1;

} else ndelx++;

}

/* pick the maximum score; we're favoring * mis over any del and deli over defy */

...nw Table 1 (cony) ...nw id=xx-yy+Ienl-1;
if (mis > = delx && mis > = dely[yy]) coll[yy] = mis;
else ff (delx > = dely[yy]) {
coll[yy] = deli;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (ldna ~ ~ (ndelx > = MA3~TMP
&& xx > dx[id].jp.x[ij]+M30 ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXrMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx(id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);

dx[id].jp.n[ij] = ndelz;
dx[id].jp.x[ij] = xx; , dx[id].score = delx;
else {
coll(py] = dely[yy];
ij = dx[id].ijmp;
~5 if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MJQ ~ ~ mis > dx[id]acore+DINSO)) {
dx[id].ijmp++;
if (++ij > = MA?fJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);

dx[id].jp.n[ij] _ -ndely[yy];
dx[id].jp.x[ij] = zx;
dx[id].score = dely[yy];
if (xx == lei && yy < lent) {
. /* last col */
if (endgaps) coll[yy] -= ins0+insl*(lenl-yy);
if (coll[yy] > smax) {
smax = coll[yy];
dmaz = id;

if (endgaps && xx < len0) toll[yy-1] -= ins0+insl*(len0-xx);
if (coll[yy-1] > smax) {
smax = coil[yy-1];
dmax = id;
tmp = col0; col0 = coil; cull = tmp;

(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)coll); }

Table 1 (cony) /*
*
* print() -- only routine visible outside this module * static;
* getmatQ -- trace back best path, count matches: printQ
* pr alignQ - print alignment of described in array p[]; print0 * dumpblockQ -- dump a block of lines with numbers, stars: pr align() * nums() - put out a number line: dumpblockQ
* putline0 -- put out a line (name, [num], seq, [num]): dumpblock0 * stars0 - -put a line of stars: dumpblockQ
* stripnameQ - strip any path and prefix from a seqname *1 ZS #include "nw.h"
#define SPC 3 #detlne P LINE 256 I* maximum output line *I
#defme P SPC 3 /* space between name or num and seq */
-extern _day[26][26j;
int olen; /* set output line length */
FILE *fx; /* output file */
print print() ' {
int lx, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup(I);
fprintf(fx, "<first sequence: %s (length = 96d)1n", namex[Oj, len0);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[Ij, lenl);
olen = 60;
Ix = len0;
ly = lent;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x *I
pp[0].spc = firstgap = lenl - dmax - 1;
ly _= pP[0].spc;
else if (dmax > lenl - 1) { /* leading gap in y */
pp[1].spc = firstgap = dmax - (lenl - 1);
Ix -= pp[I].spc;
if (dmax0 < len0 - I) { /* trailing gap in x *!
lastgap = len0 - dmax0 -1;
lx -= lastgap;
SO ) else if (dmax0 > IenO - 1) { /* trailing gap in y */
lastgap = dmax0 - (len0 - I);
ly -= lastgap;
getmat(Ix, ly, firstgap, lasigap);
pr align();

Table 1 (cony) /*
* trace back the best path, count matches */
static S getmat(lx, ly, firstgap, lastgap) getlriat int lx, ly; /* "core" (minus endgaps) *!
int firstgap, lastgap; /* leading trailing overlap */
{
int nm, i0, il> siz0, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
IS /* get total matches, score */
i0 = il = siz0 = sizl = 0;
p0 = seqx[0] + pp[I].spc;
pl = seqx[1] + pp[0].spc;
n0 = pp[1].spc + 1;
nl = pp[0].spc + 1;
nm = 0;
while ( *p0 && *pl ) {
if (siz0) {
pl++;
nl + +;
siz0-;
else if (sizl) {
p0++;
n0++;
sizl--;
else {
if (xbm(*p0-'A']&xbm(*pl-'A']) nm++;
if (n0++ == pp[0].x[i0]) siz0 = pp[0].n[i0++];
if (nl++ _= pp[1].x[il]) sizl = pp[1].n[il++];
p0++;
pl++;
1* pct homology:
* if penalizing endgaps, base is the shorter seq * else, (mock off overhangs and take shorter core */
if (endgaps) lx = pen0 < lenl)? len0 : lenl;
else lx = (lx < ly)? lx : ly;
pct = 100. *(double)nm/(double)lx;
' fprintf(fx, non");
fprintf(fx, " < % d match %s in an overlap of % d: % .2f percent similarityln", ~ =0 1)? °" : n~"~. 17C, pCt);

Table 1 (cony) fprintf(fx, " < gaps in first sequence: 'Y d", gapx); ... getfnAt if (gapx) {
(void) sprintf(outx, " (%d %s%s)", ngapx, (dna)? "base":"residue", (ngapx == I)? "~:"s");
fprintt(fx,"Yos", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
(gaPY) {
(void) sprintf(outx, " (%d ~s%s)", ngapy, (dna)? "base":"residue", (ngapy == I)7 "":"s");
fprintf(fx," ~s", outx);
if (dna) fprintf(fx, "\n<score: ~d (match = 96d, mismatch = ~d, gap penalty = ~d + 9bd per base)\n", smax, DMAT, DMIS, DINSO, DINSI);
else fprintf(fx, "\n<score: ?6d (Dayhoff PAM 250 matrix, gap penalty = ~d + hod per residue)\n", smax, PINSO, PINSl);
if (endgaps) fPT~~.
"<endgaps penalized. left endgap: %d %s~s, right endgap: ~d 96s96s\n", firstgap, (dna)? "base" : "residue", (ftrstgap == I)? "" : "s", lastgap, (dna)7 "base" : "residue", (lastgap == 1)? "" : "s");
else fprintf(&, "<endgapsnotpenalizedln");
static nm; /* matches in core -- for checlang */

static Imax; /* lengths of stripped file names */

static ij[2]; /* jmp index for a path */

static nc[2]; . /* number at start of current line *!

static ni[2]; /* current elem number -- for gapping */

static siz[2];

static *ps[2]; /* ptr to current element */
char static *po[2]; /* ptr to next output char slot */
char static out[2][P_LINE]; l* output line *!
char static star[P_LINE]; /* set by stars() */
char /*

* print of described in struct path pp[]
alignment *l static pr align()13P ahgtl int nn; /* char count */

i~ more;

register i;

for (i 0, lmax = 0; i < 2; i++) {
=

nn = stripname(namex[i]);

if (nn > lmax) lmax = nn;

nc[iJ = I;
ni[i] = l; .
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[iJ = out[i]; }

Table 1 (cony) for (nn = nm = 0, more = 1; more; ) { ...pr align for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence?
*/
if (!*ps[i]) continue;
more++;
if (pp[i].spc) { /* leading space */
*po[i]++ _ ' ';
PPfi].sPc--;
1$ }
else if (siz[i]) { /* in a gap */
*po[i]++ _ , siz[i]--;
else { l* we're putting a seq element */
*po[i] _ *ps[i];
if (islower(*ps(i])) *ps[i] = toupper(*ps[i]);
po[i]++;
ps(i]++;
/*
* are we at next gap for this seq?
*/
(~[7 ° ° PP[i].x[ij[i]]) {
/*
* we need to merge all gaps * at this location */
siz[i] = pp[i].n[ij[i]++];
while (ni[i] _= pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++];
ni[i]++;
}
if (++nn == olen ~ ~ !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++) po[i] = out[i];
nn = 0;
}
}
SO }
/*
* dump a block of lines, including numbers, stars: pr align */ .
static dumpblockQ dumpblock {
register i;
f0 for (i = 0; i < 2; i++) . *po[i]- _ '\0';

Table 1 (cony) ... dumpb]ock (void) putc('\n', fx);

for (i = 0; i < 2; i++) {

If .(*out[i] &&. (*out[i] !_ ' ' I I *(po[il) ! _ ' ')) {

if (i == 0) nums(i);

if (i == 0 && *out[1]) stars0;

putiine(i);

if (i == 0 && *out[1P

fprintf(fx, star);

if (i == 1) nums(i); _ }

}

}

/*

* put out a number line:
dumpbloclcQ

*/

static nums(ix)IlilrilS

int ix; /* index in out[] holding seq line */

{

char nline[P_LINE];

register i, j;

register char *pn, *px, *py;

for (pn = mine, i = 0; i < lmax+P SPC; i++, pn++) *pn = , ~;

for (i = ncjix], py = out[ix]; *py; py++, pn++) {

~ (*~Y = _ ' ' I I *~Y = _ '-') *pn = ~ ~, else {

if (i % 10 == 0 I I (i == 1 && nc[ix] ! = 1)) {

j = (i < 0)? -i : i;

for (px = pn; j; j /= 10, px--) *px = j9~10 + '0';

if (i < o) *px = , , ;

}

else *pn _-. ~ ~:

i++;

}

}

*pn = ~\0~.
~

nc[ix] = i;

for (pn = mine; *pn; pn++) (void) putc(*pn, fx);

(void) putc('1n', fx);

/*

* put out a line (name, [num], seq, [num]):
dumpblockp */

static putline(ix) puthlle int ix; {

Table 1 (coast') ...putline int i;

register char *px;

for (px = namex[ix], i = 0; *px && *px ! _ ':'; px++, i++) (void) putc(*px, fx);

for (; i < lmax+P SPC; i++) (void) putc(' ', fx);

/* these count from i: ' * nib is current element (from 1) * nc[] is number at start of current line */ .

for (px = out[ix]; *px; px++) (void) putc(*px&Ox7F, fx);

(void) putc('\n', fx);

}

I*

* line of stars (seqs always in out[O], out[1]): dumpblock0 put a */

static stars() stars {

int i;

register char *p0, *pl, cx, *px;

if (!*out(0] I I (*out[0] _ _ ' ' &8c *(po[o]) _ _ ' ') I I

!*OUt(1] I I (*OUt[1] __ ' ' BLBC *(p0[1]) _- ' ')) return;

px = star;

for (i = lmax+P SPC; i; i--) *px++ _ ' ', for (p0 = out(0], pl = out[1]; *p0 && *pl; p0++, pl++) {

if (isalpha(*p0) && isalpha(*pl)) {

if (xbm[*p0-'A']&xbm[*pl-'A']) {

cx = '*';

nm++;

}

else if (!dna && day[*p0-'A'][*pl-'A'] > 0) cx = '.~;

else cx = ";

}

else cx = , *px++ = cx;

}

*px++ _ '\n';

*px = '\0';

}

Table 1 (cony) /*
* strip path or prefix from pn, return len: pr alignQ
*%
static S stripname(pn) stripname char *pn; I * file name (may be path) *I
register char *px, *py;
py = 0;
for (px = pn; *px; px++) if (*px -- '/') py=px+l;
(PY) 1$ (void) strcpy(pn, py);
return(strlen(pn));

Table 1 (cony) /*
* cleanupQ - cleanup any tmp file * getseqQ - read in seq, set dna, len, maxlen * g callocp -- callocp with error checldn $ * readjmpsQ -- get the good jmps; from tmp file if necessary * writejmpsQ - write a filled array of jmps to a tmp file: nwQ
*/
/finclude "nw.h"
#include < syslfile.h >
char *jname = "/tmp/homg3~7LX3fX"; . . . /* ~p ~e for jmps */
FILE *fj;
int cleanup(); l* cleanup tmp file *I
long lseelcQ;
/*
* remove any tmp file if we blow */
cleanup(i) cleanup int i;
{
if (fj) (void) unlinl.(jname);
ezit(i);

/*
* read, return ptr to seq, set dna, len, maiden * skip lines starting with ';', ' <', or ' >' * seq in upper or lower case */
char getseq(file, len) getSe(1 3S char *file; /* file name */
int *len; /* seq len */
f char , line[1024], *pseq;
register char *px, *py;
40 int natgc, lien;
FILE *fp;
if ((fp = fopen(fde, "r")) _ = 0) {
fprintf(stderr,"%"s: can't read %s~n", prog, file);
45 exit(1);

lien = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =_ '~' ~ ~ *line == ' <' ~ ~ *line =_ ' >') S0 continue;
for (px = line; *px ! _ '\n'; px++) if (isupper(*px) ~ ~ islower(*px)) lien++;

55 if ((pseq = malloc((unsigned)(tlen+G))) _= 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for ~s\n", prog, lien+6, file);
exit(1);
60 Pseq[0] = pseq[1] = pseq(2] = pseq[3] _ '\0';

Table 1 (cony) ...getseq py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 102,4, fp)) {
if (*line =- ' ~ ~ *line =- ' <' ~ ~ *line =- ' >') continue;
for (px = line; *px ! _ '\n'; px++) {
if (isupper(*px)) *py++ = *px;
else if (islower(*px)) *py++ = toupper(*px);
if (index("ATGCU",*(py-1))) natgc+ +;
*py++ _ '\o';
*Py = '\0,;
(void) fclose(fp);
dna = natgc > (tlen/3); ' return(pseq+4);
char g~calloc(msg, nx, sz) g-CallOC
char *msg; /* program, calling routine */
int nx, sz; /* number and size of elements */
{
char *px, *calloc0; ' if ((px = calloc((unsigned)nx, (unsigned)sz)) _ = 0) {
if (*msg) {
fprintf(stderr, "%s: g-callocQ failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(1);
return(px);
j /*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: mainQ
*l readjmps() readjmps {
int fd = -1;
int siz, i0, il;
register i, j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname, O ItDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't openQ %s\n", prog, jname);
cleanup(1);
}
for (i = i0 = il = 0, dmax0 = dmax, xx = len0; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j-) , Table 1 (cony) ...readjmps if (j < 0 && dx[dmax].offset && fj) {
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp))~
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
}
else break;
}
if (i > = JMPS) { .
fprintf(stderr, "%s: too many gaps in alignmentln", prog);
cleanup(1);
1 s if (j > = o) {
siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x[jl;
dmax += siz;
if (siz < 0) { /* gap in second seq */
pp[1].n[il] _ -siz;
xx += siz;
/*id=xx-yy+lenl-1 */
Pa[1]++11] = xx - dmax + lenl - 1;
g PY
~aPY -= s~>
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~ ) endgaps)? -siz : MAXGAP;
il+'+;
}
else if (siz > 0) { /* gap in first seq */
pp[0].n[i0] = siz;
PP[0] ~ x[i0] = xx;
gapx++;
ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
}
}
else break; , }
/* reverse the order of jmps */
for (j = 0, i0--; j < i0; j++, i0--) {
i = PP[OI.nU]; PP[Ol.nLll = PP[0].n[i0]; pP[0].n[i0] = i;
' - PP[0].xClI;.PP[0].xLl] = PP[0].x[i0]; pPCOI.x[i0] = i;
}
for (j = 0, il--; j < il; j++, il--) {
i = pp[1].n[j]; pp[1].n[j] = pp(1].n[il]; pp[1].n[il] = i;
i = pp[1].x[j]; pp[1].x[j] = pp[1].x[il]; pp[1].x[il] = i;
~ (fd > = o) (void) close(fd);
~(f){
(void) unlink(jname);
fj = 0;
offset = 0;
} }

Table 1 (cont'1 /*
* write a filled jmp struct offset of the prev one (if any): nwQ
*!
S writejmps(ix) wTitejmps int ix;
char *mktempQ;
if (!fj) f if (mlrtemp(jname) < 0) {
fprintt(stderr, "96s: can't mlttemp0 ~s~n", prog, jname);
cleanup(1);
if ((fj = fopen(jname, "w")) _ = 0) f fprintf(stderr, "%s: can't write ~s~n", prog, jname);
exit(1);
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, C7);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);

Table 2 PRO XXXXXX?~XXXX (Length = 15 amino acids) Comparison Protein XX~~~'YYYYYYY (Length = 12 amino acids) % amino acid sequence identify =
(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 15 = 33.3 Table 3 PRO XXXXXXXXXX (Length = 10 amino acids) Comparison Protein XXXXX7~~YYYYZZYZ (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%
Table 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides) % nucleic acid sequence identity =
(the number of identically 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 NNNNLLLW (Length = 9 nucleotides) ~ nucleic acid sequence identity =
(the number of identically 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) _ 4 divided by 12 = 33.3 3&
II. Compositions and Methods of the Invention A. F~11-Length 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
polypeptides have been identified and isolated, as disclosed in farther detail in the Examples 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 all further 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 cDNA clones have bin deposited with the ATCC, The actual nucleotide sequences of those clones can readily be determined by the skilled 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 routine skill. For the PRO polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time.
B. PRO Polypepdde Variants In addition to the full-length native sequence PRO pokypeptides 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, and/or 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 glycosykation sites or altering the membrane anchoring characteristics.
. Variations in the native full-kength 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 instance, 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 more 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 S comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural 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 S 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 full-length or mature native sequence.
PRO polypepdde fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein.
Certain fragments lack amino acid residues that are not essential for a desired biologicai activity of the PRO
1S 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 an enzyme lmown to cleave proteins at sites defined by particular amino 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). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, PRO polypeptide fragments share at least one biological and/or immunological activity with the native PRO polypeptide disclosed herein.
In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading 2S of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.

WO O1/G88.~8 PCT/USO1/06520 Table 6 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (I~ gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;
phe;

norleucine leu Leu (L) norleucine; ile; val;

met; ala; phe ile Lys (I~ arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; leu tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser Ser Trp (R~ tyr; phe tyr Tyr ('S~ trp; phe; thr; ser phe Val (V) ile; leu; met; phe;

ala; norleucine leu Substantial modifications in function or immunological identity of the PRO
polypeptide are accomplished by selecting substitutions that differ significantly in their effect 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 (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: 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 influence 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-mediated (site directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.
Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (I985)], restriction selection mutagenesis [Wells et al., Philos.
Traps. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA
to produce the PRO variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is 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 main-chain conformation of the variant [G~mningham 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.H. Freeman & Co., N.Y.);
Chothia, 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 bifunctional agents is useful, for instance, for crosslinking PRO to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO antibodies, and vice-versa. Commonly used cmsslinlang agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1, 8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspariyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of Beryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, 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 the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern"
is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO
(either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO.
In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence PRO (for O-linked glycosylation sites). The PRO amino acid sequence may optionally be altered through changes at the DNA Level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons 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
87/05330 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 pokypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Haldmuddin, et al., Arch. Biochem. Bioohvs., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et ak., Meth. E ,nz~mol., 138:350 (1987).
Another type of covalent modification of PRO comprises linking 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, heterologous 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 purification 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 well known in the art. 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., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et ak., Molecular and Cellular Bioloey, 5:3610-3616 (1985)]; and the Herpes Simplex virus gkycoprotein D (gD) tag and its antibody [Paborsky et al., Protein En ing eJg, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al. , BioTechnolo~v, 6_:1204-1210 ( 1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an a-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane 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 immunoglobukin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995.
D. Preyaration 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 known in the art, may be employed to prepare PRO. For instance, 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 may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO.
1. Isolation of DNA Encoding 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 cDNA library prepared from human tissue, such as described in the Examples. The PRO-encoding gene may also be obtained from a genomic library or by known 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 Laboratory 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., ' su ra; Dieffenbach et al., PCR Primer: A Laboratory.Manual (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 if 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'ZP-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
Sequences identiEed in such library screening methods can be compared and aligned to other lmown 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 molecule 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 cDNA 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., supra, to detect precursors and WO O1ZG8848 PCTlUSOIl06520 processing intermediates of mRNA that may not have been reverse-izanscribed into eDNA.
2. Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for PRO
production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, oz amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. Tn general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical A,.poroach, M. Butler, ed. (IRL
Press, 1991) and Sambrook et al., su ra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaPO~, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., ssunra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens 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 )rb, Virolo , 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in 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, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymoloev, 185:527-537 (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 eukaryote 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. coIi K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC
31,537); E. coti strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include F.nterobacteriaceae such as Escheric)tia, e.g., E. coll, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhirnurium, Serratia, e.g., Serratia rnarcescans, and Shigella, as well as Bacillt such as B.
subtilis and B. licheniformis (e.g., B, Zlchenifonnis 41P disclosed in DD
266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. 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 fermentations. Preferably, the host cell secretes minimal 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 1A2, which has the complete genotype tonA ; E. coli W3110 strain 9FA~, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC

55,244), which has the complete genotype tonA ptr3 phoA E15 (argF lac)169 degP
ompT kan'; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA EI S (argF lac)169 degP ompT rbs7 ilvG kan';
E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No.
4,946,783 issued 7 August 1990.
Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors. Saccharonryces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981];
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No.
4,943,529; Fleer et al., BiolTechnolo~y, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technolosy, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]);
Candida; Trichoderma reesia (EP
244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwannionryces occidentalis (EP 394,538 published 31 October 1990);
and 6lamentous fungi such as, e.g., Neurospora, Penicilliutn, Tolypocladium (VJO 91/00357 published 10 January 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 (1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).~ Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methvlotrophs, 269 (1982).
Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms.
Examples of invertebrate cells include insect cells such as DrosQphila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC
CCL51). The selection of the appropriate host cell is deemed to be within the skill in the art.
3. Selection and Use of a Re licable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid,, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an WO 01/68848 PCT1i7S01/06520 appropriate restriction endonuclease sites) using techniques known in the art.
Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination 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 signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be 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, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C.
albicans glucoamylase leader (EP
362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypepddes 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 plasnud pBR322 is suitable for most Gram-negative bacteria, the 2tc 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 media, 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 the CHO cell line deficient 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 trill gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);
Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)].
The trill gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No.
44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning 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 well known. Promoters suitable for use with prokaryotic hosts include the (3-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 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sei. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding PRO.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate ldnase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enz~ne Re~~, 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 ldnase, triosephosphate isomerase, phosphoglucose isomerase, and glucoldnase.
Other yeast promoters, which are inducible promoters 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, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further 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 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PRO by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhaneer on the late side of the replication origin (bp 100-270), the cytomegalovirus 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' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO.
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantel et al., Nature, 281:40-46 (I979); EP 117,060; and EP 117,058.
4. Detecting Gene AmplificationlExpression wo oo~xx-tx PcT/uso~/o~;2u Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.
Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. .
Gene expression, alternatively, may be measured by immunological methods, such as inununohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO
DNA and encoding a specific antibody epitope.
5. Purification of Polypeptide Forms of PRO may be recovered from culture medium or from host cell lysates.
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 chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
It may be desired to purify PRO from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAF;
chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75;
protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the PRO. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in EnzymOlo~y, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification steps) selected will depend, for example, on the nature of the production process used and the particular PRO produced.
E. Uses for PRO
Nucleotide sequences (or their complement) encoding PRO have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. PRO nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein.
The full-length native sequence PRO gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length PRO cDNA or to isolate still other cDNAs (for instance, those encoding *-t:rademarit ~$

naturally-occurring variants of PRO or PRO from other species) which have a desired sequence identity to the native PRO sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases.
The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence PRO. By way of example, a screening method will comprise isolating the coding region of the PRO 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'~P or 355, 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 mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
Other useful fragments of the PRO nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target PRO mRIVA (sense) or PRO DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of PRO DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from 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 (Cancer Res. 48:2659, 1988) and van tier Krol et al. (BioTechniques 6:958, 1988).
Binding of antisense 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, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
The antisense oligonucleotides thus may be used to block expression of PRO
proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating 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 nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaP04-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Burr virus.
In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the marine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCTSA, DCTSB and DCTSC (see WO
90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytoldnes, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with 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 antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/ 10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
Antisense or sense RNA or DNA molecules are generally at least about 5 bases in length, about 10 bases in length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 bases in length, 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 is length, about 90 bases in length, about 95 bases in length, about 100 bases in length, or more.
The probes may 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 mapping the gene which encodes that PRO and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known 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 binding 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 fmd lead compounds that mimic the biological activity of a native PRO ox a receptor for PRO. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, 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 animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA 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 established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding PRO. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the 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 animals that include a copy of a transgene encoding PRO introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding PRO. Such animals 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 the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of PRO can be used to construct a PRO
"knock out" 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 example, cDNA encoding PRO can be used to clone genomic 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 which 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 Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA
has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)].
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J.
Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and 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 animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO
polypeptide.
Nucleic acid encoding the PRO polypeptides may also be used in gene therapy.
In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example fox replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which 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.
It has already been shown that short antisense oligonuckeotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane.
(Zamecnik et al., Proc. Natl. Acad. Sci. USA 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, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in BiotechnoloQy 11, 205-210 [1993]). In some 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 cell surface membrane protein or the target cell, a kigand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem.
262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and 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 sequences may be used for recombinantly expressing those markers.
The nucleic acid molecules encoding the PRO.polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking 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 molecules 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 fmd use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
The PRO polypeptides described herein may also be employed as therapeutic agents. The PRO
polypeptides of the present invention can be formulated according to lmown methods to prepare pharmaceutically useful compositions, whereby the PRO product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Reminaton's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrofidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, PLURONICS'~"'' or PEG.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The route of administration is in accord with lmown methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional mutes, topical administration, or by sustained release systems.
Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy, hzterspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W.
"The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al. , Eds., Pergamon Press, New York 1989, pp. 42-96.
When in vivo administration of a PRO polypeptide or agonist or antagonist thereof is employed, normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more .per day, preferably about 1 ~g/kg/day to 10 mg/kg/day, depending upon the 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. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
Where sustained-release administration 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, microencapsulation of the PRO polypeptide is contemplated.
Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-3S (rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio/Technolosy. 8:755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in Vaccine Desire Subunit WO 01/688:18 PCT/USO1/06520 and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO
97/03692, WO 96/40(?72, WO 96/07399; and U.S. Pat. No. 5,654,010.
The sustained-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 degradation products of PLGA; lactic and glycolic acids, can be cleared quickly within the human body. Moreover, the degradability of this polymer can be adjusted 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.), Biodegradable Polymers as Dru De~li'very Systems (Marcel Deld<er: 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 compounds that bind or complex 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 chenucal 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, immunoassays, and cell-based assays, which are well characterized in the art.
All assays for antagonists are common in 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 interact.
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the PRO polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the PRO polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal anh'body, speck 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-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
If the candidate compound interacts with but does not bind to a particular PRO
polypeptide encoded by a gene identified herein, its .interaction with that polypeptide can be assayed by methods well lmown for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature lLondon~, 340:245-246 (1989); Chien et al., Pros.
Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. 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 other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GALA, and another, in which candidate activating proteins are fused to the activation domain.
The expression of a GALL-lacZ reporter gene under control of a GAIA-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for p-galactosidase. A complete Idt (MATCHMAKERT"') for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
Compounds that interfere with the interaction of a gene encoding a PRO
polypeptide identified herein and other infra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the infra- or extracellular component under conditions and for a time 1S allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. 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 infra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reactions) but not in the reaction mixture 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, the PRO polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO
polypeptide. Alternatively, 2S antagonists may 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 labeled, 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 panning and FACS sorting. Coligan et al., C~rrrent Protocols in Immun., 1(2): Chapter 5 (1991).
Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the PRO polypeptide 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 the PRO polypeptide.
Transfected cells that are grown on glass slides are exposed to labeled PRO polypeptide. The PRO polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single, clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PRO
polypeptide can be photoaffmity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing.
The amino acid sequence obtained S from micro- sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA
library to identify the gene encoding the putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled PRO polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with PRO polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
Alternatively, a potential 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, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature PRO polypeptides herein, is used to design an antisense RNA oligoiiucleotide of from about 10 to 40 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., Nucl.
Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of the PRO polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the PRO polypeptide (antisense -Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC
Press: Boca Raton, PL, 1988).
The oligonucleotides described above can 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 translation-initiation site, e.g., between about -10 and + 10 positions of the target gene nucleotide sequence, are preferred.
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 blocking the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
ltibozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.

Ribozymes act by sequence-speck hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques.
For further details see, e.g., Rossi, Current Bioloay, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. 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 duplex. For further details see, e.g., PCT publication No. WO
97/33551, supra.
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well lmown for those 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 Antibodies The present invention further provides anti-PRO antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
1. Polyclonal Antibodies The anti-PRO antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include 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 mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The immunization protocol may be selected by one skilled in the art without undue experimentation.
2. Monoclonal Antibodies The anti-PRO antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the PRO polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs ") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (I986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells: For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are marine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in 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 radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [coding, su ra . Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention canbe readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of marine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous marine sequences [U.S.
Patent No. 4,816,567; Morrison et al., su ra or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well Imown in the art. For example, one method involves recombinant expression of immunoglobulin 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 crosslinldmg. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinldng.
IO In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques kaown in the art.
3. Human and Humanized Antibodies The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., marine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Pab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include human itnmunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human itnmunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (I986); Riechmann et al., Nature, 332:323-329 (1988); and Presto, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often refezred to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human anri'body. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No.
s9 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Maria et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al, and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes IO have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, 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/Technolo~y 10, 779-783 (1992); Lonberg et ad., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et 1S al., Nature Biotechnoloev 14, 845-51 (1996); Neuberger, Nature Biotechnolo~y 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
The antibodies may also be affinity matured using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or 20 human) from which the matured antibody is prepared.
4. Bispecific Antibodies Bispecii~ic antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding speci~cities is for the PRO, 25 the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are lrnown in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and G~ello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a 30 potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affuuty chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., fiMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can 35 be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzvmoloav, 121:210 (1986).
According to another approach described in VJO 96/27011, the interface between a pair of antibody molecules can be engineered to maacimize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies 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, bispecific antibodies can be prepared can be prepared using chemical linkage. Brennan et al. , Science 229: 81 ( 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Ex~. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')z molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers.
Kostelny et al. , J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444.-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V,~ by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary WO 01!68848 PCT/US01I06520 V~ and VH 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, trispeci~c antibodies can be prepared.
Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein.
Alternatively, an anti-PRO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc~yR), such as Fc~yRI (CD64), FcyRII (GD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide. These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the PRO polypeptide and further binds tissue factor (TF). .
5. Heteroconj_gate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.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 synthetic protein chemistry, including those involving crosslinldng agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
6. Effector Function Engineering It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residues) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated call lolling and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. , J. ExP Med. , 176:
1191-1195 (1992) and Shopes, J. Immunol., 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 thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design.
3: 219-230 (1989).
' 7. Imtnunoconju ag tes The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e. g. , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful 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 (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of zadionuclides are available for the production of radioconjugated antibodies. Examples include zl2Bi, '31I, '3~In, 9°Y, and lssRe.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,S-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
8. Immunoliposomes The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods laiown in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci.
USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.
4,485,045 and 4,544,S4S. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG
PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., 1. Biol. Chem. , 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al. ,1. National Cancer Inst., 81(19):
1484 (1989).

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 polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also >ie used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acid. Sci. USA, 90: 7889-7893 (1993). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary .activities that do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytoldne, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S.
Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT ~ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

G. Uses for anti-PRO Antibodies The anti-PRO antibodies of the invention have various utilities. For example, anti-PRO antibodies may be used in diagnostic assays for PRO, e.g., detecting its expression (and in some cases, differential expression) in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, lnc.
(1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 'H,'4C,'zP,'sS, or'ZSI, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistrv, 13:1014 (1974); Pain et al., J.
Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
Anti-PRO antibodies also are useful for the affinity purification of PLO from recombinant cell culture or natural sources. In this process, the antibodies against PRO are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the PRO to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the PRO, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the PRO from the antibody.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
EXAMPLES
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, vA.
EXAMPLE 1: Extracellular Domain Homoloay Screening to Identify Novel Poly~peptides and cDNA Encoding Therefor The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST databases. The EST
databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST-2 (Altschul et al., Methods in Enzvmology, 266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation 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 with the program "phrap" (Phil Green, University of Washington, Seattle, WA).
Using this extracellular domain homology screen, consensus DNA sequences were assembled relative S to the other identified EST sequences using phrap. In addition, the consensus DNA sequences obtained were often (but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above.
Based upon the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide.
Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR
product of about 100-1000 by in length. The probe sequences are typically 40-SS by in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-l.Skbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current 1S Protocols in Molecular Bioloey, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.
The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA.
The cDNA was primed with oligo dT containing a NotI site, linked with blunt to SaII hemilanased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRICD; pRKSB is a precursor of pRKSD that does not contain the SfiI
site; see, Hohnes et al. , Science, 253:1278-1280 (1991)) in the unique XhoI and NotI sites.
EXAMPLE 2: Isolation of cDNA clones b~Amylase Screening 2S 1. Preparation of oligo dT grimed cDNA library mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2). This RNA was used to generate an oligo dT primed cDNA library in the vector pRKSD using reagents and protocols from Life Technologies, Gaithersburg, MD
(Super Script Plasmid System).
In this procedure, the double stranded cDNA was sized to greater than 1000 by and the SaII/NotI Tinkered cDNA
was cloned into XhoT/NotI cleaved vector, pRKSD is a cloning vector that has an sp6 transcription initiation site followed by an SfiI restriction enzyme site preceding the XhoI/Notl cDNA
cloning sites.
2. Preparation of random_primed cDNA library A secondary cDNA library was generated in order to preferentially represent the S' ends of the primary 3S cDNA clones. Sp6 RNA was generated from the primary library (described above), and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY.O using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above). In this procedure the double stranded cDNA was sized to 500-1000 bp, tinkered with blunt to NotI adaptors, cleaved with SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cDNA
cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sites. Thus, cDNAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies.
3. Transformation and Detection DNA from the library described in paragraph 2 above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was then electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37°C for 30 minutes. The transformants were then plated onto 20 standard 150 mm LB plates containing ampicillin and incubated fox 16 hours (37°C). Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCI-gradient. The purified DNA was then carried on to the yeast protocols below.
The yeast methods were divided into three categories: (1) Transformation of yeast with the plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones secreting amylase; and (3) PCR
amplification of the insert directly from the yeast colony and purification of the DNA for sequencing and further analysis.
The yeast strain used was HD56-5A (ATCC-90785). This strain has the following genotype: MAT
alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL+, 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 sec71 being most preferred.
Alternatively, antagonists (including antisense nucleotides and/or ligands) which interfere with the normal operation of these genes, other proteins implicated in this post translation pathway (e.g., SEC6lp, SEC72p, SEC62p, SEC63p, TDJlp or SSAlp-4p) or the complex formation of these proteins may also be preferably employed in combination with the amylase-expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al. , Nucl. Acid. Res. , 20:1425 (1992). Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and grown overnight at 30°C. The YEPD broth was prepared as described in Kaiser et al. , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The overnight culture was then diluted to about 2 x 106 cells/ml (approx. OD6oo=0.1) into fresh YEPD broth (500 ml) and regrown to 1 x 10' cells/ml (approx.
ODboo=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended into sterile water, and centrifuged again in SO ml falcon tubes at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM
Tris-HCI, 1 mM EDTA pH 7.5, 100 mM LizOOCCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells (100 pl) with freshly denatured single stranded WO O1/~8848 PCT/USO1/06520 salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1 ~,g, vol. < 10 pl) in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE
(600 pl, 40% polyethylene glycol-4000, 10 mM Tris-HCI, 1 mM EDTA, 100 mM LiZOOCCH3, pH 7.5) was added.
This mixture was gently mixed and incubated at 30°C while agitating for 30 minutes. The cells were then heat shocked at 42°C
for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspended into TE (500 pl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were then diluted into TE (1 ml) and aliquots (200 p,l) were spread onto the selective media previously prepared in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was performed using a single, large scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil (SCD-Ura) prepared as described in Kaiser et al. , 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.
The detection of colonies secreting amylase was performed by including red starch in the selective growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the procedure described by Biely et al., Anal. Biochem., 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 mM
final concentration).
The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies. Well isolated single 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 DNA by PCR Amplification When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 pl) in a 96 well plate. At this time, 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 ~.1 volume containing: 0.5 p,l Klentaq (Clontech, Palo Alto, CA); 4.0 pl 10 mM dNTP's (Perkin Elmer-Cetus);
2.5 wl Kentaq buffer (Clontech); 0.25 ~.1 forward oligo 1; 0.25 ~1 reverse oligo 2; 12.5 pl distilled water. The sequence of the forward oligonucleotide 1 was:
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID N0:611) The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID N0:612) PCR was then performed as follows:
a. Denature 92°C, 5 minutes b. 3 cycles of: Denature 92°C, 30 seconds Anneal 59°C, 30 seconds Extend 72°C, 60 seconds c. 3 cycles of: Denature 92°C, 30 seconds Anneal 57°C, 30 seconds Extend 72°C, 60 seconds d. 25 cycles of: Denature 92°C, 30 seconds Anneal 55°C, 30 seconds Extend 72°C, 60 seconds e. Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 by region from vector pSST-AMY.O
when no insert was present.
Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers. Thus, the total product of the PCR reaction from an empty vector was 343 bp. However, signal sequence-fused cDNA resulted in considerably longer nucleotide sequences.
Following the PCR, an aliquot of the reaction (5 ~,1) was examined by agarose gel electrophoresis in a 1'~ agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et ad., supra.
Clones resulting in a single strong PCR product larger than 400 by were further analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc., Chatsworth, CA).
EXAMPLE 3: Isolation of cDNA Clones UsingS_ianal Algorithm Analvsis Various polypeptide-encoding nucleic acid sequences were identified by applying a proprietary signal sequence finding algorithm developed by Genentech, Inc. (South San Francisco, CA) upon ESTs as well as clustered and assembled EST fragments from public (e.g., GenBank) and/or private (LIFESEQ~, Incyte Pharmaceuticals, Tnc., Palo Alto, CA) databases. The signal sequence algorithm computes a secretion signal score based on the character of the DNA nucleotides surrounding the first and optionally the second methionine codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under consideration. The nucleotides following the first ATG must code for at least 35 unambiguous amino acids without any stop codons. If the first ATG has the required amino acids, the second is not examined. If neither meets the requirement, the candidate sequence is not scored. In order to determine whether the 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 parameters) known to be associated with secretion signals.
Use of this algorithm resulted in the identification of numerous polypeptide-encoding nucleic acid sequences.
EXAMPLE 4: Isolation of cDNA clones Encoding Human PRO Polypeptides Using the techniques described in Examples 1 to 3 above, numerous full-length cDNA clones were identified as encoding PRO polypeptides as disclosed herein. These cDNAs were then deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Blvd., Manassas, VA
20110-2209, USA (ATCC) as shown in Table 7 below.

Table 7 Material ATCC Dep. No. Deposit Date DNA16435-1208209930 June 2, 1998 DNA23318-1211209787 April2l, 1998 DNA23322-1393203400 October 27, 1998 DNA23334-1392209918 June 2, 1998 DNA26843-1389203099 August 4, 1998 DNA 26844-1394209926 June 2, 1998 DNA30867-1335209807 April 28, 1998 DNA33470-1175209398 October 17, 1997 DNA34436-1238209523 December 10, DNA35557-1137209255 September 16, DNA35599-1168209373 October 16, 1997 DNA35668-1171209371 October 16, 1997 DNA36992-1168209382 October 16, 1997 DNA39423-1182209387 October 17, 1997 DNA39427-1179209395 October 17, 1997 DNA39510-1181209392 October 17, 1997 DNA39518-1247209529 December 10, DNA39975-1210209783 Apri121, 1998 DNA39976-1215209524 December 10, DNA39979-1213209789 April 21, 1998 DNA40594-1233209617 February 5, 1998 DNA40603-1232209486 November 21, DNA40604-1187209394 October 17, 1997 DNA40625-1189209788 April2l, 1998 DNA41225-1217209491 November 21, DNA41379-1236209488 November 21, DNA41386-1316209703 March 26, 1998 DNA44161-1434209907 May 27, 1998 DNA44179-1362209851 May 6, 1998 DNA44192-1246209531 December 10, DNA44694-1500203114 August 11, 1998 DNA45234-1277209654 March 5, 1998 DNA45409-2511203579 January 12, 1999 DNA45415-1318209810 April 28, 1998 DNA45417-1432209910 May 27, 1998 DNA45493-1349209805 April 28, 1998 WO O1JG8848 . PCT/USO1JOG520 Table 7 fcont') Material ATCC Deb Deposit Date No.

DNA46776-1284209721 March 31, 1998 DNA48296-1292209668 March 11, 1998 DNA48306-1291209911 May 27, 1998 DNA48328-1355209843 May 6, 1998 DNA48329-1290209785 Apri121, 1998 DNA48334-1435209924 June 2, 1998 DNA49141-1431203003 June 23, 1998 DNA49624-1279209655 March 5, 1998 DNA49647-1398209919 June 2, 1998 DNA49819-1439209931 June 2, 1998 DNA50911-1288209714 March 31, 1998 DNA50914-1289209722 March 31, 1998 DNA50919-1361209848 May 6, 1998 ' DNA50980-1286209717 March 31, 1998 DNA52185-1370209861 May 14, 1998 DNA53906-1368209747 April7, 1998 DNA53912-1457209870 May 14, 1998 DNA53913-1490203162 August 25, 1998 DNA53977-1371209862 May 14, 1998 DNA53978-1443209983 June 16, 1998 DNA53996-1442209921 June 2, 1998 DNA54002-1367209754 April 7, 1998 DNA55737-1345209753 Apri17, 1998 DNA56050-1455203011 June 23, 1998 DNA56052-1454203026 June 23, 1998 DNA56107-1415203405 October 27, 1998 DNA56110-1437203113 August 11, 1998 DNA56406-1704203478 November 17, 1998 DNA56409-1377209882 May 20, 1998 DNA56410-1414209923 June 2, 1998 DNA56436-1448209902 May 27, 1998 DNA56529-1647203293 September 29, 1998 DNA56855-1447203004 June 23, 1998 DNA56859-1445203019 June 23, 1998 DNA56860-1510209952 June 9, 1998 DNA56865-1491203022 June 23, 1998 Table 7 (coat') Material ATCC Dep. Deposit Date No.

DNA56868-1478203024 June 23, 1998 DNA56869-1545203161 August 25, 1998 DNA56870-1492209925 June 2, 1998 DNA57039-1402209777 April 14, 1998 DNA57253-1382209867 May 14, 1998 DNA57254-1477203289 September 29, 1998 DNA57699-1412203020 June 23, 1998 DNA57704-1452209953 June 9, 1998 DNA57710-1451203048 July 1, 1998 DNA57827-1493203045 July 1, 1998 DNA57844-1410203010 June 23, 1998 DNA58723-1588203133 August 18, 1998 DNA58727-1474203171 September 1, 1998 DNA58730-1607203221 September 15, 1998 DNA58732-1650203290 September 29, 1998 DNA58737-1473203136 August 18, 1998 DNA58743-1609203154 August 25, 1998 DNA58747-1384209868 May 14, 1998 DNA58828-1519203172 September 1, 1998 DNA58846-1409209957 June 9, 1998 DNA58848-1472209955 June 9, 1998 DNA58849-1494209958 June 9, 1998 DNA58850-1495209956 June 9, 1998 DNA58852-1637203271 September 22, 1998 DNA58853-1423203016 June 23, 1998 DNA58855-1422203018 June 23, 1998 DNA59211-1450209960 June 9, 1998 DNA59212-1627203245 September 9, 1998 DNA59213-1487209959 June 9, 1998 DNA59219-1613203220 September 15, 1998 DNA59497-1496209941 June 4, 1998 DNA59602-1436203051 July 1, 1998 DNA59603-1419209944 June 9, 1998 DNA59605-1418203005 June 23, 1998 DNA59607-1497209946 June 9, 1998 DNA59610-1556209990 June 16, 1998 Table 7 (cony) Material ATCC Dep. Deposit Date No.

DNAS9612-1466209947 June 9, 1998 DNA59613-1417203007 June 23, 1998 DNA59616-1465209991 June 16, 1998 DNA59619-1464203041 July 1, 1998 DNA59625-1498209992 ' June 16, 1998 DNA59817-1703203470 November 17, 1998 DNA59827-1426203089 August 4, 1998 DNA59828-1608203158 August 25, 1998 DNA59837-2545203658 February 9, 1999 DNAS9844-2542203650 February 9, 1999 DNA59853-1505209985 June 16, 1998 DNA59854-1459209974 June 16, 1998 DNA59855-1485209987 June 16, 1998 DNA60278-1530203170 September 1, 1998 DNA60283-1484203043 July 1, 1998 DNA60608-1577203126 August 18, 1998 DNA60611-1524203175 September 1, 1998 DNA60619-1482209993 June 16, 1998 DNA6062S-1507209975 June 16, 1998 DNA60629-1481209979 June 16, 1998 DNA60740-1615203456 November 3, 1998 DNA61608-1606203239 September 9, 1998 DNA61755-1554203112 August 11, 1998 DNA62809-1531203237 September 9, 1998 DNA62812-1594203248 September 9, 1998 DNA62813-2544203655 February 9, 1999 DNA62845-1684203361 October 20, 1998 DNA64849-1604203468 November 17, 1998 DNA64852-1589203127 August 18, 1998 DNA64863-1573203251 September 9, 1998 DNA64881-1602203240 September 9, 1998 DNA64902-1667203317 October 6, 1998 DNA64952-1568203222 September 15, 1998 DNA6S403-1565203230 September 15, 1998 DNA65413-1534203234 September 15, 1998 DNA65423-1595203227 September 15, 1998 Table 7 (cony) Material ATCC Dep. De~sit Date No.

DNA66304-1546203321 October 6, 1998 DNA66308-1537203159 August 25, 1998 DNA66511-1563203228 , September 15, 1998 DNA66512-1564203218 September 15, 1998 DNA66519-1535203236 September 15, 1998 DNA66521-1583203225 September 15, 1998 DNA66658-1584203229 September 15, 1998 DNA66660-1585203279 September 22, 1998 , DNA66669-1597203272 September 22, 1998 DNA66674-1599203281 September 22, 1998 DNA68836-1656203455 November 3, 1998 DNA68862-2546203652 February 9, 1999 DNA68866-1644203283 September 22, 1998 DNA68869-1610203164 August 25, 1998 DNA68871-1638203280 September 22, 1998 DNA68879-1631203274 September 22, 1998 DNA68880-1676203319 October 6, 1998 DNA68882-1677203318 October 6, 1998 DNA68883-1691203535 December 15, 1998 DNA68885-1678203311 October 6, 1998 DNA71180-1655203403 October 27, 1998 DNA71184-1634203266 September 22, 1998 DNA71213-1659203401 October 27, 1998 DNA71234-1651203402 October 27, 1998 DNA71269-1621203284 September 22, 1998 DNA71277-1636203285 September 22, 1998 DNA71286-1687203357 October 20, 1998 DNA71883-1660203475 November 17, 1998 DNA73401-1633203273 September 22, 1998 DNA73492-1671203324 October 6, 1998 DNA73730-1679203320 October 6, 1998 DNA73734-1680203363 October 20, 1998 DNA73735-1681203356 October 20, 1998 DNA73742-1662203316 October 6, 1998 DNA73746-1654203411 October 27, 1998 DNA73760-1672203314 October 6, 1998 Table 7 (cony) Material ATCC Dep. Deposit Date No.

DNA76393-1664203323 October 6, 1998 DNA76398-1699203474 November 17, 1998 DNA76399-1700203472 November 17, 1998 DNA76522-2500203469 November 17, 1998 DNA76533-1689203410 October 27, 1998 DNA77303-2502203479 November 17, 1998 DNA77626-1705203536 December 15, 1998 DNA77648-1688203408 October 27, 1998 DNA81754-2532203542 December 15, 1998 DNA81757-2512203543 December 15, 1998 DNA82302-2529203534 December 15, 1998 DNA82340-2530203547 December 22, 1998 DNA87991-2540203656 February 9, 1999 DNA92238-2539203602 January 20, 1999 DNA115291-2681PTA-202 June 8, 1999 DNA23336-2861PTA-1673 April 11, 2000 DNA30862-1396209920 June 2, 1998 DNA30871-1157209380 October 16, 1997 DNA32279-1131209259 September 16, 1997 DNA33206-1165209372 October 16, 1997 DNA35673-1201209418 October 28, 1997 DNA47361-1154-2209431 November 7, 1997 DNA49631-1328209806 April 28, 1998 DNA52594-1270209679 March 17, 1998 DNA55800-1263209680 March 17, 1998 DNA56531-1648203286 September 29, 1998 DNA56965-1356209842 May 6, 1998 DNA57037-1444209903 May 27, 1998 DNA57695-1340203006 June 23, 1998 DNA57834-1339209954 June 9, 1998 DNA57841-1522203458 November 3, 1998 DNA58847-1383209879 May 20, 1998 DNA59493-1420203050 July 1, 1998 DNA59586-1520203288 September 29, 1998 DNA59608-2577203870 March 23, 1999 DNA59849-1504209986 June 16, 1998 Table 7 (cont'1 Material ATCC Dep. Deposit Date No.

DNA60292-1506203540 December 15, 1998 DNA62377-1381-1203552 December 22, 1998 DNA62880-1513203097 August 4, 1998 DNA66672-1586203265 September 22, 1998 DNA67962-1649203291 September 29, 1998 DNA69555-2867PTA-1632 April 4, 2000 DNA71162-2764PTA-860 October 19, 1999 DNA71290-1630203275 September 22, 1998 DNA76401-1683203360 October 20, 1998 DNA76541-1675203409 October 27, 1998 DNA76788-2526203551 December 22, 1998 DNA77623-2524203546 December 22, 1998 DNA80136-2503203541 December 15, 1998 DNA83568-2692PTA-386 July 20, 1999 DNA84210-2576203818 March 2, 1999 DNA86576-2595203868 March 23, 1999 DNA87976-2593203888 March 30, 1999 DNA92256-2596203891 March 30, 1999 DNA92289-2598PTA-131 May 25, 1999 DNA96850-2705PTA-479 August 3, 1999 DNA96855-2629PTA-18 May 4, 1999 DNA96857-2636PTA-17 May 4, 1999 DNA96860-2700PTA-478 August 3, 1999 DNA96861-2844PTA-1436 March 2, 2000 DNA96866-2698PTA-491 August 3, 1999 DNA96870-2676PTA-254 June 22, 1999 DNA96872-2674PTA-550 August 17, 1999 DNA96878-2626PTA-23 May 4, 1999 DNA96879-2619203967 April 27, 1999 DNA96889-2641PTA-119 May 25, 1999 DNA96893-2621PTA-12 May 4, 1999 DNA96897-2688PTA-379 July 20, 1999 DNA98564-2643PTA-125 May 25, 1999 DNA107443-2718PTA-490 August 3, 1999 DNA107786-2723PTA-474 August 3, 1999 DNA108682-2712PTA-486 August 3, 1999 . Table 7 (cony) Material ATCC Dep. Deposit Date No.

DNA108684-2761PTA-653 September 14, 1999 DNA108701-2749PTA-554 August 17, 1999 DNA108720-2717PTA-511 August 10, 1999 DNA108726-2729PTA-514 August 10, 1999 DNA108728-2760PTA-654 September 14, 1999 DNA108738-2767PTA-862 October 19, 1999 DNA 108743-2722PTA-508 August 10, 1999 DNA108758-2759PTA-655 September 14, 1999 DNA108765-2758PTA-657 September 14, 1999 DNA108783-2747PTA-616 August 31, 1999 DNA108789-2748PTA-547 August 17, 1999 DNA108806-2724PTA-610 August 31, 1999 DNA108936-2719PTA-519 August 10, 1999 DNA119510-2771PTA-947 November 9, 1999 DNA119517-2778PTA-951 November 16, 1999 DNA119535-2756PTA-613 August 31, 1999 DNA119537-2777PTA-956 November 16, 1999 DNA119714-2851PTA-1537 March 21, 2000 DNA125170-2780PTA-953 November 16, 1999 DNA129594-2841PTA-1481 March 14, 2000 DNA129793-2857PTA-1733 April 18, 2000 DNA130809-2769PTA-949 November 9, 1999 DNA131639-2874PTA-1784 April 25, 2000 DNA131649-2855PTA-1482 March 14, 2000 ~

DNA131652-2876PTA-1628 April 4, 2000 DNA131658-2875PTA-1671 April 11, 2000 DNA132162-2770PTA-950 November 9, 1999 DNA136110-2763PTA-652 September 14, 1999 DNAi39592-2866PTA-1587 March 28, 2000 DNA139608-2856PTA-1581 March 28, 2000 DNA143292-2848PTA-1778 April 25, 2000 DNA144844-2843PTA-1536 March 21, 2000 DNA144857-2845PTA-1589 March 28, 2000 DNA145841-2868PTA-1678 April 11, 2000 DNA148004-2882PTA-1779 April 25, 2000 DNA149893-2873PTA-1672 April 11, 2000 Table 7 (cony) Material ATCC Dep. Deposit Date No.

DNA149930-2884 PTA-1668 April 11, 2000 DNA150157-2898 PTA-1777 April 25, 2000 DNA150163-2842 PTA-1533 March 21, 2000 DNA153579-2894PTA-1729 April 18, 2000 DNA164625-2890 PTA-1535 March 21, 2000 DNA57838-1337 203014 June 23, 1998 DNA59777-1480 203111 August 11, 1998 DNA66675-1587 203282 September 22, 1998 DNA76532-1702203473 November 17, 1998 DNA105849-2704 PTA-473 August 3, 1999 DNA83500-2506 203391 October 29, 1998 These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S.
patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC ~
122 and the Commissioner's rules pursuant thereto (including 37 CFR ~ 1.14 with particular reference to 886 OG
638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
EXAMPLE 5: Use of PRO as a hybridization probe The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe.
DNA comprising 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 human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled PRO-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium 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 filters is performed in an aqueous solution of 0. lx 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 lmown in the art.
EXAMPLE 6: Expression of PRO in E. coli This example illustrates preparation of an unglycosylated 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 enzyme 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 Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector will preferably include sequences which, encode for an antibiotic resistance gene, a tzp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU
gene.
The ligarion mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA
sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents lrnown 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. coli in a poly-His tagged form, using the following procedure. The DNA
encoding PRO is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and 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 (W3110 fuhA(tonA) lon gakE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50 mglml carbenicillin at 30°C with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)ZS04, 0.71 g sodium citrate~2H20, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, O.SS%
(w/v) glucose and 7 mM
MgS04) and grown for approximately 20-30 hours at 30°C with shaking.
Samples are removed to verify expression by SDS-PAGE analysis, and 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 in 10 volumes (w/v) in 7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulEte and sodium tetrathionate is added to make final concentrations of O.1M and 0.02 M, respectively, and the solution is stirred overnight at 4°C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization.
The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractions 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 amino acid sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycine and i mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4°C for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10%
final concentration. The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0.1 % TFA with elution with a gradient of acetonitrile from 10 to 80% . Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin.
Aggregated species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PRO polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 7: Expression of PRO in mammalian cells This example illustrates preparation of a potentially glycosylated form of FRO
by recombinant expression in mammalian cells.
The vector, pRKS (see EP 307,247, published March 15, 1989), is employed as the expression vector.
Optionally, the PRO DNA is ligated into pRKS with selected restriction enzymes to allow insertion of the PRO
DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRKS-PRO.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM
supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 wg pRKS-PRO DNA
is mixed with about 1 ~.g DNA
encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 ~sl of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaClz. To this mixture is added, dropwise, 500 pl of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaP04, and a precipitate is allowed to form for 10 minutes at 25°C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C. The culture medium is aspirated off and 2 ml of 20 % glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 ~cCiJml 35S-cysteine and 200 ~Ci/ml 35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15 SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981).
293 cells are grown to maximal density in a spinner flask and 700 wg pRKS-PRO 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 with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 ~g/ml bovine insulin and 0.1 ~.g/ml bovine transferrin. After about four days, the conditioned media 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 column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRKS-PRO can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 'SS-methionine. After determining the presence of PRO polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed PRO can then be concentrated and purified by any selected method.
Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be subcloned out of the pRKS vectox. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged PRO insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged PRO can then be concentrated and purified by any selected method, such as by NiZ+-chelate affinity chromatography.
PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO
cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgGI constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using standard techniques as described in Ausubel et al. , Current Protocols of Molecular Biolo~v, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect° (Qiagen), Dosper or Fugene° (Boehringer Mannheim). The cells are grown as described in Lucas et al., supra.
Approximately 3 x 10' cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 /cm filtered PS20 with 5% 0.2 um diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL
spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37°C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 x 105 cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Patent No. 5,122,469, issued June 16, 1992 may actually be used. A 3L production spinner is seeded at 1.2 x 106 cells/mL. On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33°C, and 30 mL of 500 g/L glucose and 0.6 mL of 10 % antifoam (e, g. , 35 % polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 ~cm filter. The filtrate was either stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA
column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified, protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4%
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM
Na phosphate buffer, pH 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 collecting 1 ml fractions into tubes containing 275 ~L of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 8: Expression of PRO in Yeast The following method describes recombinant expression of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PRO from the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO. For secretion, DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH
promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO.
Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10 % trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
Recombinant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing PRO may further be purifted using selected column chromatography resins.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 9: Expression of PRO in Baculovirus-Infected Insect Cells The following method describes recombinant expression 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. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG).
A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions. The S' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldT"'virus DNA
(Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released viruses are harvested and used for further ampliftcations. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
Expressed poly-his tagged PRO can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M
KCk), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCI, 10% glycerol, pH 7.8) and filtered through a 0.45 um filter.
A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline AZeo with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCI, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.
After reaching AZao baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Niz+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His,o tagged PRO are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be performed using lrnown chromatography techniques, including for instance, Protein A or protein G
column chromatography.
Many of the PRO polypeptides disclosed herein were successfully expressed as described above.
EXAMPLE 10: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can specifically bind PRO.
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELTSA assays to detect anti-PRO antibodies.

After a suitable antibody titer has been detected, the animals "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 (using 35 % polyethylene glycol) to a selected marine myeloma cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PRO.
Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
EXAMPLE 11: Purification of PRO Polyrx~tides Using Specific Antibodies Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-PRO polypeptide, mature PRO
polypeptide, or pre-PRO polypeptide is purified by immunoaffmity chromatography using antibodies specific for the PRO polypeptide of interest. In general, an immunoaffmity column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB
Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSET"' (Pharmacia LKB
Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
Such an immunoaffmity column is utilized in the purification of PRO
polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
A soluble PRO polypeptide-containing preparation is passed over the immunoaffmity column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/PRO polypeptide binding (e. g. , a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected.

EXAMPLE 12: Drug Screening This invention is particularly useful for screening compounds by using PRO
polypeptides or binding fragment thereof in any of a variety of drug screening techniques. The PRO
polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between PRO
polypeptide or a fragment and the agent being tested. Alternatively, one can examine the diminution in complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide-associated disease or disorder. These methods comprise contacting such an agent with an PRO
polypeptide or fragment thereof and assaying (I) 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 such competitive binding assays, the PRO polypeptide or fragment is typically labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail in WO 84/03564, published on September 13, 1984.
Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. 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 known in the art.
Purred PRO polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO
polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRO polypeptide.
EXAMPLE 13: Rational Drug Design 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 examples can be used to fashion drugs which are more active or stable forms of the PRO
polypeptide or which enhance or interfere with the function of the PRO
polypeptide in vivo (c.f., Hodgson, Bio/Technologv, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO

polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO
polypeptide must be ascertained to elucidate the structure and to determine active sites) 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 information 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 shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al., J.
Biochem., 113:742-746 (1993).
It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, Imowledge of the PRO polypeptide amino acid sequence prnvided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.
EXAMPLE 14: Identification of PRO Polypeptides That Stimulate TNF-a Release In Human Blood (Assay x28) This assay shows that certain PRO polypeptides of the present invention act to stimulate the release of TNF-a in human blood. PRO polypeptides testing positive in this assay are useful for, among other things, research purposes where stimulation of the release of TNF-a would be desired and for the therapeutic treatment of conditions wherein enhanced TNF-a release would be beneficial.
Specifically, 200 p,l of human blood supplemented with SOmM Hepes buffer (pH 7.2) is aliquoted per well in a 96 well test plate. To each well is then added 3001 of either the test PRO polypeptide in 50 mM Hepes buffer (at various concentrations) or 50 mM
Hepes buffer alone (negative control) and the plates are incubated at 37°C for 6 hours. The samples are then centrifuged and SOp,l of plasma is collected from each well and tested for the presence of TNF-a by ELISA
assay. A positive in the assay is a higher amount of TNF-a in the PRO
polypeptide treated samples as compared to the negative control samples.
The following PRO polypep2ides tested positive in this assay:
PR01079, PR0827, PR0791, PR01131, PR01316, PR01183, PR01343, PR01760, PR01567, and PR04333.
EXAMPLE 15: Promotion of Chondrocvte Redifferentiation (Assay 129) This assay is designed to determine whether PRO polypeptides of the present invention show the ability to induce the proliferation and/or redifferentiation of chondrocytes in culture. PRO polypeptides testing positive in this assay would be expected to be useful for the therapeutic treatment of various bone and/or cartilage WO ll1/GSfi48 PCT/USlll/IICS20 disorders such as, for example, sports injuries and arthritis.
Porcine chondrocytes are isolated by overnight collagenase digestion of articular cartilage of the metacarpophalangeal joint of 4-6 month old female pigs. The isolated cells are then seeded at 25,000 cells/em2 in Ham F-12 containing 10% FBS and 4 ~,g/ml gentamycin. The culture media is changed every third day. On day 12, the cells are seeded in 96 well plates at 5,000 cells/well in 100,1 of the same media without serum and 100 ~I of either serum-free medium (negative control), staurosporin (final concentration of 5 nM; positive control) or the test PRO polypeptide are added to give a final volume of 200 ~,1/well.
After.5 days at 37°C, 22 p,l of media comtaining 100~.g/ml Hoechst 33342 and 50 pg/ml 5-CFDA is added to each well and incubated for an additional 10 minutes at 37°C. A picture of the green fluorescence is taken for each well and the differentiation state of the chondrocytes is calculated by morphometric analysis. A positive result in the assay is obtained when the >50% of the PRO polypeptide treated cells are differentiated (compared to the background obtained by the negative control).
PR06029 polypeptide tested positive in this assay.
EXAMPLE 16: Microarrav Analysis to Detect Overezpression of PRO Polypentides in Cancerous Tumors ' Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate eDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is lvaown. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sample is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified. The implication of this result is that an overexpressed protein in a diseased tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition.
The methodology of hybridization of nucleic acids and microarray technology is well known in the art.
In the present example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions In the present example, cancerous tumors derived from various human tissues were studied for PRO
polypeptide-encoding gene expression relative to non-cancerous human tissue in an attempt to identify those PRO
polypeptides which are overexpressed in cancerous tumors. Two sets of experimental data were generated. In one set, cancerous human colon tumor tissue and matched non-cancerous human colon tumor tissue from the same patient ("matched colon control") were obtained and analyzed for PRO
polypeptide expression using the above described microarray technology. In the second set of data, cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a "universal"
epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung, mRNA
isolated from the pooled tissues represents a mixture of expressed gene products from these different tissues.
Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:control detection) within each experiment. The normalized ratios from various experiments were then compared and used to identify clustering of gene expression. Thus, the pooled "universal control" sample not only alkowed effective relative gene expression determinations in a simple 2-sample comparison, it also allowed mufti-sample comparisons across several experiments.
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 microarray and RNA from the tumor tissues listed above were used for the hybridization thereto. A value based upon the normalized ratio:experimental ratio was designated as a "cutoff ratio". Only values that were above this cutoff ratio were determined to be significant. Table 8 below shows the results of these experiments, demonstrating that various PRO polypeptides of the preent invention are significantly overexpressed in various human tumor tissues as compared to a non-cancerous human tissue control. As described above, these data demonstrate that the PRO polypeptides of the present invention are useful not only as diagnostic markers for the presence of one or more cancerous tumors, but also serve as therapeutic targets for the treatment of those tumors.
Table 8 Molecule is overexgessed in: as compared to:

PR0276 lung tumor universal normal control PR0284 colon tumor universal normal control PR0284 lung tumor universal normal control PR0284 breast tumor universal normal control PR0193 cplon tumor universal normal control PR0193 lung tumor universal normal control PR0193 breast tumor universal normal control PR0193 prostate tumor universal normal control PR0190 colon tumor universal normal control PR0190 lung tumor universal normal control PR0190 breast tumor universal normal control PR0180 colon tumor universal normal control PR0180 kung tumor universal normal control PR0180 breast tumor universal normal control PR0194 colon tumor universal normal control PR0194 lung tumor universal normal control PR0194 breast tumor universal normal control PR0194 cervical tumor universal normal control PR0218 colon tumor universal normal control PR0218 lung tumor universal normal control PR0260 colon tumor universal normal control PR0260 lung tumor universal normal control PR0260 breast tumor universal normal control PR0260 rectal tumor universal normal controk PR0233 colon tumor universal normal control PR0233 lung tumor universal normak control PR0233 breast tumor universal normal control Table. 8 (cony) Molecule is overexpressed in: as compared to:

PR0234 colon tumor universal normal control PR0234 lung tumor universal normal control PR0234 breast tumor universal normal control PR0234 liver tumor universal normal control PR0236 colon tumor universal normal control PR0236 lung tumor universal normal control PR0236 breast tumor universal normal control PR0244 colon tumor universal normal control PR0244 lung tumor universal normal control PR0262 colon tumor universal normal control PR0262 lung tumor universal normal control PR0262 breast tumor universal normal control PR0271 colon tumor universal normal control PR0271 lung tumor universal normal control PR0268 colon tumor universal normal control PR0268 lung tumor universal normal control PR0268- breast tumor universal normal control PR0270 colon tumor universal normal control PR0270 lung tumor universal normal control PR0270 breast tumor universal normal control PR0270 liver tumor universal normal control PR0355 lung tumor universal normal control PR0355 breast tumor universal normal control PR0355 prostate tumor universal normal control PR0298 colon tumor universal normal control PR0298 lung tumor universal normal control PR0298 breast tumor universal normal control PR0299 colon tumor universal normal control PR0299 lung tumor universal normal control PR0299 breast tumor universal normal control PR0296 colon tumor universal normal control PR0296 breast tumor universal normal control PR0329 colon tumor universal normal control PR0329 lung tumor universal normal control PR0329 breast tumor universal normal control PR0330 colon tumor universal normal control PR0330 lung tumor universal normal control PR0294 lung tumor universal normal control PR0294 breast tumor universal normal control PR0300 colon tumor universal normal control PR0300 lung tumor universal normal control PR0300 breast tumor universal normal control PR0307 lung tumor universal normal control PR0334 colon tumor universal normal control PR0334 lung tumor universal normal control PR0334 breast tumor universal normal control PR0334 prostate tumor universal normal control PR0352 colon tumor universal normal control PR0352 lung tumor universal normal control PR0352 breast tumor universal normal control PR0352 liver tumor universal normal control PR0710 breast tumor universal normal control PR0873 colon tumor universal normal control PR0873 lung tumor universal normal control Table 8 (cont'1 Molecule is overexpressed in: as compared to:

PR0873 breast.tumor universal normal control PR0873 prostate tumor universal normal control PR0354 colon tumor universal normal control PR0354 lung tumor universal normal control PR0354 breast tumor universal normal control PRO1ISI lung tumor universal normal control PR01151 breast tumor universal normal control PR0382 colon tumor universal normal control PR0382 lung tumor universal normal control PR0382 breast tumor universal normal control PR01864 lung tumor universal normal control PR01864 breast tumor universal normal control PR01864 liver tumor universal normal control PR0386 colon tumor universal normal control PR0386 lung tumor universal normal control PR0386 prostate tumor universal normal control PR0541 colon tumor universal normal control PR0541 lung tumor universal normal control PR0541 breast tumor universal normal control PR0852 breast tumor universal normal control PR0700 colon tumor universal normal control PR0700 lung tumor universal normal control PR0700 breast tumor universal normal control PR0700 rectal tumor universal normal control ~PR0708 colon tumor universal normal control PR0708 lung tumor universal normal control PR0708 breast tumor universal normal control PR0707 colon tumor universal normal control PR0707 lung tumor universal normal control PR0864 colon tumor universal normal control PR0864 lung tumor universal normal control PR0864 breast tumor universal normal control PR0706 colon tumor universal normal control PR0706 lung tumor universal normal control PRb706 breast tumor universal normal control PR0706 liver tumor universal normal control PR0732 lung tumor universal normal control PR0732 breast tumor universal normal control PR0732 cervical tumor universal normal control PR0537 colon tumor universal normal control PR0537 lung tumor universal normal control PR0537 breast tumor universal normal control PR0545 lung tumor universal normal control PR0545 breast tumor universal normal control PR0718 lung tumor universal normal control PR0718 breast tumor universal normal control PR0872 lung tumor universal normal control PR0872 breast tumor universal normal control PR0872 liver tumor universal normal control PR0704 colon tumor universal normal control PR0704 lung tumor universal normal control PR0704 ~ breast tumor universal normal control PR0705 lung tumor universal normal control PR0705 breast tumor universal normal control WO O1/G8848 PCTlUSOI/OG520 Table 8 (cony) Molecule is overex~ressed in: as compared to:

PR0871 lung tumor universal normal control PR0871 breast tumor universal normal control PR0871 liver tumor universal normal control PR0702 lung tumor universal normal control PR0944 colon tumor universal normal control PR0944 lung tumor universal normal control PR0944 rectal tumor universal normal control PR0739 lung tumor universal normal control PR0739 breast tumor universal normal control PR0739 prostate tumor universal normal control PR0941 colon tumor universal normal control PR0941 lung tumor universal normal control PR0941 breast tumor universal normal control PR0941 rectal tumor universal normal control PR01082 lung tumor universal normal control PR01082 breast tumor universal normal control PR01133 colon tumor universal normal control PR01133 lung tumor universal normal control PR0983 colon tumor universal normal control PR0983 lung tumor universal normal control PR0983 breast tumor universal normal control PR0784 colon tumor universal normal control PR0784 lung tumor universal normal control PR0784 breast tumor universal normal control PR0784 prostate tumor universal normal control PR0783 colon tumor universal normal control PR0783 lung tumor universal normal control PR0783 breast tumor universal normal control PR0783 liver tumor universal normal control PR0940 colon tumor universal normal control PR0940 lung tumor universal normal control PR0940 breast tumor universal normal control PR0768 colon tumor universal normal control PR0768 lung tumor universal normal control PR0768 breast tumor universal normal control PR01079 colon tumor universal normal control PR01079 lung tumor universal normal control PR01079 breast tumor universal normal control PR01079 rectal tumor universal normal control PR01078 colon tumor universal normal control PR01078 lung tumor universal normal control PR01018 colon tumor universal normal control PR01018 lung tumor universal normal control PR01018 breast tumor universal normal control PR0793 colon tumor universal normal control PR0793 lung tumor universal normal control PR0793 breast tumor universal normal control PR0793 rectal tumor universal normal control PR01773 colon tumor universal normal control PR01773 lung tumor universal normal control PR01773 prostate tumor universal normal control PR01014 lung tumor universal normal control PR01014 breast tumor universal normal control PR01013 colon tumor universal normal control Table 8~cont~

Molecule is oyerexpressed in: as comQ,ared to:

PR01013 lung tumor universal normal control PR01013 breast tumor universal normal control PR01013 liver tumor universal normal control PR0937 colon tumor universal normal control PR0937 lung tumor universal normal control PR0937 breast tumor universal normal control PR0937 cervical tumor universal normal control PR0937 rectal tumor universal normal control PR01477 lung tumor universal normal control PR01477 breast tumor universal normal control PR01477 rectal tumor universal normal control PR0842 colon tumor universal normal control PR0842 lung tumor universal normal control PR0842 breast tumor universal normal control PR0839 colon tumor universal normal control PR01180 colon tumor universal normal control PR01180 lung tumor universal normal control PR01180 liver tumor universal normal control PR01134 lung tumor universal normal control PR01134 breast tumor universal normal control PR01134 prostate tumor universal normal control PR01115 colon tumor universal normal control PRO1115 lung tumor universal normal control PRO1115 breast tumor universal normal control PR01277 colon tumor universal normal control PR01277 lung tumor universal normal control PR01135 lung tumor universal normal control PR01135 breast tumor universal normal control PR01135 cervical tumor universal normal control PR0827 colon tumor universal normal control PR0827 lung tumor universal normal control PR0827 prostate tumor universal normal control PR0827 cervical tumor universal normal control PR01057 lung tumor universal normal control PR01057 breast tumor universal normal contxol PR01113 colon tumor universal normal control PR01113 lung tumor universal normal control PR01006 colon tumor universal normal control PR01006 lung tumor universal normal control PR01006 breast tumor universal normal control PR01006 rectal tumor universal normal control PR01074 lung tumor universal normal control PR01074 rectal tumor universal normal control PR01073 lung tumor universal normal control PR01073 breast tumor universal normal control PR01136 colon tumor universal normal control PR01136 lung tumor universal normal control PR01136 breast tumor universal normal control PR01004 lung tumor universal normal control PR01344 colon tumor universal normal control PR01344 lung tumor universal normal control PR01344 breast tumor universal normal control PR01344 rectal tumor universal normal control PRO111.0 colon tumor universal normal control WO 01/68848 PCTlUS01/06520 Table 8 fcont') Molecule is overexpressed in: as compared to:

PRO1110 lung tumor universal normal control PRO1110 breast tumor universal normal control PR01378 colon tumor universal normal control PR01378 lung tumor universal normal control PR01378 prostate tumor universal normal control PR01378 cervical tumor universal normal control PR01481 colon tumor universal normal control PR01481 lung tumor universal normal control PRO 1109 lung tumor universal normal control PR01109 breast tumor universal normal control PR01383 colon tumor universal normal control PR01383 lung tumor universal normal control PR01383 breast tumor universal normal control PR01072 lung tumor universal normal control PR01189 colon tumor universal normal control PR01189 lung tumor universal normal control PR01189 breast tumor universal normal control PR01189 prostate tumor universal normal control PR01003 colon tumor universal normal control PR01003 lung tumor universal normal control PR01003 breast tumor universal normal control PR01003 liver tumor universal normal control PR01003 rectal tumor universal normal control PR01108 colon tumor universal normal control PR01108 lung tumor universal normal control PR01108 breast tumor universal normal control PR01137 colon tumor universal normal control PR01137 lung tumor universal normal control PR01137 breast tumor universal normal control PR01138 colon tumor w niversal normal control PR01138 lung tumor universal normal control PR01138 breast tumor universal normal control PR01415 colon tumor universal normal control PR01415 lung tumor universal normal control PR01415 prostate tumor universal normal control PR01054 lung tumor universal normal control PR01054 breast tumor universal normal control PR0994 colon tumor universal normal control PR0994 lung tumor universal normal control PR0994 rectal tumor universal normal control PR01069 lung tumor universal normal control PR01069 breast tumor universal normal control PR01411 colon tumor universal normal control PR01411 lung tumor universal normal control PR01129 lung tumor universal normal control PR01129 rectal tumor universal normal control PR01359 colon tumor universal normal control PR01359 lung tumor universal normal control PR01359 breast tumor universal normal control PR01359 prostate tumor universal normal control PR01139 lung tumor universal normal control PR01065 lung tumor universal normal control PR01028 colon tumor universal normal control PR01028 lung tumor universal normal control Table 8 (cony) Molecule is overexeressed in: as co~ared to:

PR01028 breast tumor universal normal control PR01028 cervical tumor universal normal control PR01027 colon tumor universal normal control PR01027 lung tumor universal normal control PR01027 breast tumor universal normal control PR01140 colon tumor universal normal control PR01140 breast tumor universal normal control PR01291 colon tumor universal normal control PR01291 breast tumor universal normal control PR01105 colon tumor universal normal control PRO1105 lung tumor universal normal control PR01026 lung tumor universal normal control PR01026 prostate tumor universal normal control PR01104 colon tumor universal normal control PR01104 lung tumor universal normal control PR01104 breast tumor universal normal control PRO1100 colon tumor universal normal control PRO1100 lung tumor universal normal control PRO1100 breast tumor universal normal control PRO1100 rectal tumor universal normal control PR01141 lung tumor universal normal control PR01772 colon tumor universal normal control PR01772 lung tumor universal normal control PR01772 breast tumor universal normal control PR01772 cervical tumor universal normal control PR01064 colon tumor universal normal control PR01064 lung tumor universal normal control PR01379 colon tumor universal normal control PR01379 lung tumor universal normal control PR01379 cervical tumor universal normal control PR03573 lung tumor universal normal control PR03573 breast tumor universal normal control PR03566 colon tumor universal normal control PR03566 lung tumor universal normal control PR01156 lung tumor universal normal control PR01156 breast tumor universal normal control PR01156 prostate tumor universal normal ~ control PR01098 colon tumor universal normal control PR01098 lung tumor universal normal control PR01098 rectal tumor universal normal control PR01128 colon tumor universal normal control PR01128 lung tumor universal normal control PR01128 breast tumor universal normal control PR01248 lung tumor universal normal control PR01248 breast tumor universal normal control PR01127 colon tumor universal normal control PR01127 lung tumor universal normal control PR01127 breast tumor universal normal control PR01316 colon tumor . universal normal control PR01316 lung tumor universal normal control PR01316 breast tumor universal normal control PR01197 colon tumor , universal normal control PR01197 lung tumor universal normal control PR01197 breast tumor universal normal control WO 01/68848 PCT/USO1/06s20 Table 8 lcont') Molecule is overexpressed in: as compared to:

PR01125 lung tumor universal normal control PR01158 breast tumor universal normal control PR01124 colon tumor universal normal control PR01124 lung tumor universal normal control PR01380 colon tumor universal normal control PR01380 lung tumor universal normal control PR01380 breast tumor universal normal control PR01380 liver tumor universal normal control PR01377 colon tumor universal normal control PR01377 lung tumor universal normal control PR01287 lung tumor universal normal control PR01287 breast tumor universal normal control PR01249 lung tumor universal normal control PR01249 breast tumor universal normal control PR01335 colon tumor universal normal control PR01335 lung tumor universal normal control PR01335 breast tumor universal normal control PR03572 lung tumor universal normal control PR01599 colon tumor universal normal control PR01599 lung tumor universal normal control PR01599 breast tumor universal normal control PR01374 lung tumor universal normal control PR01374 breast tumor universal normal control PR01345 lung tumor universal normal control PR01345 breast tumor universal normal control PR01311 lung tumor universal normal control PR01311 breast tumor universal normal control PR01357 colon tumor universal normal control PR01357 lung tumor universal normal control PR01557 colon tumor universal normal control PR01557 lung tumor universal normal control PR01557 breast tumor universal normal control PROI305 colon tumor universal normal control PR01305 lung tumor universal normal control PR01305 breast tumor universal normal control PR01302 colon tumor universal normal control PR01302 lung tumor universal normal control PR01302 breast tumor universal normal control PR01302 rectal tumor universal normal control PR01266 colon tumor universal normal control PR01336 colon tumor universal normal control PR01336 lung tumor universal normal control PR01336 breast tumor universal normal control PR01278 colon tumor universal normal control PR01278 lung tumor universal normal control PR01270 breast tumor universal normal control PR01298 colon tumor universal normal control PR01298 lung tumor universal normal control PR01301 lung tumor universal normal control PR01301 breast tumor universal normal control PR01268 colon tumor universal normal control PR01268 breast tumor universal normal control PR01327 lung tumor universal normal control PR01327 breast tumor universal normal control Table 8 (cony) Molecule is overexpressed in: as compared to:

PR01328 colon tumor universal normal control PR01328 lung tumor universal normal control PR01328 breast tumor universal normal control PR01329 colon tumor universal normal control PR01329 lung tumor universal normal control PR01329 breast tumor universal normal control PR01339 colon tumor universal normal control PR01339 lung tumor universal normal control PR01342 colon tumor ~ w niversal normal control PR01342 lung tumor universal normal control PR01342 breast tumor universal normal control PR01342 rectal tumor universal normal control PR01487 colon tumor universal normal control PR01487 breast tumor universal normal control PR03579 lung tumor universal normal control PR03579 breast tumor universal normal control PR01472 colon tumor universal normal control PR01472 lung tumor universal normal control PR01385 lung tumor universal normal control PR01385 breast tumor universal normal control PR0146I colon tumor universal normal . control PR01461 lung tumor universal normal control PR01461 breast tumor universal normal control PR01429 colon tumor universal normal control PR01429 lung tumor universal normal control PR01429 breast tumor universal normal control PR01568 lung tumor universal normal control PR01568 breast tumor universal normal control PR01569 colon tumor universal nozmal control PR01569 lung tumor universal normal control PR01569 breast tumor universal normal control PR01753 colon tumor universal normal control PR01753 lung tumor universal normal control PR01570 colon tumor universal normal control PR01570 lung tumor universal normal control PR01570 breast tumor universal normal control PR01570 prostate tumor universal normal control PR01570 rectal tumor universal normal control PR01559 colon tumor universal normal control PR01559 lung tumor universal normal control PROI559 breast tumor universal normal control PR01486 lung tumor universal normal control PR01486 breast tumor universal normal control PR01433 colon tumor universal normal control PR01433 lung tumor universal normal control PR01433 breast tumor universal normal control PR01433 rectal tumor universal normal control PR01490 lung tumor universal normal control PR01490 breast tumor universal normal control PR01482 lung tumor universal normal control PR01482 breast tumor universal normal control PR01409 colon tumor universal normal control PR01409 lung tumor universal normal control SS PR01409 breast tumor universal normal control Table 8 (cony) Molecule is overexpressed in: as compared to:

PR01446 colon tumor universal normal control PR01446 lung tumor universal normal control PR01446 breast tumor universal normal control PR01446 prostate tumor universal normal control PR01604 colon tumor universal normal control PR01604 lung tumor universal normal control PR01604 breast tumor universal normal control PR01491 colon tumor universal normal control PR01491 lung tumor universal normal control PR01491 breast tumor universal normal control PR01431 colon tumor universal normal control PR01431 lung tumor universal normal control PR01563 colon tumor universal normal control PR01563 lung tumor universal normal control PR01563 breast tumor universal normal control PRO1S71 colon tumor universal normal control PR01571 lung tumor universal normal control PR01571 breast tumor universal normal control PR01572 lung tumor universal normal control PR01572 prostate tumor universal normal control PR01573 lung tumor universal normal control PR01573 breast tumor universal normal control PR01508 lung tumor universal normal control PR01508 breast tumor ~ universal normal control PR01485 colon tumor universal normal control PR01485 lung tumor universal normal control PR01564 colon tumor universal normal control PR01564 lung tumor universal normal control PR01564 breast tumor universal normal control PRO15S0 colon tumor universal normal control PR01550 lung tumor universal normal control PR01550 breast tumor universal normal control PR01757 lung tumor universal normal control PR01757 breast tumor universal normal control PR01757 prostate tumor universal normal control PR01758 lung tumor universal normal control PR01781 colon tumor universal normal control PR01781 lung tumor universal normal control PR01781 breast tumor universal normal control PR01606 lung tumor universal normal control PR01606 breast tumor universal normal control PR01784 colon tumor universal normal control PR01784 lung tumor universal normal control PR01784 breast tumor universal normal control PR01774 colon tumor universal normal control PR01774 lung tumor universal normal control PR01774 breast tumor universal normal control PR01605 colon tumor universal normal control SO PR01605 lung tumor universal normal control PR01605 prostate tumor universal normal control PR01928 colon tumor universal normal control PR01928 lung tumor universal normal control PR01928 cervical tumor universal normal control PR0186S lung tumor universal normal control Table 8 (cont~

Molecule is overex~essed in: as compared to:

PR01865 liver tumor universal normal control PR01925 lung tumor universal normal control PR01926 liver tumor universal normal control PR02630 colon tumor universal normal control PR02630 lung tumor universal normal control PR02630 breast tumor universal normal control PR02630 liver tumor universal normal control PR03443 colon tumor universal normal control PR03443 lung tumor universal normal control PR03443 breast tumor universal normal control PR03301 colon tumor universal normal control PR03301 lung tumor universal normal control PR03301 breast tumor universal normal control PR03301 rectal tumor universal normal control PR03442 colon tumor universal normal control PR03442 lung tumor universal normal control PR03442 rectal tumor universal normal control PR04978 colon tumor universal normal control pR04978 lung tumor universal normal control PR04978 breast tumor universal normal control PR04978 rectal tumor universal normal control PR05801 colon tumor universal normal control PR05801 ' breast tumor universal normal control PR019630 colon tumor universal normal control PR0203 colon tumor universal normal control PR0204 colon tumor universal normal control PR0204 lung tumor universal normal control PR0204 breast tumor . universal normal control PR0204 prostate tumor universal normal control PR0210 colon tumor universal normal control PR0210 lung tumor universal normal control PR0223 lung tumor universal normal control PR0223 breast tumor universal normal control PR0247 colon tumor universal normal control PR0247 lung tumor universal normal control PR0247 breast ~ universal normal control PR0358 lung tumor universal normal control PR0358 breast tumor universal normal control PR0358 prostate tumor universal normal control PR0724 lung tumor universal normal control PR0868 colon tumor universal normal control PR0868 lung tumor universal normal control PR0868 prostate tumor universal normal control PR0868 rectal tumor universal normal control pR0740 colon tumor universal normal control PR01478 colon tumor universal normal control PR01478 lung tumor universal normal control PR0162 colon tumor universal normal control PR0162 lung tumor universal normal control PR0162 breast tumor universal normal control PR0828 colon tumor universal normal control PR0828 lung tumor universal normal control PR0828 breast tumor universal normal control PR0828 cervical tumor universal normal control Table 8 (cont'1 Molecule is overexpressed in: as compared to:

PR0828 liver tumor universal normal control PR0819 lung tumor universal normal control PR0819 breast tumor universal normal control PR0819 rectal tumor universal normal control PR0813 colon tumor universal normal control PR0813 lung tumor universal normal control PR0813 breast tumor universal normal control PR0813 prostate tumor universal normal control PR01194 colon tumor universal normal control PR01194 lung tumor universal normal control PR01194 breast tumor universal normal control PR0887 colon tumor universal normal control PR0887 lung tumor universal normal control PR0887 rectal tumor universal normal control PR01071 colon tumor universal normal control PR01071 lung tumor universal normal control PR01071 breast tumor universal normal control PR01029 colon tumor universal normal control PR01029 lung tumor universal normal control PROI029 breast tumor universal normal control PR01190 lung tumor universal normal control PR01190 breast tumor universal normal control PR04334 lung tumor universal normal control PROi155 colon tumor universal normal control PR01155 lung tumor universal normal control PR01157 breast tumor universal normal control PROI157 cervical tumor universal normal control PR01122 lung tumor universal normal control PR01122 breast tumor universal normal control PR01183 colon tumor universal normal control PR01183 lung tumor universal normal control PR01183 breast tumor universal normal control PR01337 colon tumor universal normal control PR01337 lung tumor universal normal control PR01337 breast tumor universal normal control PR01480 colon tumor universal normal control PR01480 lung tumor universal normal control PR01480 breast tumor universal normal control PR019645 colon tumor universal normal control PR09782 colon tumor universal normal control PR01419 colon tumor universal normal control PR01575 colon tumor universal normal control PR01575 lung tumor universal normal control PR01567 colon tumor universal normal control PR01567 lung tumor universal normal control PR01567 breast tumor universal normal control PR01891 colon tumor universal normal control PR01889 colon tumor universal normal control PR01889 lung tumor universal normal control PR01785 lung tumor universal normal control PR01785 prostate tumor universal normal control PR06003 colon tumor universal normal control PR04333 colon tumor universal normal control PR04356 colon tumor universal normal control Table 8 (cony) Molecule is overexpressed in: as compared to:

PR04352 colon tumor universal normal control PR04354 colon tumor universal normal control PR04354 lung tumor universal normal control PR04354 prostate tumor universal normal control PR04369 colon tumor universal normal control PR06030 colon tumor universal normal control PR04433 colon tumor universal normal control PR04424 colon tumor universal normal control PR04424 breast tumor universal normal control PR06017 colon tumor universal normal control PR019563 colon tumor universal normal control PR06015 colon tumor universal normal control PR05779 colon tumor universal normal control PR05776 colon tumor universal normal control PR04430 lung tumor universal normal control PR04421 colon tumor universal normal control PR04499 colon tumor universal normal control PR04423 colon tumor universal normal control PR05998 colon tumor universal normal control PR05998 lung tumor universal normal control PR04501 colon tumor universal normal control PR06240 colon tumor universal normal control PR06245 colon tumor universal normal control 2S PR06175 colon tumor universal normal control PR09742 colon tumor universal normal control PR07179 colon tumor universal normal control PR06239 colon tumor universal normal control PR06493 colon tumor universal normal control PR09741 colon tumor universal normal control PR09822 colon tumor universal normal control PR06244 colon tumor universal normal control PR09740 colon tumor universal normal control PR09739 colon tumor universal normal control PR07177 colon tumor universal normal control PR07178 colon tumor universal normal control PR06246 colon tumor universal normal control PR06241 colon tumor universal normal control PR09835 colon tumor universal normal control PR09857 colon tumor universal normal control PR07436 colon tumor universal normal control PR09856 colon tumor universal normal control PR019605 colon tumor universal normal control PR09859 colon tumor universal normal control PR012970 colon tumor universal normal control PR019626 colon tumor universal normal control PR09883 colon tumor universal normal control PR019670 colon tumor universal normal control PR019624 colon tumor universal normal control PR019680 colon tumor universal normal control PR019675 colon tumor universal normal control PR09834 colon tumor universal normal control PR09744 colon tumor universal normal control PR019644 colon tumor universal normal control SS PR019625 colon tumor universal normal control Table 8 (coot') Molecule is overex~essed in: as comRared to:

PR019597 colon tumor universal normal control PR016090 colon tumor universal normal control PR019576 colon tumor w niversal normal control PR019646 colon tumor universal normal control PR019814 colon tumor universal normal control PR019669 colon tumor universal normal control PR019818 colon tumor universal normal control PR020088 colon tumor universal normal control PR016089 colon tumor ~ ~ wniversal normal control PR020025 colon tumor universal normal control PR020040 colon tumor universal normal control PR01760 adrenal tumor universal normal control PR01760 breast tumor universal normal control PR01760 cervical tumor universal normal control PR01760 colon tumor universal normal control PR01760 liver tumor universal normal control PR01760 lung tumor universal normal control PR01760 prostate tumor universal normal control PR01760 rectal tumor universal normal control PR06029 adrenal tumor universal normal control PR06029 colon tumor universal nozxnal control PR06029 prostate tumor universal normal control PRO1801 colon tumor universal normal control PR01801 lung tumor universal normal control LA PRESENTS P:~RTIE DE CETTE DEI~L~.NDE OU CE BREVETS
CO1~IPREND PLUS D'U'N TOIYIE.
CECI EST LE TOiYIE ~ DE
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des Brevets.
~UlVI~~ APPEICATIOiVS / PATENTS
TIiIS SECTION OF THE APPLICATION / P ATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUI~tE OF
NOTE: For additional volumes please contact the Canadian Patent Office.

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:456.

2. Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence shown in SEQ ID NO:455.

3. Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the nucleotide sequence shown in SEQ ID NO:455.

4. Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full-length coding sequence of the DNA deposited under ATCC accession number PTA-1632.

5. A vector comprising the nucleic acid of any one of Claims 1 to 4.

6. A host cell transformed with the vector of Claim 5.

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

8. The host cell of Claim 6, wherein said cell is an E. coli.

9. The host cell of Claim 6, wherein said cell is a yeast cell.

10. A process for producing a PRO19645 (SEQ ID NO:456) polypeptide comprising culturing the host cell of Claim 6 under conditions suitable for expression of said PRO19645 polypeptide and recovering said PRO19645 polypeptide from the cell culture.

11. An isolated polypeptide having at least 80 % amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:456.

12. An isolated polypeptide having at least 80% amino acid sequence identity to an amino acid sequence encoded by the full-length coding sequence of the DNA
deposited under ATCC accession number PTA-1632.

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

14. The chimeric molecule of Claim 13, wherein said heterologous amino acid sequence is an epitope tag sequence.

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

16. An antibody which specifically binds to a polypeptide according to Claim 11.

17. The antibody of Claim 16, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody.

18. Isolated nucleic acid having at least 80% nucleic acid sequence identity to:
(a) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:456, lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in SEQ ID NO:456, with its associated signal peptide; or (c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in SEQ ID NO:456, lacking its associated signal peptide.

19. 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:456, lacking its associated signal peptide;
(b) an amino acid sequence of an extracellular domain of the polypeptide shown in SEQ ID NO:456, with its associated signal peptide; or (c) an amino acid sequence of an extracellular domain of the polypeptide shown in SEQ ID NO:456, lacking its associated signal peptide.

20. A method for detecting the presence of tumor in an mammal, said method comprising comparing the level of expression of PRO19645 polypeptide (SEQ ID
NO:456) 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 PRO19645 polypeptide in the test sample as compared to the control sample is indicative of the presence of tumor in said mammal.

21. The method of Claim 20, wherein said tumor is adrenal tumor, lung tumor, colon tumor, breast tumor, prostate tumor, rectal tumor, cervical tumor or liver tumor.

22. An oligonucleotide probe derived from the nucleotide sequence shown in SEQ
ID
NO:455.